On the Road to Digital Pathology in Denmark-National Survey and Interviews.

Digital pathology (DP) is changing pathology departments dramatically worldwide, yet globally, few departments are presently digitalized for the full diagnostic workflow. Denmark is also on the road to full digitalization countrywide, and this study aim to cover experiences during the implementation process in a national context. Thus, quantitative questionnaires were distributed to all pathology departments in Denmark (n = 13) and distributed to all professions including medical clinical directors, medical doctors (MD) and biomedical laboratory scientists (BLS). For a qualitative perspective, we interviewed four employees representing four professions. Data were collected in 2019-2020. From the questionnaire and interviews, we found strategies differed at the Danish departments with regards to ambitions, technological equipment, workflows, and involvement of type of professions. DP education was requested by personnel. Informants were in general positive toward the digital future but mainly had concerns regarding the political pressure to integrate DP before technological advances are sufficient for maintaining rational budgets, workflows, and for sustaining diagnostic quality. This study is a glance on the Danish implementation process in its early stages from personnel's point of view. It shows the complexity when large new workflow processes are to be implemented countrywide and with a large diversity of stakeholders like managers, MD, BLS, IT-professionals, and authorities. To ensure best technological and economical solutions and to maintain-or even optimize-diagnostic quality with DP and workflow alignment, we suggest superior inter- and intradepartmental communication. When implementing DP countrywide, a national working group is warranted with the variety of stakeholders represented.

Utilizing structure based drug design and metabolic soft spot identification to optimize the in vitro potency and in vivo pharmacokinetic properties leading to the discovery of novel reversible Bruton's tyrosine kinase inhibitors.

Bruton's tyrosine kinase (BTK) is an essential node on the BCR signaling in B cells, which are clinically validated to play a critical role in B-cell lymphomas and various auto-immune diseases such as Multiple Sclerosis (MS), Pemphigus, and rheumatoid arthritis (RA). Although non-selective irreversible BTK inhibitors have been approved for oncology, due to the emergence of drug resistance in B-cell lymphoma associated with covalent inhibitor, there an unmet medical need to identify reversible, selective, potent BTK inhibitor as viable therapeutics for patients. Herein, we describe the identification of Hits and subsequence optimization to improve the physicochemical properties, potency and kinome selectivity leading to the discovery of a novel class of BTK inhibitors. Utilizing Met ID and structure base design inhibitors were synthesized with increased in vivo metabolic stability and oral exposure in rodents suitable for advancing to lead optimization.

Optimization of novel reversible Bruton's tyrosine kinase inhibitors identified using Tethering-fragment-based screens.

Since the approval of ibrutinib for the treatment of B-cell malignancies in 2012, numerous clinical trials have been reported using covalent inhibitors to target Bruton's tyrosine kinase (BTK) for oncology indications. However, a formidable challenge for the pharmaceutical industry has been the identification of reversible, selective, potent molecules for inhibition of BTK. Herein, we report application of Tethering-fragment-based screens to identify low molecular weight fragments which were further optimized to improve on-target potency and ADME properties leading to the discovery of reversible, selective, potent BTK inhibitors suitable for pre-clinical proof-of-concept studies.

Neurophysiology versus clinical genetics in Rett syndrome: A multicenter study.

