T cells isolated from patients with checkpoint inhibitor-resistant melanoma are functional and can mediate tumor regression.

Background: Almost half of the patients with metastatic melanoma obtain only short-term or no benefit at all from checkpoint inhibitor (CPI) immunotherapy. In this study, we investigated whether the immune system of patients progressing following CPI treatment was able to generate functional tumor-specific immune responses. Materials and methods: Tumor-infiltrating lymphocytes (TILs) were isolated and expanded from metastatic melanoma lesions which progressed during or after anti-programmed cell death protein 1 (PD)-1 and anti-Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) treatment. Tumor-specific immune responses were assessed with co-culture assays of TILs and autologous tumor cells. Results: TILs from 23 metastases of individual patients could be assessed for T cells recognition of autologous tumor cells. All metastases were progressive on or following anti-PD-1 (23/23, 100%), and the majority also after anti-CTLA-4 (17/23, 74%). Functional antitumor immune responses were detected in 19/23 patients (83%). Both CD8+ (in 18/23 patients, 78%) and CD4+ (in 16/23 patients, 70%) TILs were able to recognize autologous tumors. A large fraction of CD8+ TILs (median 23%, range 1.0%-84%) recognized tumor cells. This is similar to the cohorts of unselected patient populations with metastatic melanoma presented in previous studies. The localization of intratumoral immune infiltrates was heterogeneous among samples. In a phase I/II clinical trial, TILs were administered with lymphodepleting chemotherapy, pegIFNα2b and interleukin-2 to 12 patients with CPI-resistant melanoma. Out of 12 patients who previously failed CPI therapy, treatment with TILs resulted in two partial responses, of which one is ongoing. Conclusions: Tumor-reactive T cells appear to heavily infiltrate the tumor microenvironment of patients who failed previous CPI treatment. These patients can still respond to an infusion of unselected autologous TILs. Our results warrant further testing of novel immune re-activation strategies in melanoma patients who failed multiple CPI therapy.

The headache under-response to treatment (HURT) questionnaire, an outcome measure to guide follow-up in primary care: development, psychometric evaluation and assessment of utility.

BACKGROUND: Headache disorders are both common and burdensome but, given the many people affected, provision of health care to all is challenging. Structured headache services based in primary care are the most efficient, equitable and cost-effective solution but place responsibility for managing most patients on health-care providers with limited training in headache care. The development of practical management aids for primary care is therefore a purpose of the Global Campaign against Headache. This manuscript presents an outcome measure, the Headache Under-Response to Treatment (HURT) questionnaire, describing its purpose, development, psychometric evaluation and assessment for clinical utility. The objective was a simple-to-use instrument that would both assess outcome and provide guidance to improving outcome, having utility across the range of headache disorders, across clinical settings and across countries and cultures. METHODS: After literature review, an expert consensus group drawn from all six world regions formulated HURT through item development and item reduction using item-response theory. Using the American Migraine Prevalence and Prevention Study's general-population respondent panel, two mailed surveys assessed the psychometric properties of HURT, comparing it with other instruments as external validators. Reliability was assessed in patients in two culturally-contrasting clinical settings: headache specialist centres in Europe (n = 159) and primary-care centres in Saudi Arabia (n = 40). Clinical utility was assessed in similar settings (Europe n = 201; Saudi Arabia n = 342). RESULTS: The final instrument, an 8-item self-administered questionnaire, addressed headache frequency, disability, medication use and effect, patients' perceptions of headache "control" and their understanding of their diagnoses. Psychometric evaluation revealed a two-factor model (headache frequency, disability and medication use; and medication efficacy and headache control), with scale properties apparently stable across disorders and correlating well and in the expected directions with external validators. The literature review found few instruments linking assessment to clinical advice or suggested actions: HURT appeared to fill this gap. In European specialist care, it showed utility as an outcome measure across headache disorders. In Saudi Arabian primary care, HURT (translated into Arabic) was reliable and responsive to clinical change. CONCLUSIONS: With demonstrated validity and clinical utility across disorders, cultures and settings, HURT is available for clinical and research purposes.

Bedside ultrasound of the neck confirms endotracheal tube position in emergency intubations.

