Quantifying tumour-infiltrating lymphocyte subsets : a practical immuno-histochemical method

Background: Efficient histological quantification of tumour-infiltrating T and B lymphocyte (TIL) subsets in archival tissues would greatly facilitate investigations of the role of TIL in human cancer biology. We sought to develop such a method. Methods: Ten ×40 digital images of 4 μ sections of 16 ductal invasive breast carcinomas immunostained for CD3, CD4, CD8, and CD20 were acquired (a total of 640 images). The number of pixels in each image matching a partition of Lab colour space corresponding to immunostained cells were counted using the ‘Color range’ and ‘Histogram’ tools in Adobe Photoshop 7. These pixel counts were converted to cell counts per mm2 using a calibration factor derived from one, two, three or all 10 images of each case/antibody combination. Results: Variations in the number of labelled pixels per immunostained cell made individual calibration for each case/antibody combination necessary. Calibration based on two fields containing the most labelled pixels gave a cell count minimally higher (+ 5.3%) than the count based on 10-field calibration, with 95% confidence limits − 14.7 to + 25.3%. As TIL density could vary up to 100-fold between cases, this accuracy and precision are acceptable. Conclusion: The methodology described offers sufficient accuracy, precision and efficiency to quantify the density of TIL sub-populations in breast cancer using commonly available software, and could be adapted to batch processing of image files

Heart failure-associated anemia: bone marrow dysfunction and response to erythropoietin

Heart failure (HF)-associated anemia is common and has a poor outcome. Because bone marrow (BM) dysfunction may contribute to HF-associated anemia, we first investigated mechanisms of BM dysfunction in an established model of HF, the transgenic REN2 rat, which is characterized by severe hypertrophy and ventricular dilatation and SD rats as controls. Secondly, we investigated whether stimulation of hematopoiesis with erythropoietin (EPO) could restore anemia and BM dysfunction. After sacrifice, erythropoietic precursors (BFU-E) were isolated from the BM and cultured for 10 days. BFU-E were quantified and transcript abundance of genes involved in erythropoiesis were assayed. Number of BFU-E were severely decreased in BM of REN2 rats compared to SD rats (50 ± 6.2 vs. 6.4 ± 1.7, p < 0.01). EPO treatment increased hematocrit in the SD-EPO group (after 6 weeks, 49 ± 1 vs. 58 ± 1%, p < 0.01); however, in the mildly anemic REN2 rats, there was no effect (43 ± 1 vs. 44 ± 1%). This was paralleled by a 67% decrease in BFU-E in BM of REN2 rats compared to SD (p < 0.01). EPO significantly improved BFU-E in both SD and REN2 but could not restore this to control levels in the REN2 rats. Expression of several genes involved in differentiation (LMO2), mobilization (SDF-1), and iron incorporation (transferrin receptor) of the BM were differentially expressed in REN2 rats compared to SD rats, and EPO did not normalize this. Altogether, these results suggest that BM dysfunction is an important contributor to HF-associated anemia and that EPO is not an effective agent to treat HF-associated anemia

The relationship of objectively measured physical activity and sedentary behaviour with gestational weight gain and birth weight

OBJECTIVE: To evaluate the relationship of physical activity (PA) and sedentary behaviour with gestational weight gain (GWG) and birth weight. DESIGN: Combined data from two prospective studies: (1) nulliparous pregnant women without BMI restrictions and (2) overweight and obese pregnant women at risk for gestational diabetes. METHODS: Daily PA and sedentary behaviour were measured with an accelerometer around 15 and at 32-35 weeks of gestation. The association between time spent in moderate-to-vigorous PA (MVPA) and in sedentary activities with GWG and birth weight was determined. Main outcome measures were GWG between 15 and 32 weeks of gestation, average GWG per week, and birth weight. RESULTS: We studied 111 women. Early in pregnancy, 32% of women spent ≥ 30 minutes/day in at least moderate PA versus 12% in late pregnancy. No significant associations were found between time spent in MVPA or sedentary behaviour with GWG or birth weight. CONCLUSIONS: We found no relation between MVPA and sedentary behaviour with GWG or birth weight. The small percentage of women meeting the recommended levels of PA indicates the need to inform and support pregnant women to maintain regular PA, as there seems to be no adverse effect on birth weight and maintaining PA increases overall health.Anneloes E. Ruifrok, Ellen Althuizen, Nicolette Oostdam, Willem van Mechelen, Ben Willem Mol, Christianne J. M. de Groot and Mireille N. M. van Poppe