Many studies have attempted to establish the genotype-phenotype correlation in Rett syndrome (RTT). Cardiorespiratory measurements provide robust objective data, to correlate with each of the different clinical phenotypes. It has important implications for the management and treatment of this syndrome. The aim of this study was to correlate the genotype with the quantitative cardiorespiratory data obtained by neurophysiological measurement combined with a clinical severity score. This international multicenter study was conducted in four European countries from 1999 to 2012. The study cohort consisted of a group of 132 well-defined RTT females aged between 2 and 43 years with extended clinical, molecular, and neurophysiological assessments. Diagnosis of RTT was based on the consensus criteria for RTT and molecular confirmation. Genotype-phenotype analyses of clinical features and cardiorespiratory data were performed after grouping mutations by the same type and localization or having the same putative biological effect on the MeCP2 protein, and subsequently on eight single recurrent mutations. A less severe phenotype was seen in females with CTS, p.R133C, and p.R294X mutations. Autonomic disturbances were present in all females, and not restricted to nor influenced by one specific group or any single recurrent mutation. The objective information from non-invasive neurophysiological evaluation of the disturbed central autonomic control is of great importance in helping to organize the lifelong care for females with RTT. Further research is needed to provide insights into the pathogenesis of autonomic dysfunction, and to develop evidence-based management in RTT. © 2016 Wiley Periodicals, Inc.

Variant of Rett syndrome and CDKL5 gene: clinical and autonomic description of 10 cases.

UNLABELLED: Rett syndrome (RTT) is a severe neurodevelopmental disorder affecting almost exclusively females. The Hanefeld variant, or early-onset seizure variant, has been associated with mutations in CDKL5 gene. AIMS: In recent years more than 60 patients with mutations in the CDKL5 gene have been described in the literature, but the cardiorespiratory phenotype has not been reported. Our aim is to describe clinical and autonomic features of these girls. METHODS: 10 girls with CDKL5 mutations and a diagnosis of Hanefeld variant have been evaluated on axiological and clinical aspects. In all subjects an evaluation of the autonomic system was performed using the Neuroscope. RESULTS: Common features were gaze avoidance, repetitive head movements and hand stereotypies. The autonomic evaluation disclosed eight cases with the Forceful breather cardiorespiratory phenotype and two cases with the Apneustic breather phenotype. CONCLUSIONS: The clinical picture remains within the RTT spectrum but some symptoms are more pronounced in addition to the very early onset of seizures. The cardiorespiratory phenotype was dominated by Forceful breathers, while Feeble breathers were not found, differently from the general Rett population, suggesting a specific behavioral and cardiorespiratory phenotype of the RTT the Hanefeld variant.

[Digital pathology].

Digitalisation of pathology slides allows pathologists to make diagnoses using a high-resolution computer screen instead of a conventional microscope. In 2020/21, the four pathology departments in the Region of Southern Denmark implemented digital pathology for all histologic samples. Going digital necessitated optimisation of workflows and training of pathologists, avoiding a reduction in diagnostic quality. This review describes the process for realisation of digital pathology and its future perspectives, including artificial intelligence algorithms to be implemented.

Discovery of a potent and highly selective PDK1 inhibitor via fragment-based drug discovery.

We report the use of a fragment-based lead discovery method, Tethering with extenders, to discover a pyridinone fragment that binds in an adaptive site of the protein PDK1. With subsequent medicinal chemistry, this led to the discovery of a potent and highly selective inhibitor of PDK1, which binds in the 'DFG-out' conformation.

Statin treatment prevents increased cardiovascular and all-cause mortality associated with clarithromycin in patients with stable coronary heart disease.

In the CLARICOR trial, significantly increased cardiovascular (CV) and all-cause mortality in stable patients with coronary heart disease were observed after a short course of clarithromycin. We report on the impact of statin treatment at entry on the CV and all-cause mortality. The multicenter CLARICOR trial randomized patients to oral clarithromycin (500 mg daily; n = 2172) versus matching placebo (daily; n = 2201) for 2 weeks. Patients were followed through public databases. In the 41% patients on statin treatment at entry, no significant effect of clarithromycin was observed on CV (hazard ratio [HR], 0.68, 95% confidence interval [CI], 0.38-1.22; P = 0.20) or all-cause mortality (HR, 1.08; 95% CI, 0.71-1.65; P = 0.72) at 2.6-year follow up. In the patients not on statin treatment at entry, clarithromycin was associated with a significant increase in CV (HR, 1.90; 95% CI, 1.34-2.67; P = 0.0003; statin-clarithromycin interaction P = 0.0029) and all-cause mortality (HR, 1.33; 95% CI, 1.05-1.67; P = 0.016; statin-clarithromycin interaction P = 0.41). Multivariate analysis and 6-year follow up confirmed these results. Concomitant statin treatment in stable patients with coronary heart disease abrogated the observed increased CV mortality associated with 2 weeks of clarithromycin.

