Automated quantification and architectural pattern detection of hepatic fibrosis in NAFLD.

Accurate detection and quantification of hepatic fibrosis remain essential for assessing the severity of non-alcoholic fatty liver disease (NAFLD) and its response to therapy in clinical practice and research studies. Our aim was to develop an integrated artificial intelligence-based automated tool to detect and quantify hepatic fibrosis and assess its architectural pattern in NAFLD liver biopsies. Digital images of the trichrome-stained slides of liver biopsies from patients with NAFLD and different severity of fibrosis were used. Two expert liver pathologists semi-quantitatively assessed the severity of fibrosis in these biopsies and using a web applet provided a total of 987 annotations of different fibrosis types for developing, training and testing supervised machine learning models to detect fibrosis. The collagen proportionate area (CPA) was measured and correlated with each of the pathologists semi-quantitative fibrosis scores. Models were created and tested to detect each of six potential fibrosis patterns. There was good to excellent correlation between CPA and the pathologist score of fibrosis stage. The coefficient of determination (R2) of automated CPA with the pathologist stages ranged from 0.60 to 0.86. There was considerable overlap in the calculated CPA across different fibrosis stages. For identification of fibrosis patterns, the models areas under the receiver operator curve were 78.6% for detection of periportal fibrosis, 83.3% for pericellular fibrosis, 86.4% for portal fibrosis and >90% for detection of normal fibrosis, bridging fibrosis, and presence of nodule/cirrhosis. In conclusion, an integrated automated tool could accurately quantify hepatic fibrosis and determine its architectural patterns in NAFLD liver biopsies.

New decolorization method produces more information from tissue sections stained with hematoxylin and eosin stain and masson-trichrome stain.

Histological analysis is a fundamental and principal method used in biological research and even for disease diagnosis. The result shows the status of cells and tissues in organs and enables us to infer the condition of the whole body. The tissue staining method known as hematoxylin and eosin staining (HE) is one of the most general methods of investigating the status of cells and tissues. Hematoxylin stains the nucleus violet and eosin stains cytosol pink. HE staining shows the unique morphologies of tissues and cells. However, after being stained with HE, tissues are very difficult to use in another histological analysis because hematoxylin is hard to remove from the sections due to its stain stability. Therefore, serial sections of the tissue are used to obtain more information through another staining, including immunohistochemistry. The adjacent tissue section is not the same as the HE-stained section, however, so the results from the adjacent sections can cause confusion or ambiguity. The present study showed that our decolorization solution can decolor the hematoxylin or iron hematoxylin stain from stained structures, including the nucleus, and the decolored section could be stained again in another staining, including immunohistochemistry. This decolorization method is very valuable, in that it can determine the accurate distribution of substances and features in cells and tissues, and thus it can improve the robustness of the resulting data.

Modified Cajal's trichrome stain as a diagnostic aid in the study of epithelial pathology.

BACKGROUND: Diagnosis of initial epithelial pathology maybe difficult in Squamous Cell Carcinoma (SCC), Carcinoma In Situ and other atypical epithelial malignancies, under routine Haematoxylin and Eosin (H and E) stain. The detection of minor basement membrane alterations in doubtful cases is both time consuming and confusing. AIMS: To evaluate efficacy of Modified Cajal's Trichrome Stain (CTS) in relation to Haematoxylin and Eosin for study of epithelial dysplasia, carcinoma in situ, micro invasive SCC, frank SCC, and SCC in lymph nodes. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tissue blocks of mild epithelial dysplasia (n = 2), moderate epithelial dysplasia (n = 2), severe epithelial dysplasia (n = 4), carcinoma in situ (n = 1), micro-invasive SCC (n = 4), verrucous carcinoma (n = 1), and frank OSCC (n = 5) were stained with CTS and H&E. The sections were compared based on set histopathological criteria. RESULTS AND CONCLUSION: In SCC cases stained with CTS, invasion into connective tissue and keratin pearls were strikingly evident. Depth of invasion could be more accurately determined. Tumour cells in lymph node were intensely contrasted and easily discernible. Thus, CTS is a good differential stain, clearly delineating the epithelial elements from the connective tissue elements visually. This helps in tracing the basement membrane very clearly. It is an economic, rapid and easy to use method which cannot replace Haematoxylin and Eosin stain in cancer diagnosis, but can definitely be used adjunctive to it. Prompt diagnosis is crucial to effective treatment, and this stain assists in early and rapid diagnosis of cancer.