Visual parameter optimisation for biomedical image processing

Background: Biomedical image processing methods require users to optimise input parameters to ensure high quality output. This presents two challenges. First, it is difficult to optimise multiple input parameters for multiple input images. Second, it is difficult to achieve an understanding of underlying algorithms, in particular, relationships between input and output. Results: We present a visualisation method that transforms users’ ability to understand algorithm behaviour by integrating input and output, and by supporting exploration of their relationships. We discuss its application to a colour deconvolution technique for stained histology images and show how it enabled a domain expert to identify suitable parameter values for the deconvolution of two types of images, and metrics to quantify deconvolution performance. It also enabled a breakthrough in understanding by invalidating an underlying assumption about the algorithm. Conclusions: The visualisation method presented here provides analysis capability for multiple inputs and outputs in biomedical image processing that is not supported by previous analysis software. The analysis supported by our method is not feasible with conventional trial-and-error approaches

A metabolite-derived protein modification integrates glycolysis with KEAP1-NRF2 signalling.

Mechanisms that integrate the metabolic state of a cell with regulatory pathways are necessary to maintain cellular homeostasis. Endogenous, intrinsically reactive metabolites can form functional, covalent modifications on proteins without the aid of enzymes1,2, and regulate cellular functions such as metabolism3-5 and transcription6. An important 'sensor' protein that captures specific metabolic information and transforms it into an appropriate response is KEAP1, which contains reactive cysteine residues that collectively act as an electrophile sensor tuned to respond to reactive species resulting from endogenous and xenobiotic molecules. Covalent modification of KEAP1 results in reduced ubiquitination and the accumulation of NRF27,8, which then initiates the transcription of cytoprotective genes at antioxidant-response element loci. Here we identify a small-molecule inhibitor of the glycolytic enzyme PGK1, and reveal a direct link between glycolysis and NRF2 signalling. Inhibition of PGK1 results in accumulation of the reactive metabolite methylglyoxal, which selectively modifies KEAP1 to form a methylimidazole crosslink between proximal cysteine and arginine residues (MICA). This posttranslational modification results in the dimerization of KEAP1, the accumulation of NRF2 and activation of the NRF2 transcriptional program. These results demonstrate the existence of direct inter-pathway communication between glycolysis and the KEAP1-NRF2 transcriptional axis, provide insight into the metabolic regulation of the cellular stress response, and suggest a therapeutic strategy for controlling the cytoprotective antioxidant response in several human diseases

The erythropoietin receptor expressed in skeletal muscle is essential for mitochondrial biogenesis and physiological exercise

Erythropoietin (EPO) is a haematopoietic hormone that regulates erythropoiesis, but the EPO-receptor (EpoR) is also expressed in non-haematopoietic tissues. Stimulation of the EpoR in cardiac and skeletal muscle provides protection from various forms of pathological stress, but its relevance for normal muscle physiology remains unclear. We aimed to determine the contribution of the tissue-specific EpoR to exercise-induced remodelling of cardiac and skeletal muscle. Baseline phenotyping was performed on left ventricle and m. gastrocnemius of mice that only express the EpoR in haematopoietic tissues (EpoR-tKO). Subsequently, mice were caged in the presence or absence of a running wheel for 4 weeks and exercise performance, cardiac function and histological and molecular markers for physiological adaptation were assessed. While gross morphology of both muscles was normal in EpoR-tKO mice, mitochondrial content in skeletal muscle was decreased by 50%, associated with similar reductions in mitochondrial biogenesis, while mitophagy was unaltered. When subjected to exercise, EpoR-tKO mice ran slower and covered less distance than wild-type (WT) mice (5.5 ± 0.6 vs. 8.0 ± 0.4 km/day, p < 0.01). The impaired exercise performance was paralleled by reductions in myocyte growth and angiogenesis in both muscle types. Our findings indicate that the endogenous EPO-EpoR system controls mitochondrial biogenesis in skeletal muscle. The reductions in mitochondrial content were associated with reduced exercise capacity in response to voluntary exercise, supporting a critical role for the extra-haematopoietic EpoR in exercise performance. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00424-021-02577-4