PURPOSE: In controlled environments such as the operating room, bedside ultrasound (BUS) of the neck has shown high accuracy for distinguishing endotracheal (ETI) from esophageal intubations. We sought to determine the accuracy of BUS for endotracheal tube (ETT) position in the emergency department (ED) setting. MATERIALS AND METHODS: We assessed the utility of BUS in a single-center observational study in an ED setting. BUS was performed either simultaneously with ED intubation (S/ED), within < 3 minutes of ED intubation (A/ED), or in < 3 minutes of patient's ED arrival after pre-hospital intubation (A/EMS). Trained ED providers performed BUS; intubators were blinded to ultrasound findings. We used Cormack and Lehane categories (CL) to classify intubation attempts as "easy" (CL-I/II), "moderate" (CL-III) and "difficult" (CL-IV). Additional data included the diagnostic accuracy of the sonographer and intubator compared to the clinical outcome, anatomy identified by sonography and time to diagnosis. RESULTS: During a 10-month period, 89 subjects with 115 intubation attempts were included in the study, and 86 patients/101 attempts with complete data were used in the study (63-easy, 19-moderate, 19-difficult). The sonographers achieved 100 % accuracy with respect to determining the correct ETT position utilizing an anterior neck approach, while the intubators' accuracy in assessing correct tube location was 97 % compared to the clinical outcome. A blinded review of sonography findings confirmed all BUS anatomical findings. A sonographically empty esophagus was 100 % specific for endotracheal intubation, and a "double trachea sign" was 100 % sensitive and 91 % specific for esophageal intubation. The sonographic time to diagnosis was significantly faster than the intubator time to diagnosis ("easy" p < 0.001; n = 47; "moderate" p = 0.001; n = 15; "difficult" p < 0.001; n = 19); Wilcoxon test; A/EMS cases excluded). CONCLUSION: In this emergency setting, ultrasound determined ETT locations rapidly with 100 % accuracy and independently of the CL-category.

Multi-disciplinary assessment of the entrustable professional activities of surgery residents.

Purpose: Medicine is practiced in a collaborative and interdisciplinary manner. However, medical training and assessment remain largely isolated in traditional departmental silos. Two Entrustable Professional Activities (EPAs) developed by the American Board of Surgery are multidisciplinary in nature and offer a unique opportunity to study interdisciplinary assessment. Methods: EPA microassessments were collected from Surgery and Emergency Medicine (EM) faculty between July 2018 and May 2020. Differences in feedback provided by faculty were assessed using natural language processing (NLP) techniques, (1) automated algorithms; and (2) topic modeling. Summative content analysis was used to identify themes in text feedback. We developed automated coding algorithms for these themes using regular expressions. Topic modeling was performed using latent Dirichlet allocation. Results: 549 assessments were collected for two EPAs: 198 for GS Consultation and 351 for Trauma. 27 EM and 27 Surgery faculty provided assessments for 71 residents. EM faculty were significantly more likely than Surgery faculty to submit feedback coded as Communication, Demeanor, and Timeliness, (all chi-square test p-values < 0.01). No significant differences were found for Clinical Performance, Skill Level, or Areas for Improvement. Similarly, topic modeling indicated that assessments submitted by EM faculty focused on communication, timeliness, and interpersonal skills, while those submitted by Surgery faculty focused on the residents' abilities to effectively gather information and correctly diagnose the underlying pathology. Conclusions: Feedback from EM and Surgery faculty differed significantly based on NLP analyses. EPA assessments should stem from multiple sources to avoid assessment gaps and represent a more holistic picture of performance.

Modulation of keratinocyte gene expression and differentiation by PPAR-selective ligands and tetradecylthioacetic acid.

Peroxisome proliferator-activated receptors (PPARs) are pleiotropic regulators of growth and differentiation of many cell types. We have performed a comprehensive analysis of the expression of PPARs, transcriptional cofactors, and marker genes during differentiation of normal human keratinocytes using a combination of reverse transcriptase polymerase chain reaction, Northern and Western blotting, and immunohistochemistry. PPARdelta was the predominant PPAR subtype in human keratinocytes and highly expressed in basal cells and suprabasal cells. Induction of PPARalpha and PPARgamma expression was linked to differentiation, and accordingly, expression of PPARalpha and PPARgamma was in essence confined to suprabasal cells. Differentiation was not accompanied by significant changes in the expression of the coactivators CREB-binding protein, p300, steroid receptor coactivator 1, or the corepressors nuclear receptor corepressor and silence mediator for retinoid and thyroid hormone receptors. We critically evaluated the effects of selective PPAR ligands and a synthetic fatty acid analog, tetradecylthioacetic acid. Tetradecylthioacetic acid activated all human PPAR subtypes in the ranking order PPARdelta >> PPARalpha > PPARgamma. All selective PPAR ligands marginally induced transglutaminase-1 expression with the PPARdelta-selective ligand L165041 being the most potent. The PPARalpha- and PPARgamma-selective ligands Wy14643 and BRL49653 had negligible effect on involucrin expression, whereas a dose-dependent induction was observed with L165041. Simultaneous addition of L165041 and BRL49653 synergistically induced strong involucrin expression. Additionally, L165041 potently induced CD36 mRNA expression. Administration of tetradecylthioacetic acid resulted in a dramatic decrease in proliferation and a robust upregulation of the expression of involucrin and transglutaminase. Our results indicate that tetradecylthioacetic acid may affect keratinocyte gene expression and differentiation via PPAR-dependent and PPAR-independent pathways, and that the latter play an important role.

