Development and prognostic relevance of a histologic grading and staging system for alcohol-related liver disease.

BACKGROUND & AIMS: The SALVE Histopathology Group (SHG) developed and validated a grading and staging system for the clinical and full histological spectrum of alcohol-related liver disease (ALD) and evaluated its prognostic utility in a multinational cohort of 445 patients. METHODS: SALVE grade was described by semiquantitative scores for steatosis, activity (hepatocellular injury and lobular neutrophils) and cholestasis. The histological diagnosis of steatohepatitis due to ALD (histological ASH, hASH) was based on the presence of hepatocellular ballooning and lobular neutrophils. Fibrosis staging was adapted from the Clinical Research Network staging system for non-alcoholic fatty liver disease and the Laennec staging system and reflects the pattern and extent of ALD fibrosis. There are 7 SALVE fibrosis stages (SFS) ranging from no fibrosis to severe cirrhosis. RESULTS: Interobserver κ-value for each grading and staging parameter was >0.6. In the whole study cohort, long-term outcome was associated with activity grade and cholestasis, as well as cirrhosis with very broad septa (severe cirrhosis) (p <0.001 for all parameters). In decompensated ALD, adverse short-term outcome was associated with activity grade, hASH and cholestasis (p = 0.038, 0.012 and 0.001, respectively), whereas in compensated ALD, hASH and severe fibrosis/cirrhosis were associated with decompensation-free survival (p = 0.011 and 0.001, respectively). On multivariable analysis, severe cirrhosis emerged as an independent histological predictor of long-term survival in the whole study cohort. Severe cirrhosis and hASH were identified as independent predictors of short-term survival in decompensated ALD, and also as independent predictors of decompensation-free survival in compensated ALD. CONCLUSION: The SALVE grading and staging system is a reproducible and prognostically relevant method for the histological assessment of disease activity and fibrosis in ALD. LAY SUMMARY: Patients with alcohol-related liver disease (ALD) may undergo liver biopsy to assess disease severity. We developed a system to classify ALD under the microscope by grading ALD activity and staging the extent of liver scarring. We validated the prognostic performance of this system in 445 patients from 4 European centers.

Diagnostic and therapeutic ERß, HER2, BRCA biomakers in the histological subtypes of lung adenocarcinoma according to the IASLC/ATS/ERS classification.

Several studies revealed that non-small cell lung cancers (NSCLCs) frequently express ER, PR, HER2 and carry BRCA mutation. However, these markers in histological subtypes of lung adenocarcinoma have not been thoroughly investigated. We retrospectively evaluated a total of 640 lung adenocarcinoma samples for ERα, ERß, PR and HER2 expression by immunohistochemistry and western-blotting, for EGFR and BRCA mutation by real-time PCR and sequencing. Furthermore, HER2 amplification and mutation were explored in samples harboring immunopositivity HER2 using fluorescence in situ hybridization and real-time PCR, respectively. The micropapillary and invasive mucinous predominant adenocarcinoma were frequently detected the higher level of cytoplasmic ERß (64.9% and 56.6%), HER2 (68.1% and 60.1%) protein expression. But, amplification of HER2 was detected in only three cases (3/110, 2.7%) and 26 HER2 mutations in 110 cases were identified (23.6%) in the HER2 immunopositivity patients. Logistic regression analysis showed that cytoplasmic ERß (P = 0.032) and HER2 (P = 0.015) expression were independently associated with EGFR mutation. 8 patients (8/640, 1.25%) harbored pathogenic BRCA mutations, 6 with germline BRCA mutations and 2 with somatic BRCA1 mutations were detected with lacking ERß, PR and HER2 expression. Acinar predominant adenocarcinoma had the higher percentage of BRCA mutations than other subtypes. A systematic examination of ERß, HER2 and BRCA biomarkers could potentially be useful to diagnosis and identify patients with the histological subtypes of lung adenocarcinoma, who might benefit from the further individualized treatment of anti-hormone, anti-HER2 and/or PARP inhibitors therapeutics.

Medical Devices; Hematology and Pathology Devices; Classification of a Cervical Intraepithelial Neoplasia Test System. Final order.

The Food and Drug Administration (FDA or we) is classifying the cervical intraepithelial neoplasia (CIN) test system into class II (special controls). The special controls that apply to the device type are identified in this order and will be part of the codified language for the CIN test system's classification. We are taking this action because we have determined that classifying the device into class II (special controls) will provide a reasonable assurance of safety and effectiveness of the device. We believe this action will also enhance patients' access to beneficial innovative devices, in part by reducing regulatory burdens.

