A deep-learning-based model for assessment of autoimmune hepatitis from histology: AI(H).

Histological assessment of autoimmune hepatitis (AIH) is challenging. As one of the possible results of these challenges, nonclassical features such as bile-duct injury stays understudied in AIH. We aim to develop a deep learning tool (artificial intelligence for autoimmune hepatitis [AI(H)]) that analyzes the liver biopsies and provides reproducible, quantifiable, and interpretable results directly from routine pathology slides. A total of 123 pre-treatment liver biopsies, whole-slide images with confirmed AIH diagnosis from the archives of the Institute of Pathology at University Hospital Basel, were used to train several convolutional neural network models in the Aiforia artificial intelligence (AI) platform. The performance of AI models was evaluated on independent test set slides against pathologist's manual annotations. The AI models were 99.4%, 88.0%, 83.9%, 81.7%, and 79.2% accurate (ratios of correct predictions) for tissue detection, liver microanatomy, necroinflammation features, bile duct damage detection, and portal inflammation detection, respectively, on hematoxylin and eosin-stained slides. Additionally, the immune cells model could detect and classify different immune cells (lymphocyte, plasma cell, macrophage, eosinophil, and neutrophil) with 72.4% accuracy. On Sirius red-stained slides, the test accuracies were 99.4%, 94.0%, and 87.6% for tissue detection, liver microanatomy, and fibrosis detection, respectively. Additionally, AI(H) showed bile duct injury in 81 AIH cases (68.6%). The AI models were found to be accurate and efficient in predicting various morphological components of AIH biopsies. The computational analysis of biopsy slides provides detailed spatial and density data of immune cells in AIH landscape, which is difficult by manual counting. AI(H) can aid in improving the reproducibility of AIH biopsy assessment and bring new descriptive and quantitative aspects to AIH histology.

Deficient DNA base-excision repair in the forebrain leads to a sex-specific anxiety-like phenotype in mice.

BACKGROUND: Neuropsychiatric disorders, such as schizophrenia (SZ) and autism spectrum disorder (ASD), are common, multi-factorial and multi-symptomatic disorders. Ample evidence implicates oxidative stress, deficient repair of oxidative DNA lesions and DNA damage in the development of these disorders. However, it remains unclear whether insufficient DNA repair and resulting DNA damage are causally connected to their aetiopathology, or if increased levels of DNA damage observed in patient tissues merely accumulate as a consequence of cellular dysfunction. To assess a potential causal role for deficient DNA repair in the development of these disorders, we behaviourally characterized a mouse model in which CaMKIIa-Cre-driven postnatal conditional knockout (KO) of the core base-excision repair (BER) protein XRCC1 leads to accumulation of unrepaired DNA damage in the forebrain. RESULTS: CaMKIIa-Cre expression caused specific deletion of XRCC1 in the dorsal dentate gyrus (DG), CA1 and CA2 and the amygdala and led to increased DNA damage therein. While motor coordination, cognition and social behaviour remained unchanged, XRCC1 KO in the forebrain caused increased anxiety-like behaviour in males, but not females, as assessed by the light-dark box and open field tests. Conversely, in females but not males, XRCC1 KO caused an increase in learned fear-related behaviour in a cued (Pavlovian) fear conditioning test and a contextual fear extinction test. The relative density of the GABA(A) receptor alpha 5 subunit (GABRA5) was reduced in the amygdala and the dorsal CA1 in XRCC1 KO females, whereas male XRCC1 KO animals exhibited a significant reduction of GABRA5 density in the CA3. Finally, assessment of fast-spiking, parvalbumin-positive (PV) GABAergic interneurons revealed a significant increase in the density of PV+ cells in the DG of male XRCC1 KO mice, while females remained unchanged. CONCLUSIONS: Our results suggest that accumulation of unrepaired DNA damage in the forebrain alters the GABAergic neurotransmitter system and causes behavioural deficits in relation to innate and learned anxiety in a sex-dependent manner. Moreover, the data uncover a previously unappreciated connection between BER deficiency, unrepaired DNA damage in the hippocampus and a sex-specific anxiety-like phenotype with implications for the aetiology and therapy of neuropsychiatric disorders.

Identification of disease-promoting stromal components by comparative proteomic and transcriptomic profiling of canine mammary tumors using laser-capture microdissected FFPE tissue.