Induction, purification and characterization of a novel manganese peroxidase from Irpex lacteus CD2 and its application in the decolorization of different types of dye.

Manganese peroxidase (MnP) is the one of the important ligninolytic enzymes produced by lignin-degrading fungi which has the great application value in the field of environmental biotechnology. Searching for new MnP with stronger tolerance to metal ions and organic solvents is important for the maximization of potential of MnP in the biodegradation of recalcitrant xenobiotics. In this study, it was found that oxalic acid, veratryl alcohol and 2,6-Dimehoxyphenol could stimulate the synthesis of MnP in the white-rot fungus Irpex lacteus CD2. A novel manganese peroxidase named as CD2-MnP was purified and characterized from this fungus. CD2-MnP had a strong capability for tolerating different metal ions such as Ca2+, Cd2+, Co2+, Mg2+, Ni2+ and Zn2+ as well as organic solvents such as methanol, ethanol, DMSO, ethylene glycol, isopropyl alcohol, butanediol and glycerin. The different types of dyes including the azo dye (Remazol Brilliant Violet 5R, Direct Red 5B), anthraquinone dye (Remazol Brilliant Blue R), indigo dye (Indigo Carmine) and triphenylmethane dye (Methyl Green) as well as simulated textile wastewater could be efficiently decolorized by CD2-MnP. CD2-MnP also had a strong ability of decolorizing different dyes with the coexistence of metal ions and organic solvents. In summary, CD2-MnP from Irpex lacteus CD2 could effectively degrade a broad range of synthetic dyes and exhibit a great potential for environmental biotechnology.

Sonic hedgehog protein is decreased and penile morphology is altered in prostatectomy and diabetic patients.

Erectile dysfunction (ED) is a debilitating medical condition and current treatments are ineffective in patients with cavernous nerve (CN) injury, due to penile remodeling and apoptosis. A critical regulator of penile smooth muscle and apoptosis is the secreted protein sonic hedgehog (SHH). SHH protein is decreased in rat prostatectomy and diabetic ED models, SHH inhibition in the penis induces apoptosis and ED, and SHH treatment at the time of CN injury suppresses smooth muscle apoptosis and promotes regeneration of erectile function. Thus SHH treatment has significant translational potential as an ED therapy if similar mechanisms underlie ED development in patients. In this study we quantify SHH protein and morphological changes in corpora cavernosal tissue of control, prostatectomy and diabetic patients and hypothesize that decreased SHH protein is an underlying cause of ED development in prostatectomy and diabetic patients. Our results show significantly decreased SHH protein in prostatectomy and diabetic penis. Morphological remodelling of the penis, including significantly increased apoptotic index and decreased smooth muscle/collagen ratio, accompanies declining SHH. SHH signaling is active in human penis and is altered in a parallel manner to previous observations in the rat. These results suggest that SHH has significant potential to be developed as an ED therapy in prostatectomy and diabetic patients. The increased apoptotic index long after initial injury is suggestive of ongoing remodeling that may be clinically manipulatable.

The first case of Enterocytozoon bieneusi infection in Poland.

Microsporidia are intracellular parasites that cause opportunistic infections in humans of various immunological status. Only a few case reports exist on microsporidial infection in solid organ transplant recipients worldwide. The presented study demonstrates the first case in Poland of Enterocytozoon bieneusi infection in a liver transplant patient. Parasites were diagnosed in stool samples using both modified trichrome staining and PCR.

Evidence for a regional specificity in the density and distribution of noradrenergic varicosities in rat cortex.