Deep UV autofluorescence microscopy for cell biology and tissue histology.

BACKGROUND INFORMATION: Autofluorescence spectroscopy is a powerful tool for molecular histology and for following metabolic processes in biological samples as it does not require labelling. However, at the microscopic scale, it is mostly limited to visible and near infrared excitation of the samples. Several interesting and naturally occurring fluorophores can be excited in the UV and deep UV (DUV), but cannot be monitored in cellulo nor in vivo due to a lack of available microscopic instruments working in this wavelength range. To fulfil this need, we have developed a synchrotron-coupled DUV microspectrofluorimeter which is operational since 2010. An extended selection of endogenous autofluorescent probes that can be excited in DUV, including their spectral characteristics, is presented. The distribution of the probes in various biological samples, including cultured cells, soft tissues, bone sections and maize stems, is shown to illustrate the possibilities offered by this system. In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology. RESULTS: To fulfil this need, we have developed a synchrotron-coupled DUV microspectrofluorimeter which is operational since 2010. An extended selection of endogenous autofluorescent probes that can be excited in DUV, including their spectral characteristics, is presented. The distribution of the probes in various biological samples, including cultured cells, soft tissues, bone sections and maize stems, is shown to illustrate the possibilities offered by this system. In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology. CONCLUSIONS: In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology.

Handcrafted Histological Transformer (H2T): Unsupervised representation of whole slide images.

Diagnostic, prognostic and therapeutic decision-making of cancer in pathology clinics can now be carried out based on analysis of multi-gigapixel tissue images, also known as whole-slide images (WSIs). Recently, deep convolutional neural networks (CNNs) have been proposed to derive unsupervised WSI representations; these are attractive as they rely less on expert annotation which is cumbersome. However, a major trade-off is that higher predictive power generally comes at the cost of interpretability, posing a challenge to their clinical use where transparency in decision-making is generally expected. To address this challenge, we present a handcrafted framework based on deep CNN for constructing holistic WSI-level representations. Building on recent findings about the internal working of the Transformer in the domain of natural language processing, we break down its processes and handcraft them into a more transparent framework that we term as the Handcrafted Histological Transformer or H2T. Based on our experiments involving various datasets consisting of a total of 10,042 WSIs, the results demonstrate that H2T based holistic WSI-level representations offer competitive performance compared to recent state-of-the-art methods and can be readily utilized for various downstream analysis tasks. Finally, our results demonstrate that the H2T framework can be up to 14 times faster than the Transformer models.

Gut lumen formation defect can cause intestinal atresia: evidence from histological studies of human embryos and intestinal atresia septum.

Intestinal atresia (IA), a common cause of neonatal intestinal obstruction, is a developmental defect, which disrupts the luminal continuity of the intestine. Here, we investigated (i) the process of lumen formation in human embryos; and (ii) how a defective lumen formation led to IA. We performed histological and histochemical study on 6-10 gestation week human embryos and on IA septal regions. To investigate the topology of embryonic intestine development, we conducted 3D reconstruction. We showed that a 6-7th gestation week embryonic gut has no lumen, but filled with mesenchyme cells and vacuoles of a monolayer of epithelial cells. A narrow gut lumen was formed by gestation week-9, the gut was filled with numerous vacuoles of different sizes, some vacuoles were merging with the developing embryonic gut wall. At gestation week-10, a prominent lumen was developed, only few vacuoles were present and were merging with the intestine wall. At IA septal regions, vacuoles were located in the submucous layer, covered by a single layer of epithelium without glandular structure, and surrounded with fibrous tissue. The mucosal epithelium was developed with lamina propria and basement membrane, but the submucosa and the longitudinal smooth muscle layers were not properly developed. Hence, the vacuoles in IA septum could represent a remnant of vacuoles of embryonic gut. In conclusion, the fusion of vacuoles with the developing intestine wall associates with the disappearance of vacuoles and gut lumen formation in human embryos, and perturbation of these developmental events could lead to IA.

Protein arginine methyltransferase 3 promotes glycolysis and hepatocellular carcinoma growth by enhancing arginine methylation of lactate dehydrogenase A.