Allosteric inhibition of protein tyrosine phosphatase 1B.

Obesity and type II diabetes are closely linked metabolic syndromes that afflict >100 million people worldwide. Although protein tyrosine phosphatase 1B (PTP1B) has emerged as a promising target for the treatment of both syndromes, the discovery of pharmaceutically acceptable inhibitors that bind at the active site remains a substantial challenge. Here we describe the discovery of an allosteric site in PTP1B. Crystal structures of PTP1B in complex with allosteric inhibitors reveal a novel site located approximately 20 A from the catalytic site. We show that allosteric inhibitors prevent formation of the active form of the enzyme by blocking mobility of the catalytic loop, thereby exploiting a general mechanism used by tyrosine phosphatases. Notably, these inhibitors exhibit selectivity for PTP1B and enhance insulin signaling in cells. Allosteric inhibition is a promising strategy for targeting PTP1B and constitutes a mechanism that may be applicable to other tyrosine phosphatases.

Discovery of N-(1-Acryloylazetidin-3-yl)-2-(1H-indol-1-yl)acetamides as Covalent Inhibitors of KRASG12C.

KRAS regulates many cellular processes including proliferation, survival, and differentiation. Point mutants of KRAS have long been known to be molecular drivers of cancer. KRAS p.G12C, which occurs in approximately 14% of lung adenocarcinomas, 3-5% of colorectal cancers, and low levels in other solid tumors, represents an attractive therapeutic target for covalent inhibitors. Herein, we disclose the discovery of a class of novel, potent, and selective covalent inhibitors of KRASG12C identified through a custom library synthesis and screening platform called Chemotype Evolution and structure-based design. Identification of a hidden surface groove bordered by H95/Y96/Q99 side chains was key to the optimization of this class of molecules. Best-in-series exemplars exhibit a rapid covalent reaction with cysteine 12 of GDP-KRASG12C with submicromolar inhibition of downstream signaling in a KRASG12C-specific manner.

Clarithromycin for 2 weeks for stable coronary heart disease: 6-year follow-up of the CLARICOR randomized trial and updated meta-analysis of antibiotics for coronary heart disease.

OBJECTIVES: We have reported increased 2.6-year mortality in clarithromycin- versus placebo-exposed stable coronary heart disease patients, but meta-analysis of randomized trials in coronary heart disease patients showed no significant effect of antibiotics on mortality. Here we report the 6-year mortality of clarithromycin- versus placebo-exposed patients and updated meta-analyses. METHODS: Centrally randomized, placebo controlled multicenter trial. All parties were blinded. Analyses were by intention to treat. Meta-analyses followed the Cochrane Collaboration methodology. RESULTS: We randomized 4,372 patients with stable coronary heart disease to clarithromycin 500 mg (n = 2,172) or placebo (n = 2,200) once daily for 2 weeks. Mortality was followed through public register. Nine hundred and twenty-three patients (21.1%) died. Six-year mortality was significantly higher in the clarithromycin group (hazard ratio 1.21, 95% confidence interval 1.06-1.38). Adjustment for entry characteristics (sex, age, prior myocardial infarction, center, and smoking) did not change the results (1.18, 1.04-1.35). Addition of our data to that of other randomized trials on antibiotics for patients with coronary heart disease versus placebo/no intervention (17 trials, 25,271 patients, 1,877 deaths) showed a significantly increased relative risk of death from antibiotics of 1.10 (1.01-1.20) without heterogeneity. CONCLUSIONS: Our results stress the necessity to consider carefully the strength of the indication before administering antibiotics to patients with coronary heart disease.