The brainstem nucleus locus coeruleus (LC) is the sole source of norepinephrine (NE)-containing fibers in the mammalian cortex. Previous studies suggest that the density of noradrenergic fibers in rat is relatively uniform across cortical regions and that cells in the nucleus discharge en masse. This implies that activation of the LC results in equivalent release of NE throughout the cortex. However, it is possible that there could be differences in the density of axonal varicosities across regions, and that these differences, rather than a difference in fiber density, may contribute to the regulation of NE efflux. Quantification of dopamine ß-hydroxylase (DßH)-immunostained varicosities was performed on several cortical regions and in the ventral posterior medial (VPM) thalamus by using unbiased sampling methods. The density of DßH varicosities is greater in the prefrontal cortex than in motor, somatosensory, or piriform cortices, greater in superficial than in deep layers of cortex, and greater in the VPM than in the somatosensory cortex. Our results provide anatomical evidence for non-uniform release of NE across functionally discrete cortical regions. This morphology may account for a differential, region-specific, impact of LC output on different cortical areas.

Obesity suppresses circulating level and function of endothelial progenitor cells and heart function.

BACKGROUND AND AIM: This study tested the hypothesis that obesity suppresses circulating number as well as the function of endothelial progenitor cells (EPCs) and left ventricular ejection fraction (LVEF). METHODS: High fat diet (45 Kcal% fat) was given to 8-week-old C57BL/6 J mice (n = 8) for 20 weeks to induce obesity (group 1). Another age-matched group (n = 8) were fed with control diet for 20 weeks as controls (group 2). The animals were sacrificed at the end of 20 weeks after obesity induction. RESULTS: By the end of study period, the heart weight, body weight, abdominal fat weight, serum levels of total cholesterol and fasting blood sugar were remarkably higher in group 1 than in group 2 (all p<0.01). The circulating level of EPCs (C-kit/CD31, Sca-1/KDR, CXCR4/CD34) was significantly lower in group 1 than in group 2 (p<0.03) at 18 h after critical limb ischemia induction. The angiogenesis and migratory ability of bone marrow-derived EPCs was remarkably impaired in group 1 compared to that in group 2 (all p<0.01). The repair ability of aortic endothelium damage by lipopolysaccharide was notably attenuated in group 1 compared with that in group 2 (p<0.01). Collagen deposition (Sirius red staining) and fibrotic area (Masson's Trichrome staining) in LV myocardium were notably increased in group 1 compared with group 2 (p<0.001). LVEF was notably lower, whereas LV end-diastolic and end-systolic dimensions were remarkably higher in group 1 than in group 2 (all p<0.001). CONCLUSIONS: Obesity diminished circulating EPC level, impaired the recovery of damaged endothelium, suppressed EPC angiogenesis ability and LVEF, and increased LV remodeling.

Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm.

In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre-formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non-healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.

The role of Phe329 in binding of cationic triarylmethane dyes to human butyrylcholinesterase.

Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25°C in 50mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K(i) values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 µM, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K(i) values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme.

Trichrome Mallory's stain may indicate differential rates of RNA synthesis in eutopic and ectopic endometrium.

Mallory's triple staining is a histochemical technique used mainly for analysing connective tissues and glands and other tissues. We have described the differences in the nuclear staining between eutopic and ectopic endometrium as well as endometrial hyperplasia and adenocarcinoma using the Mallory's method. The ultrastructural differences between eutopic and ectopic endometrium have been detected. In normal and hyperplastic endometrium the presence of stromal cell nuclei with an increased affinity to aniline blue has been observed. The affinity has disappeared after digestion of tissues with RNase. In cases of endometriosis, independently of cell types, the nuclei have shown affinity to orange G. Similar effects in adenocarcinoma have been noted. The ultrastructural studies have shown that in normal endometrium the stroma contained cells with euchromatic and low electron density cell nuclei. In endometriosis heterochromatic cell nuclei present both in the stroma and within glands have been detected. The results indicate that the Mallory's technique may be a useful tool for recognizing the differences between eutopic and ectopic endometrium. The affinity for aniline blue in normal and hyperplastic endometrium occurs most likely due to increased RNA synthesis. Based on Mallory's staining a similarity between hyperplasia and unchanged endometrium in contrast to similar results of the staining obtained in cases of adenocarcinoma and endometriosis may be suggested.

Assessment of the joint effect of laccase and cellobiose dehydrogenase on the decolouration of different synthetic dyes.