Cancer-associated stroma (CAS) profoundly influences progression of tumors including mammary carcinoma (mCA). Canine simple mCA represent relevant models of human mCA, notably also with respect to CAS. While transcriptomic changes in CAS of mCA are well described, it remains unclear to what extent these translate to the protein level. Therefore, we sought to gain insight into the proteomic changes in CAS and compare them with transcriptomic changes in the same tissue. To this end, we analyzed CAS and matched normal stroma using laser-capture microdissection (LCM) and LC-MS/MS in a cohort of 14 formalin-fixed paraffin embedded (FFPE) canine mCAs that we had previously characterized using LCM-RNAseq. Our results reveal clear differences in protein abundance between CAS and normal stroma, which are characterized by changes in the extracellular matrix, the cytoskeleton, and cytokines such as TNF. The proteomics- and RNAseq-based analyses of LCM-FFPE show a substantial degree of correlation, especially for the most deregulated targets and a comparable activation of pathways. Finally, we validate transcriptomic upregulation of LTBP2, IGFBP2, COL6A5, POSTN, FN1, COL4A1, COL12A1, PLOD2, COL4A2, and IGFBP7 in CAS on the protein level and demonstrate their adverse prognostic value for human breast cancer. Given the relevance of canine mCA as a model for the human disease, our analysis substantiates these targets as disease-promoting stromal components with implications for breast cancer in both species.

Differential stromal reprogramming in benign and malignant naturally occurring canine mammary tumours identifies disease-modulating stromal components.

While cancer-associated stroma (CAS) in malignant tumours is well described, stromal changes in benign forms of naturally occurring tumours remain poorly characterized. Spontaneous canine mammary carcinomas (mCA) are viewed as excellent models of human mCA. We have recently reported highly conserved stromal reprogramming between canine and human mCA based on transcriptome analysis of laser-capture-microdissected FFPE specimen. To identify stromal changes between benign and malignant mammary tumours, we have analysed matched normal and adenoma-associated stroma (AAS) from 13 canine mammary adenomas and compared them to previous data from 15 canine mCA. Our analyses reveal distinct stromal reprogramming even in small benign tumours. While similarities between AAS and CAS exist, the stromal signature clearly distinguished adenomas from mCA. The distinction between AAS and CAS is further substantiated by differential enrichment in several hallmark signalling pathways as well as differential abundance in cellular composition. Finally, we identify COL11A1, VIT, CD74, HLA-DRA, STRA6, IGFBP4, PIGR, and TNIP1 as strongly discriminatory stromal genes between adenoma and mCA, and demonstrate their prognostic value for human breast cancer. Given the relevance of canine CAS as a model for the human disease, our approach identifies disease-modulating stromal components with implications for both human and canine breast cancer.

Next-generation RNA sequencing of FFPE subsections reveals highly conserved stromal reprogramming between canine and human mammary carcinoma.

Spontaneous canine simple mammary carcinomas (mCA) are often viewed as models of human mCA. Cancer-associated stroma (CAS) is central for initiation and progression of human cancer, and is likely to play a key role in canine tumours as well. However, canine CAS lacks characterisation and it remains unclear how canine and human CAS compare. Formalin-fixed paraffin embedded (FFPE) tissue constitutes a valuable resource of patient material, but chemical crosslinking has largely precluded its analysis by next-generation RNA sequencing (RNAseq). We have recently established a protocol to isolate CAS and normal stroma from archival FFPE tumours using laser-capture microdissection followed by RNAseq. Using this approach, we have analysed stroma from 15 canine mCA. Our data reveal strong reprogramming of canine CAS. We demonstrate a high-grade molecular homology between canine and human CAS, and show that enrichment of upregulated canine CAS genes strongly correlates with the enrichment of an independently derived human stromal signature in the TCGA breast tumour dataset. Relationships between different gene signatures observed in human breast cancer are largely maintained in the canine model, suggesting a close interspecies similarity in the network of cancer signalling circuitries. Finally, we establish the prognostic potential of the canine CAS signature in human samples, emphasising the relevance of studying canine CAS as a model of the human disease. In conclusion, we provide a proof-of-principle to analyse specific subsections of FFPE tissue by RNAseq, and compare stromal gene expression between human and canine mCA to reveal molecular drivers in CAS supporting tumour growth and malignancy.

An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.