Defining functional drug-interaction domains on topoisomerase II by exploiting mechanistic differences between drug classes.

Topoisomerase II is the primary cellular target for a variety of antineoplastic drugs that are active against human cancers. These drugs exert their cytotoxic effects by stabilizing covalent topoisomerase II-cleaved DNA complexes that are fleeting intermediates in the catalytic cycle of the enzyme. Despite this common feature of drug action, a number of mechanistic differences between drug classes have been described. These mechanistic differences (including effects on DNA cleavage/religation, DNA strand passage, and adenosine triphosphate hydrolysis) were used as the basis for a series of competition experiments to determine whether different compounds share a common site of action on topoisomerase II or interact at distinct sites. Results of the present study strongly suggest that at least four structurally disparate antineoplastic drugs, etoposide, amsacrine, genistein, and the quinolone CP-115,953, share an overlapping interaction domain on the enzyme.

[Fetal chylothorax]. / Føtal chylothorax.

Foetal chylothorax was diagnosed in female foetus by ultrasound scanning on account of suspected twin pregnancy at the 34th week. No other signs of hydrops foetalis were found. The chylothorax reformed rapidly following intrauterine thoracocentesis which was therefore repeated immediately before Cesarean section at the 38th week in order to facilitate the perinatal cardiopulmonary adjustment. Pulmonary maturation was found to be normal. Postnatally, marked chylous effusion in the pleural cavity continued. Conservative treatment with pleural drainage and total parenteral nutrition was attempted initially. On account of the absence of response and supervening infection, it was decided to operate after the elapse of three weeks. Pleural decortication was performed with good result.

Impact of age and gender on blood pressure and low-density lipoprotein cholesterol reduction: results of a pooled analysis.

OBJECTIVES: To compare the simultaneous reduction of blood pressure (BP) to below 150 mmHg and low-density lipoprotein cholesterol (LDL-C) after treatment with single-pill amlodipine/atorvastatin (SPAA) among younger (<65 years), older (≥65 years) and elderly (≥75 years) men and women with hypertension and dyslipidemia. METHODS: Data from five, 14-20-week, open-label, multi-national studies (GEMINI US, GEMINI-Australia, Asia, Latin-America, Africa/Middle-East [AALA], JEWEL 1, JEWEL 2, and the Clinical Utility of Caduet in Simultaneously Achieving Blood Pressure and Lipid End Points [CAPABLE]) were pooled. In these studies, SPAA (5/10 to 10/80 mg/mg) was electively titrated to achieve study-specific targets. Reductions in BP and LDL-C, and changes in renal and liver function tests, fasting glucose and adverse event (AE) rates were compared across the three age groups. RESULTS: A total of 3613 patients (65%) were <65 years, 1946 (35%) were ≥65 years and 441 (8%) were ≥75 years. Baseline mean systolic BP tended to increase with age and diastolic BP and LDL-C decreased, p<0.001. Final mean SPAA dose was similar (7.2/23.9, 7.1/24.3, 7.1/24.0 mg/mg). Final mean BP in the younger/older/elderly groups was 128.1/79.9, 131.3/75.0, 132.8/73.4 mmHg (adjusted BP reductions -20.2/-10.4, -18.6/-12.7, -17.7/-13.2 mmHg, p<0.001). Final mean LDL-C was 91, 87, 87 mg/dl (2.4, 2.3, 2.3 mmol/l) p<0.001; adjusted %LDL-C reductions -27.1, -26.8, -26.4, p<0.001. Estimated glomerular filtration rate increased in the younger group but decreased in the older and elderly groups (p=0.005). Small increases in liver function tests and fasting glucose were observed. Discontinuations due to AEs tended to increase with age but were low in all groups (6.2%, 7.9%, 8.8%, p=0.045). Study limitations include post hoc analysis and short duration of follow-up. CONCLUSIONS: Simultaneous reduction of BP to below 150 mmHg and LDL-C using SPAA is both effective and well-tolerated among younger and older men and women, including those aged≥75 years. Clinicians may be reassured by the low proportion of AEs that led to discontinuation in all groups suggesting that older patients were not disadvantaged by this treatment.