In this paper the efficiency of the combined action of laccase and cellobiose dehydrogenase (CDH) to decolourise different synthetic dyes such as Remazol Brilliant Blue R (RBBR), Methyl Green (MG), Direct Violet (DV), Ponceau Xylidine (PX), Bismark Brown (BB) and Poly R-478 (PR) was assessed. It was found that the use of CDH could be a promising alternative to the utilisation of the expensive and poisonous chemical mediators such as HOBT although much research on this topic remains still to be done.

Neural cell death is induced by neutralizing antibody to nerve growth factor: an in vivo study.

The central nervous system (CNS) of vertebrates originates from neuroepithelial cells located within the embryonic neural tube. Coincidental with the processes of proliferation, migration and differentiation in the developing CNS, cell death is also a major phenomenon during normal development. The investigation of neural cell death in development has focused on the role of target-derived survival factors such as nerve growth factor (NGF). In this study, the effects of anti-NGF antibody on neural cell death in the cerebral cortex have been investigated. Injection of anti-NGF antibody into the cisterna magnum of mouse pups increased the number of neural cell deaths and resulted in thinning of the cerebral cortex compared with a control group. It is concluded that endogenous NGF is essential for cortical cell survival in the cerebral cortex of the newborn mouse. Moreover, this method may be applied to the other factors and different CNS regions, allowing identification of molecules and signals involved in neural cell survival.

Localized expression of specific P2X receptors in dystrophin-deficient DMD and mdx muscle.

Using a combination of molecular and immunohistochemical methods, we have obtained evidence for a distinctive change in the expression patterns of ATP-gated (P2X) receptor subunits in dystrophic muscle from both Duchenne muscular dystrophy (DMD) patients and the mdx mouse model. In control myofibres there was no staining for any P2X subtype studied here, although P2X1 stained the smooth muscle of the blood vessels and P2X6 nerves and the tunica intima in small arteries. In contrast, P2X1 and P2X6 were co-expressed strongly in small regenerating muscle fibres in the dystrophic muscles, whereas this expression decreased in fully regenerated fibres. Moreover, immunoreactivity for the P2X2 receptor re-appeared in dystrophic muscle, where it co-localised with the Type 1 fibres. There is, thus, a burst of production of several P2X receptor subtypes in regenerating dystrophic muscle, which may have implications for drug targets for this muscle pathology.

Extracellular matrix protein expression during mouse detrusor development.

BACKGROUND/PURPOSE: Extracellular matrix proteins are implicated in regulating cell proliferation and differentiation. The authors systematically analysed the expression of elastin; collagen types I, III, IV; laminin; and fibronectin during mouse detrusor muscle development, a period during which downregulation of detrusor proliferation and increasing smooth muscle differentiation is known to occur. METHODS: Embryonic days 14 (E14) and 18 (E18), and postnatal day 1 (D1) and week 6 (6wk) were examined, a period spanning the inception of the bladder to postnatal maturity. Immunohistochemistry of whole bladders was used to immunolocalise protein expression, and Western blot of dissected detrusor layers was used to semiquantify soluble protein expression. RESULTS: All proteins were detected at all 4 stages. Statistically significant increases were documented for elastin (E14 to 6wk), collagen type I (E18 to 6wk), collagen type III (D1 to 6wk) and laminin (E14 to 6wk). Fibronectin levels were relatively high up to D1, after which levels declined significantly. Collagen type IV levels decreased significantly (E18 to 6wk). CONCLUSIONS: The authors postulate that changing levels of laminin and fibronectin have opposing effects on the transition from proliferating primitive mesenchymal cells to differentiated detrusor muscle. Furthermore, changes in collagen type III and elastin may be important for bladder compliance.

Insights into UV-induced apoptosis: ultrastructure, trichrome stain and spectral imaging.