BACKGROUND: Protein arginine methylation has emerged a pivotal role in cancer progression. However, the role of protein arginine methyltransferase 3 (PRMT3) in hepatocellular carcinoma (HCC) remains unknown. METHODS: The expression pattern of PRMT3 in HCC was analysed using quantitative real-time-polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry assays. Loss- and gain-of-function experiments were carried out to determine the oncogenic role of PRMT3 in HCC. Glucose consumption and lactate production assays, seahorse bioscience, mass spectrometry, co-immunoprecipitation, metabonomic analysis and site-specific mutation experiments were used to explore the underlying molecular mechanisms. Furthermore, a xenograft mouse model was established to investigate the effects of PRMT3 and its inhibitor, SGC707, treatment on tumour growth in vivo. RESULTS: The expression of PRMT3 was significantly upregulated in HCC, with high expression of which correlated with poor prognosis. PRMT3 knockdown led to the decrease in proliferation, glycolysis of HCC cells and tumour growth, whilst its overexpression showed opposite results. The catalytic activity of PRMT3 was important in mediating these biological processes. Mechanistically, our data showed that PRMT3 interacted with and mediated asymmetric dimethylarginine (ADMA) modification of lactate dehydrogenase A (LDHA) at arginine 112 (R112). Compared with LDHA-wild-type (LDHA-WT) cells, LDHA-R112K-mutant-expressing HCC cells exhibited a decrease in lactate dehydrogenase (LDH) activity, HCC cell glycolysis and proliferation. Furthermore, the administration of SGC707, a selective inhibitor of PRMT3, disrupted the PRMT3-mediated LDHA methylation and abolished PRMT3-induced HCC glycolysis and tumour growth. CONCLUSIONS: Our results suggested a novel oncogenic role of PRMT3 in HCC, and it could be a promising therapeutic target for HCC by linking post-translational modification and cancer metabolism.

PINK1/PRKN-dependent mitophagy in the burn injury model.

Burn injury leads to mitochondrial dysfunction and autophagy, also known as mitophagy. The alleviation of mitochondrial damage may be a potential method for the treatment of burn injury and complications. In this animal study, we analyzed the expression of mitochondrial damage- and mitophagy-related factors, specifically PINK1 and PRKN. The results showed mitochondria damage in the skin; compared with the normal control group, genes involved in the mitochondrial damage, such as Nrf-1, UQCRC2, CYC1, and NDUFA9, as well as in the mitophagy, including PINK1, PRKN, MFN1, and USP30, were differentially expressed. Furthermore, PINK1 interacted with PRKN and participated in mitophagy in the skin. In conclusion, our data reveal more about the mechanism underlying mitophagy in burns, providing a potential clinical treatment.

Phytostat for continuous culture and automatic sampling of plant-cell suspensions.

An apparatus for growing plant cells in suspension culture is described; it may be used for continuous or batch culture, and is equipped with a valve for automatic collection of samples. Aeration is by continuous bubbling of air into the culture through fritted glass. Normal culture-duplication times are from 30 to 35 hours.

Transmissible spongiform encephalopathies: assessment of exposure risk in the histological working environment using GC-MS detection of fatty acids as marker for central nervous tissues.

To elucidate the risk of occupational exposure to the agent of transmissible spongiform encephalopathies (TSE) in the histological working environment, we assessed the principal suitability of three analytical methods for the detection of tissues of the central nervous system (CNT). We tested a neuron-specific enolase (NSE) Western blot, a glial fibrillary acidic protein (GFAP) ELISA and the GC-MS detection of some CNT typical fatty acids (FAs): omega9-tetracosenic acid, omega7-tetracosenic acid, lignoceric acid, and cerebronic acid. Histological sample processing (formalin fixation, dehydration, paraffin embedding) affected both of the immunochemical approaches considerably. The NSE Western blot produced negative results without exception. The results for the GFAP ELISA were better but still far too insensitive. Thus, both methods were judged to be unsuitable in their present form without major analytical adjustment. GC-MS sensitivity remained unaffected by the formalin fixation process. Sensitivity was reduced in the course of the final dehydration step using xylene in the histological sample processing. However, this reduction was found to be rather moderate (range 42-59%) when compared to the immunochemical methods. Overall, we judged GC-MS to be a promising analytical approach for the assessment of a potential TSE exposure risk via airborne CNT particles in the histological working environment. All the FAs we tested showed very low but detectable baseline contents. Thus, cut-off values must be used in the present GC-MS approach. The most suitable FA turned out to be omega9-tetracosenic acid due to the greatest difference between its content in histological CNT samples and the respective cut-off value (689:1). Preliminary results by GC-MS monitoring of CNT via omega9-tetracosenic acid (and other FAs) on filters of routinely used vacuum cleaners and on filters after air sampling indicate that the airborne CNT/TSE exposure risk in the histological laboratory is minor if existing at all. However, further in depth studies will have to validate our preliminary findings and assess these results in the light of possible future data on human oral and/or pulmonary TSE susceptibility.