Admission rates in a general practitioner-based versus a hospital specialist based, hospital-at-home model: ACCESS, an open-labelled randomised clinical trial of effectiveness.

BACKGROUND: Hospital at home (HaH) is an alternative to acute admission for elderly patients. It is unclear if should be cared for a primarily by a hospital intern specialist or by the patient's own general practitioner (GP). The study assessed whether a GP based model was more effective than a hospital specialist based model at reducing number of hospital admissions without affecting the patient's recovery or number of deaths. METHODS: Pragmatic, randomised, open-labelled multicentre parallel group trial with two arms in four municipalities, four emergency departments and 150 GPs in Southern Denmark, including + 65 years old patients with an acute medical condition that required acute hospital in-patient care. The patients were randomly assigned to hospital specialist based model or GP model of HaH care. Five physical and cognitive performance tests were performed at inclusion and after 7 days. Primary outcome was number of hospital admissions within 7 days. Secondary outcomes were number of admissions within 14, 21 and 30 days, deaths within 30 and 90 days and changes in performance tests. RESULTS: Sixty seven patients were enrolled in the GP model and 64 in the hospital specialist model. 45% in the hospital specialist arm versus 24% in the GP arm were admitted within 7 days (effect size 2.7, 95% CI 1.3-5.8; p = 0.01) and this remained significant within 30 days. No differences were found in death or changes in performance tests from day 0-7 days between the two groups. CONCLUSIONS: The GP based HaH model was more effective than the hospital specialist model in avoiding hospital admissions within 7 days among elderly patients with an acute medical condition with no differences in mental or physical recovery rates or deaths between the two models. REGISTRATION: No. NCT02422849 Registered 27 March 2015. Retrospectively registered.

Facial burning in women with leprosy, physiological or pathological?

INTRODUCTION: Eight peri-menopausal women, five with borderline lepromatous leprosy and three with borderline tuberculoid leprosy, self-referred complaining of 'burning of the face. Four were seen in 1993, three having been treated as 'menopausal'without betterment and four were seen in 1997. METHODS: Eight peri-menopausal women who self-referred because of facial burning', and seven women who self-referred for other problems had a careful review of clinical records and were assessed fully for leprosy including graded sensory skin testing of the face, and standard nerve function tests. RESULTS: On examination three in each group of four complaining of facial burning were found to have major loss of facial sensation and one had generalised neuritis without significant facial involvement. Treatment with antileprotics and steroids resulted in recovery of facial sensation, although one later became blind. Of the seven who self-referred without facial burning, five had no facial sensory loss and two had slight loss of facial sensation.

Growth and development of children of mothers with leprosy and healthy controls.

INTRODUCTION: In the pre-sulphone and early sulphone years children of leprous parents had been followed in a few prospective studies to observe the development of leprosy. No studies were made of the growth and development of these children. METHODS: A prospective, open-ended, cohort study began in 1975 with follow-up of both mothers and their children until 2003. 156 pregnancies were studied consisting of 36 non-leprous (NL), 25 tuberculoid and borderline tuberculoid leprosy (TT&BT) (released-from-treatment), 18 with TT&BT (active), 42 borderline lepromatous leprosy (BL) and 35 lepromatous leprosy (LL). RESULTS: Babies of mothers with leprosy had lower birth weight, smaller placentae, grew more slowly, had more infections and higher infant mortality than those of non-leprous mothers. The findings were most marked in babies of LL mothers. Growth in childhood was uneventful, infants of LL mothers catching up by age 3.6 years. Childhood infections were common in all groups but more serious for children of lepromatous mothers. The puberty skeletal growth spurt, and, for the girls, menarche was delayed for children studied compared with a new healthy control group, with catch-up by late teens. These findings were most marked in children of lepromatous, especially LL, mothers. CONCLUSION: Impaired growth in utero and infancy is probably due to immunological factors but we could find no explanation for the delayed growth in adolescent children of LL mothers.