PINCH is an independent prognostic factor in rectal cancer patients without preoperative radiotherapy - a study in a Swedish rectal cancer trial of preoperative radiotherapy

<p>Abstract</p> <p>Background</p> <p>The clinical significance between particularly interesting new cysteine-histidine rich protein (PINCH) expression and radiotherapy (RT) in tumours is not known. In this study, the expression of PINCH and its relationship to RT, clinical, pathological and biological factors were studied in rectal cancer patients.</p> <p>Methods</p> <p>PINCH expression determined by immunohistochemistry was analysed at the invasive margin and inner tumour area in 137 primary rectal adenocarcinomas (72 cases without RT and 65 cases with RT). PINCH expression in colon fibroblast cell line (CCD-18 Co) was determined by western blot.</p> <p>Results</p> <p>In patients without RT, strong PINCH expression at the invasive margin of primary tumours was related to worse survival, compared to patients with weak expression, independent of TNM stage and differentiation (<it>P </it>= 0.03). No survival relationship in patients with RT was observed (<it>P </it>= 0.64). Comparing the non-RT with RT subgroup, there was no difference in PINCH expression in primary tumours (invasive margin (<it>P </it>= 0.68)/inner tumour area (<it>P </it>= 0.49). In patients with RT, strong PINCH expression was related to a higher grade of LVD (lymphatic vessel density) (<it>P </it>= 0.01)</p> <p>Conclusions</p> <p>PINCH expression at the invasive margin was an independent prognostic factor in patients without RT. RT does not seem to directly affect the PINCH expression.</p

Study protocol: differential effects of diet and physical activity based interventions in pregnancy on maternal and fetal outcomes—individual patient data (IPD) meta-analysis and health economic evaluation

Abstract Background Pregnant women who gain excess weight are at risk of complications during pregnancy and in the long term. Interventions based on diet and physical activity minimise gestational weight gain with varied effect on clinical outcomes. The effect of interventions on varied groups of women based on body mass index, age, ethnicity, socioeconomic status, parity, and underlying medical conditions is not clear. Our individual patient data (IPD) meta-analysis of randomised trials will assess the differential effect of diet- and physical activity-based interventions on maternal weight gain and pregnancy outcomes in clinically relevant subgroups of women. Methods/design Randomised trials on diet and physical activity in pregnancy will be identified by searching the following databases: MEDLINE, EMBASE, BIOSIS, LILACS, Pascal, Science Citation Index, Cochrane Database of Systematic Reviews, Cochrane Central Register of Controlled Trials, Database of Abstracts of Reviews of Effects, and Health Technology Assessment Database. Primary researchers of the identified trials are invited to join the International Weight Management in Pregnancy Collaborative Network and share their individual patient data. We will reanalyse each study separately and confirm the findings with the original authors. Then, for each intervention type and outcome, we will perform as appropriate either a one-step or a two-step IPD meta-analysis to obtain summary estimates of effects and 95% confidence intervals, for all women combined and for each subgroup of interest. The primary outcomes are gestational weight gain and composite adverse maternal and fetal outcomes. The difference in effects between subgroups will be estimated and between-study heterogeneity suitably quantified and explored. The potential for publication bias and availability bias in the IPD obtained will be investigated. We will conduct a model-based economic evaluation to assess the cost effectiveness of the interventions to manage weight gain in pregnancy and undertake a value of information analysis to inform future research. Systematic review registration PROSPERO 2013: CRD4201300380

Neuronal Deletion of Caspase 8 Protects against Brain Injury in Mouse Models of Controlled Cortical Impact and Kainic Acid-Induced Excitotoxicity

system. mice demonstrated superior survival, reduced seizure severity, less apoptosis, and reduced caspase 3 processing. Uninjured aged knockout mice showed improved learning and memory, implicating a possible role for caspase 8 in cognitive decline with aging.Neuron-specific deletion of caspase 8 reduces brain damage and improves post-traumatic functional outcomes, suggesting an important role for this caspase in pathophysiology of acute brain trauma