BACKGROUND: Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult. METHODS: We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step. RESULTS: We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well. CONCLUSIONS: We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.

Analysis of Gene Expression Signatures in Cancer-Associated Stroma from Canine Mammary Tumours Reveals Molecular Homology to Human Breast Carcinomas.

Cancer-associated stroma (CAS) plays a key role in cancer initiation and progression. Spontaneously occurring canine mammary carcinomas are viewed as excellent models of human breast carcinomas. Considering the importance of CAS for human cancer, it likely plays a central role in canine tumours as well. So far, however, canine CAS lacks characterisation, and it remains unclear whether the biology between CAS from canine and human tumours is comparable. In this proof-of-principle study, using laser-capture microdissection, we isolated CAS and normal stroma from 13 formalin-fixed paraffin embedded canine simple mammary carcinomas and analysed the expression of seven known human CAS markers by RT-qPCR (Reverse Transcription quantitative PCR) and validated some targets by immunohistochemistry. We found that Col1a1 (Collagen1α1), αSMA (alpha Smooth Muscle Actin), FAP (Fibroblast activation protein), PDGFRß (Platelet-derived growth factor receptor beta), and Caveolin-1 were significantly upregulated in canine CAS, and the expression of CXCL12 (Stromal cell derived factor 1) significantly decreased, whereas MMP2 (Matrix Metalloproteinase 1) and IL6 (Interleukin 6) did not change. Our results suggest strong similarities in CAS biology in canine and human mammary carcinomas but also reveal some differences. To the best of our knowledge, this is the first report to provide a comprehensive expression analysis of the most important CAS markers in canine simple mammary carcinomas and further supports the validity of the dog as model for human cancer.

The effect of acupuncture on pain score after open kidney surgery.

BACKGROUND: The pain after nephrectomy, just as any other surgery, is one of the problems that surgeons confront. The aim of this study was to evaluate the relieving effect of acupuncture on the pain experienced after nephrectomy. METHODS: This was a clinical trial that was performed on 30 patients with mean age of 40.8 ± 12.54 who were candidates for nephrectomy. After matching for gender and age, the patients were divided into either acupuncture or control group. In acupuncture group, four points were stimulated for 30 minutes, and in control group, other points were stimulated ineffectively for 30 min. Then, the pain experienced in the first six hours after the surgery was registered by a blind observer based on visual analog scale. Data were analyzed using SPSS software and the t-test. RESULTS: Our results showed that the severity of pain in the acupuncture group was significantly lower than that in the control group. Also, there were six patients who required opioids in the acupuncture group, but there were 12 such patients in the control group. The mean opiate used in the control group was significantly greater than the mean for the acupuncture group. CONCLUSIONS: Acupuncture can reduce the severity of pain and the demand for opioids in patients who have undergone nephrectomy.

The Incidence of Intravascular Needle Entrance during Inferior Alveolar Nerve Block Injection.

BACKGROUND AND AIMS: Dentists administer thousands of local anesthetic injections every day. Injection to a highly vascular area such as pterygomandibular space during an inferior alveolar nerve block has a high risk of intravascular needle entrance. Accidental intravascular injection of local anesthetic agent with vasoconstrictor may result in cardiovascular and central nervous system toxicity, as well as tachycardia and hypertension. There are reports that indicate aspiration is not performed in every injection. The aim of the present study was to assess the incidence of intravascular needle entrance in inferior alveolar nerve block injections. MATERIALS AND METHODS: Three experienced oral and maxillofacial surgeons performed 359 inferior alveolar nerve block injections using direct or indirect techniques, and reported the results of aspiration. Aspirable syringes and 27 gauge long needles were used, and the method of aspiration was similar in all cases. Data were analyzed using t-test. RESULTS: 15.3% of inferior alveolar nerve block injections were aspiration positive. Intravascular needle entrance was seen in 14.2% of cases using direct and 23.3% of cases using indirect block injection techniques. Of all injections, 15.8% were intravascular on the right side and 14.8% were intravascular on the left. There were no statistically significant differences between direct or indirect block injection techniques (P = 0.127) and between right and left injection sites (P = 0.778). CONCLUSION: According to our findings, the incidence of intravascular needle entrance during inferior alveolar nerve block injection was relatively high. It seems that technique and maneuver of injection have no considerable effect in incidence of intravascular needle entrance.