Health profile of highland Ethiopians in a small town in the south-western part of the country.

BACKGROUND: In conjunction with an investigation of nerve function tests of highland Ethiopians in an area of low endemicity of leprosy for 25-30 years, a comprehensive health survey was performed. METHODS: We assessed 236 subjects, 118 females and 118 males, 177 students and 59 adults (Others) aged 10-75 years from Chencha woreda. We used a detailed medical, personal and social history, anthropomorphic measurements and complete physical examination. RESULTS: We identified six areas of medical concern: Intestinal parasite and diarrhoeal diseases affected 62%, despite treatment of all students and symptomatic adults the recurrence rate was high; skin diseases/conditions affected 88%; eye injections affected 26%; dental decay affected 33%; 20% had hypertension 14% requiring treatment; 11% gave a personal and/or family history of tuberculosis, 44% gave a history of BCG immunisation, while 34% had BCG scars, the lowest percentage (11%) being in students from Daramallo woreda. RECOMMENDATIONS: Supplies of clean water, effective disposal of human excreta and control of flies would greatly reduce intestinal, skin and eye infections. Regular tooth cleaning and possibly fluoridation of drinking water should improve dental health, with diet and exercise for young hypertensives. BCG immunisation in Chencha and Daramallo woredas (inaccessible areas) needs urgent attention.

Thermal threshold tester, a useful tool for detection of very early nerve damage--determination of normal values in a healthy population unexposed to Mycobacterium leprae and its application in the A9 study.

INTRODUCTION: In Ethiopia, a large percentage of leprosy patients present with established nerve damage. Present techniques for measuring nerve function impairment show no abnormality until 30% of nerve axons are destroyed. Nerve damage in leprosy occurs first in small diameter unmyelinated fibres, then in small myelinated fibres, and much later in large myelinated fibres. The Thermal Threshold Tester (TTT) was used to measure function in nerves carrying heat sensation (unmyelinated C fibres) and cold sensation (thinly myelinated Adelta fibres). PATIENTS: A school and community health survey, assessed 234 students and adults aged 10-75 years from Chencha Woreda, an area with low endemicity of leprosy. A group of students in Addis Ababa, exposed to leprosy, were also studied. RESULTS: The upper limits of normal were: wrist hot threshold (HT): 0.17 degrees C, wrist cold threshold (CT): 0.19 degrees C, foot HT: 0.17 degrees C and foot CT: 0.20 degrees C. Both the leprosy group and also controls in Addis Ababa showed significantly increased TTT values. CONCLUSION: The TTT detects nerve damage before clinical neuritis occurs and is a valuable tool for early diagnosis of leprosy or detecting clinical relapse of treated patients and for sequential and quantitative monitoring of small diameter nerve function in other neuropathies.

Peripheral nerves and nerve function in highland Ethiopians.

INTRODUCTION: In Ethiopia, where leprosy has been one of the commonest causes of peripheral nerve enlargement and dysfunction, nerve functions are assessed by a battery of "physical" tests. Voluntary Muscle Test (VMT) and Graded Sensory Skin Test (STG) are standard tests used for persons with leprosy. Normal values for nerve function tests (NFT) in Highland Ethiopians have not previously been determined, but have been taken from standard textbooks. In this study, normal values for NFT were determined by VMT, STG, 2-Point Discrimination Tests both static and moving, and Thermal Threshold Test. Physiological enlargement of right ulnar and radial-cutaneous nerves has been recognised by some leprologists, but we were unable to find written records in the available medical literature. MATERIALS: We assessed 236 students and adults aged 10-75 years from Chencha Woreda, an area with low endemicity of leprosy for 25-30 years. Two affected by leprosy were excluded from the analysis. RESULTS: NFT thresholds were affected variously by age, exercise and skin factors, domicile and exposure to organo-phosphates. Nerve size was affected by age, gender, exercise, skin fold thickness, body mass index. Exercise related physiological nerve enlargement has been documented. CONCLUSION: These data provide a usefull baseline for investigation of peripheral nerve function in highland Ethiopians.