A hardware approach for histological and histopathological digital image stain normalization.

Advances in technology made the migration of pathological diagnosis to digital slides possible. As the need for objectivity and automation emerged, new computer software algorithms were proposed. Computer algorithms demand accurate color and intensity values in order to provide reliable results. The tissue samples undergo several processing steps from histological preparation to digitalization, which cannot be completely standardized. Thus, non-standardized input data generates unreliable output data. In this article, we discuss a new computational normalization algorithm for histopathological stained slides that uses a hardware color marker. The marker is added to the glass slide together with the tissue section, exposed to all the processing steps and altered in the same manner as the biological material of interest, thus becoming a solid color marker for image normalization. The results of the proposed method are numerically and perceptually tested in order to prove the advantages of the method. We conclude that our combined hardware-software technique for staining normalization of digital slides is superior to the existing methods based on only software normalization, and that its implementation will tackle not only the acquisition errors but also the technical errors that may occur during the staining process.

Radioautographology general and special.

A new concept, termed "radioautographology" is advocated and its contents are reviewed. This term is the coinage synthesized from "radioautography" and "(o)logy", expressing a new science derived from radioautography. The concept of radioautographology (RAGology) is a science to localize the radioactive substances in the biological structure of the objects and to analyze and to study the significance of these substances in the biological structure. On the other hand, the old term radioautography (RAG) or autoradiography (ARG) is the technique to demonstrate the pattern of localization of various radiolabeled compounds in biological specimens. The specimens used in biology and medicine are cells and tissues. They are fixed, sectioned and made contact with the radioautographic emulsions, exposed and developed to produce metallic silver grains. Such specimens are designated as radioautographs (or autoradiographs) and the patterns of pictures made of silver grains are named radioautograms. Those people who produced radioautographs were formerly named radioautographers (or autoradiographers) who were only technicians, while those who study RAGology are not technicians but scientists and should be called as radioautographologists. The science of radioautographology was developed in the 20th century and can be divided into two parts, general radioautographology and special radioautographology, as most natural sciences usually can. The general radioautographology is the technology of RAG which consists of 3 fields of sciences, physics concerning radioactivity, histochemistry treating the cells and tissues and photochemistry dealing with the photographic emulsions. The special radioautographology, on the other hand, consists of applications of general radioautographology to various biological and medical sciences. The applications can be classified into several scientific fields, i.e., cellular molecular biology, anatomy, histology, embryology, pathology and pharmacology. Studies carried out in our laboratory were summarized and reviewed. The results obtained from the technology includes 4-dimensional structures of the organs taking the time dimension into account by labeling cells and localizing the sites of incorporation, synthesis, discharge of the labeled compounds in connection with the time lapse and aging of animals. All the results obtained from such applications should be systematized as a new filed of science in the future in the 21st century.

A simple and cheap method for removing glass coverslips from neuronal cultures and relocating identified cells.

A simple and cheap method is described for removing glass coverslips from neuronal cultures grown on collagen and embedded into Epoxy resins for electron microscopy. The method involves scoring the coverslip with a scalpel and rubbing an ice block over the scored coverslip. A thin film of water then runs between the glass and the resin, which expands during cooling and causes the coverslip to spring away. This method is superior to the application of liquid nitrogen, which often causes damage of the embedded tissue.

Histological demonstration of initial lymphatics in human skin: comparison between different extension techniques.

Initial lymphatic vessels (IL) are difficult to demonstrate histologically in excised normal skin, as they are usually completely collapsed. In this report three different methods (vacuum extension, large traction extension, and small traction extension) are described by which human skin specimens can be mechanically extended. After extension specimens were Epon enbedded and the IL counted and their diameters measured. The greatest number of lymphatic vessels was found in specimens extended by vacuum, and the smallest number in small specimens extended by traction. The highest density of lymphatic vessels was found 50-300 microns below the epidermis. Our data reveal that vacuum extension is useful for investigating questions concerning the topography of IL, whereas preparations extended by traction are more suitable for obtaining information about single lymphatic vessels.

Creating a histology-embryology free digital image database using high-end microscopy and computer techniques for on-line biomedical education.