The mitogen-activated protein kinases p38 and ERK1/2 are increased in lesional psoriatic skin.

BACKGROUND: Alterations in specific signal transduction pathways may explain the hyperproliferation and abnormal differentiation of the keratinocytes as well as the increased expression of inflammatory cytokines seen in psoriasis. Major signalling pathways used by eukaryotic cells to transduce extracellular signals into cellular responses impinge on the mitogen-activated protein kinases (MAPKs). OBJECTIVES: To investigate the expression of the MAPK p38, extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in psoriatic skin. METHODS: Keratome biopsies were taken from patients with plaque-type psoriasis. Western blot analysis was used to determine p38, ERK and JNK activity and protein levels, whereas kinase assays were used to examine the kinase activity of p38. RESULTS: We demonstrated increased levels of the phosphorylated forms of p38 and ERK1/2 in lesional psoriatic skin compared with nonlesional psoriatic skin. No abnormality was found in the activation and expression of JNK1/2. Ex vivo kinase assays confirmed the increased activation of p38, and furthermore demonstrated increased kinase activity of the p38 isoforms p38alpha, p38beta and p38delta in lesional compared with nonlesional psoriatic skin. p38gamma was not detected in the psoriatic skin. Clearance of the psoriatic lesions, induced by climatotherapy at the Dead Sea for 4 weeks, led to a normalization in the activity of both p38 and ERK1/2. CONCLUSIONS: Taken together, our results demonstrate that the activity of the MAPKs p38alpha, p38beta and p38delta and ERK1/2 are increased in lesional psoriatic skin compared with nonlesional psoriatic skin, and that clearance of psoriasis normalizes the p38 and ERK1/2 activity. Thus, p38 and ERK1/2 might be potential targets in the treatment of psoriasis.

Presynaptic events in meiocytes of Lilium longiflorum and their relation to crossing-over: a preselection hypothesis.

We are proposing a "Preselection Hypothesis" to account for the regulation of crossing-over in eukaryotic organisms. The hypothesis characterized meiosis in terms of three major physiological stages: (1) a presynaptic stage when pairs of homologous DNA stretches are selected so as to become trapped within the synaptinemal complex during synapsis, (2) an alignment of homologous chromosomes and stabilization of paired bivalents via the synaptinemal complex, and (3) a scission and rejoining of DNA stretches leading to the formation of chiasmata and crossovers. The hypothesis centers on the first stage and is based on evidence for the occurrence of significant cytological and biochemical changes prior to synapsis. The major feature of the hypothesis is that crossing-over occurs only in trapped DNA stretches. Thus, potential crossing-over sites, though not crossing-over itself, are determined well before chromosomes pair. Since, to a large degree, crossovers are distributed randomly along the length of each chromosome, the preselection process must result in a random assortment of trapped DNA stretches, the assortment differing from one meiocyte to another.

Quinolones share a common interaction domain on topoisomerase II with other DNA cleavage-enhancing antineoplastic drugs.

Topoisomerase II is the cytotoxic target for a number of clinically relevant antineoplastic drugs. Despite the fact that these agents differ significantly in structure, a previous study [Corbett, A. H., Hong, D., & Osheroff, N. (1993) J. Biol. Chem. 268, 14394-14398] indicated that the site of action for etoposide on topoisomerase II overlaps those of other DNA cleavage-enhancing drugs. Therefore, to further define interactions between drugs and the enzyme, the functional interaction domain (i.e., interaction domain defined by drug function) for quinolones on Drosophila topoisomerase II was mapped with respect to several classes of antineoplastic agents. This was accomplished by characterizing the effects of ciprofloxacin (a gyrase-targeted antibacterial quinolone) on the ability of etoposide, amsacrine, genistein, and the antineoplastic quinolone, CP-115,953, to enhance topoisomerase II-mediated DNA cleavage. Although ciprofloxacin interacts with the eukaryotic type II enzyme, it shows little ability to stimulate DNA cleavage. Ciprofloxacin attenuated cleavage enhancement by all of the above drugs. Similar results were obtained using a related quinolone, CP-80,080, as a competitor. In addition, kinetic analysis of DNA cleavage indicated that ciprofloxacin is a competitive inhibitor of CP-115,953 and etoposide. Finally, ciprofloxacin inhibited the cytotoxic actions of CP-115,953 and etoposide in mammalian cells to an extent that paralleled its in vitro attenuation of cleavage. These results strongly suggest that several structurally disparate DNA cleavage-enhancing antineoplastic drugs share an overlapping site of action on topoisomerase II. Based on the results of drug competition and mutagenesis studies, a model for the drug interaction domain on topoisomerase II is described.