Safety at The William Quarrier Scottish Epilepsy Centre.

PURPOSE: We examined the yield from EMFIT bed alarms and staff response time to generalised seizure in a medium term residential assessment unit for epilepsy. METHODS: The Scottish Epilpesy Centre (SEC) has a Video Observation System (VOS) that provides continuous recording of all patient spaces (external and internal) and allows retention of clinically relevant events. A retrospective audit of daily EMFIT test records, nursing seizure record sheets (seizure type and EMFIT alert status), clinical incident reporting systems and the VOS database of retained clinical events was conducted for an 9 month period from April 1st 2016 till December 31st 2016. All generalized tonic clonic seizures (GTCS) were noted by patient, time and location and staff response time to GTCS was calculated. RESULTS: There were 85 people admitted during the audit period who had 61 GTCS. 50 events were in bed and EMFIT alert status was recorded. On 8 occasions the EMFIT did not alert: 5 events were not of sufficient duration or frequency, in 2 the patient fell from the bed early and 1 event the alarm did not trigger. The average response time to GTCS was 23s. The longest response time was 69s (range, 0-69s, sd 15.76.). CONCLUSIONS: The EMFIT bed alarm appears to be a valuable adjunct to safety systems. Within the novel environment of the SEC it is possible to maintain a response time to GTCS that is comparable to hospital based UK video telemetry units.

Management of a severe forceful breather with Rett syndrome using carbogen.

We have used a novel neurophysiological technique in the NeuroScope system in combination with conventional electroencephalography (EEG) to monitor both brainstem and cortical activity simultaneously in real-time in a girl with Rett syndrome. The presenting clinical features in our patient were severe sleep disturbances, irregular breathing in the awake state dominated by Valsalva's type of breathing followed by tachypnoea and very frequent attacks of seizures and vacant spells. Our novel neurophysiological data showed that the patient was a Forceful Breather according to the breathing categories in Rett syndrome. She had frequent abnormal spontaneous brainstem activation (ASBA) preceded by severe attacks of hypocapnoea, which was caused by a combination of Valsalva's type of breathing and tachypnoea and all these together were responsible for the seizures and non-epileptic vacant spells. The ASBA was not detectable in conventional EEG and there were no epileptiform changes in the EEG during the seizures and vacant spells caused by the hypocapnic attacks, therefore these were pseudo-seizures. The record of brainstem activity confirmed that these were autonomic events, a kind of "brainstem epilepsy". We successfully treated the sleep disturbance with Pipamperone, a 5-hydroxytryptophan antagonist of receptor type 2 and we prevented the severe hypocapnoea during Valsalva's type of breathing and during tachypnoea using carbogen (a mixture of 5% carbon dioxide and 95% oxygen), which we gave by inhalation. Our treatment drastically reduced the autonomic events, promoted whole night sleep and significantly improved the quality of life in our patient. She can now participate in normal family activity which was previously impossible before treatment.

Making drugs on proteins: site-directed ligand discovery for fragment-based lead assembly.

Rapid progress in genomics and proteomics has provided a wealth of new targets for the pharmaceutical industry, even as many older targets still remain challenging for small-molecule drug discovery. Fragment-based lead discovery, in which leads are built progressively by expanding or combining small fragments, is a rapidly growing field that offers potential advantages over traditional lead-discovery processes. However, identifying and assembling the fragments themselves can be challenging. Here, we review the concept of site-directed ligand discovery, in which a covalent bond is used to stabilize the interaction between a low-affinity fragment and a target protein. We also describe how this technique can facilitate fragment-based lead discovery and help overcome some of the limitations of traditional screening methods.