The development of new technology and the possibility of fast information delivery by either Internet or Intranet connections are changing education. Microanatomy education depends basically on the correct interpretation of microscopy images by students. Modern microscopes coupled to computers enable the presentation of these images in a digital form by creating image databases. However, the access to this new technology is restricted entirely to those living in cities and towns with an Information Technology (IT) infrastructure. This study describes the creation of a free Internet histology database composed by high-quality images and also presents an inexpensive way to supply it to a greater number of students through Internet/Intranet connections. By using state-of-the-art scientific instruments, we developed a Web page (http://www2.uerj.br/~micron/atlas/atlasenglish/index.htm) that, in association with a multimedia microscopy laboratory, intends to help in the reduction of the IT educational gap between developed and underdeveloped regions.

[Calculator for micrometric analysis of histological structures from negatives, photographic plates and photographs]. / Schetnoe ustroistvo dlia mikrometricheskogo analiza gistologicheskikh struktur s negativov, fotoplastinok i fotografii.

The description of a counting device for morphometric analysis of histological structures from negatives, photographic plates and photographs, assembled on the basis of the colony counter and a binocular attachment to MBC-1 microscope, is presented. The counting of dots or histological structures on a definite area is performed using an electrical pen. The results are recorded by the counter. The proposed device allows a morphometric analysis in various types of morphological investigations to be carried out rapidly and with a sufficient precision.

[Use of the microprojector MRTU 42-2216-63 for microphotography]. / Ispol'zovanie mikroproektora MRTY 42-2216-63 dlia mikrofotos'emki.

Two methods of microphotography using microprojector MPTY 42-2216-63 without using microphoto heads are proposed. In the first method, a mirror camera without objective is introduced perpendicularly to the luminous flux going from the microprojector ocular to the vertical screen. Exposure time is determined with any photoelectric exposure meter. Photographs are obtained by projection printing. In the second method, microprojector is set for demonstration of the image on a horizontal screen. Instead of the latter, a photoframe with a negative photographic plate is used. Photographs are produced by contact printing. The exposure time is determined in this method as mentioned above.

[Mobile laboratory for autopsies and histological studies]. / Peredvizhnaia laboratoraia dlia vskryti umershikh i gistologicheskikh issledovanii.

The laboratory is set up in an "Ikarus-556" bus and has two sections: histological laboratory and an autopsy room equipped with autonomous water supply and sewage systems, heating systems, and necessary medical equipment allowing one to perform autopsies of the fatal cases and histological examinations under field conditions, in district and rural hospitals, and to improve the quality of therapeutic and diagnostic work.

[Tibor Péterfi, the founder of micromanipulation]. / Péterfi Tibor, a mikrurgia megalapítója.

Tibor Péterfi (1883-1953) was an eminent and internationally renowned biologist. He made great advances in the field of experimental physiology focusing his cytological research on microscopic examination of living cells. For this task, he created a tool named micromanipulator basing the development of microsurgery and that of cell surgery as well. His histological and cytological researches took their beginning first in Kolozsvár/Cluj (then Hungary, now Romania), where he worked as an assistant of professor István Apáthy then in Budapest where he spent fruitful years under the tutorship of professor Mihály Lenhossék. His scientific career however was broken by the political persecution which followed the fall of the communist revolution in 1919. He emigrated and spent the following decades in Prague, in Jena, in Berlin and in Cambridge. The apogee however of his scientific career proved to be the period he spent in Istanbul as a guest professor of the local university. He returned home only after the war already mortally ill. His illness did not allow him to continue his activity any more. Present article evaluates Tibor Péterfi's scientific achievements based mostly on recent archival researches.

Real-time histology with the endocytoscope.

Endoscopic Imaging has progressed tremendously over the last few decades. Novel imaging technologies such as high-resolution and high-magnification white light endoscopy, narrow band imaging, optimal band imaging, autoflourescence imaging and optical coherence tomography not only aid the endoscopist in detecting malignant or pre-malignant lesions but also assist in predicting histology. Recently, the introduction of Endocytoscopy (EC) and Confocal Endomicroscopy has taken us into a new realm of diagnostic endoscopy. With the ability to magnify up to 1000 ×, cellular structures can be visualized in real-time. This advance in technology could potentially lead to a paradigm shift negating the need to obtain biopsies. EC is, however, still in the early stages of development and further research needs to be carried out before it can be accepted as standard practice. This review will focus on the diagnostic utility of the Endocytoscope.