Nuclear substructures associated with apoptosis in HeLa cells have been examined using light-microscopic morphometry, trichrome staining, spectral imaging and transmission electron microscopy. This detailed analysis reveals several sites where alterations in the normal cellular ultrastructure occur during apoptotic progression. To correlate these ultrastructural changes with the underlying molecular processes, we have characterized and quantified apoptotic cell morphology with and without inhibition of two caspases, which are key effectors of the apoptotic program. Using this analysis, early apoptotic events included: (a) the segregation of nucleolar components, a diminished granular component, and a reduction in number but increase in size of fibrillar centers, (b) an increase in the number of cytoplasmic ribosomes and (c) a very minimal increase in the amount of peripherally condensed DNA. Apoptosis progressed with: (a) an increase in the number of perichromatin granules and perichromatin fibrils, (b) a reduction in number of interchromatin granule centers concomitant with an increase in their size, (c) partial digestion and circumferential condensation of the DNA at the nuclear membrane and (d) rounding of the cytoplasm with an increase in organellar density and shrinkage in cell size. Endstage apoptotic cells showed: (a) one (or two) very large pools of incompletely digested DNA, (b) one (or two) very large interchromatin granule centers, (c) an increased number of perichromatin granules which were distanced from DNA and often closely apposed to the nucleolus, (d) formation of unusually condensed, highly coiled perinucleolar bodies and (e) blebbing of highly dense cytoplasm. In HeLa cells treated with UV and inhibitors of caspase 1 and 3, the length of time from early apoptosis to the formation of apoptotic bodies was greatly extended. Inhibiting caspase activity: (a) prevented the pooling of DNA, (b) retarded the formation of large interchromatin granule centers, (c) increased the number of perichromatin granules, (d) produced disassembly of the nucleolus, (e) prevented the formation of highly coiled perinucleolar bodies, and (f) caused vacuolization in the cell center and a unipolar blebbing of the cytoplasm. Spectral imaging in conjunction with serial section electron microscopy confirmed the staining specificities of the condensed DNA, of the large condensed interchromatin granule centers, and of the nucleoli. The results indicate that the interface between the components of the chromatin domain and the interchromatin space is a critical site of caspase activity in apoptosis, and precedes other events such as internucleosomal DNA degradation.

A colorimetric assay for high-throughput screening of inhibitors of herpes simplex virus type 1 alkaline nuclease.

Herpes simplex virus type 1 (HSV-1) encodes a deoxyribonuclease that is frequently referred to as alkaline nuclease (AN) because of its elevated pH optimum. Studies with recombinant viruses which contain deletions in the HSV-1 gene encoding AN have indicated that this enzyme is required for efficient virus replication and therefore represents a potential target for novel antiviral therapies. A simple colorimetric assay for deoxyribonuclease activity employing a DNA-methyl green substrate was adapted for use in a high-throughput screen to identify small molecule inhibitors of this enzyme. This screen identified 1,2-benzoisothiazolin-3-one as a specific inhibitor of AN, since it exhibited activity against AN but was completely inactive against bovine pancreatic DNaseI. Subsequent studies revealed that this compound most likely inhibited AN by forming disulfide linkages with one or more exposed cysteine residues on the surface of the enzyme and that AN was sensitive to sulfhydryl-group-modifying reagents in general. These results demonstrated the utility of this DNA-methyl green substrate-based assay in both the rapid identification and the characterization of novel small molecule inhibitors of the AN encoded by HSV-1 and other herpesviruses.

Voltammetric and spectroscopic studies on methyl green and cationic lipid bound to calf thymus DNA.

DNA interaction with cationic lipids promises to be a versatile and effective synthetic transfection agent. This paper presents the study on binding of a simple artificial cationic lipid, cetyltrimethylammonium bromide (CTAB), to calf thymus DNA (CT DNA) prior to the condensation process, taking methyl green (MG) as a probe. The results show that the CTAB binds to DNA through electrostatic interaction forming a hydrophobic complex, thus changing the micro-environment of duplex of DNA, so the binding state of MG and DNA is changed, and a complex CTAB-CT DNA-MG is formed. This fact suggests a new way to mediate the conformation of molecular assemblies of DNA and lipids.

Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera l).

Different modes of cell death have been revealed in the regressing hypopharyngeal glands of worker honey bees. The hypopharyngeal gland, which is well developed in young nursing bees to produce protein for larval food, was seen to regress naturally in foraging adult worker bees. A range of techniques including histology, cytochemistry, in situ TUNEL, Annexin V and Comet assays indicated that cells within the gland demonstrate progressive symptoms of apoptosis, necrosis and a vacuolar form of programmed cell death. The latter mode of cell death did not display chromatin margination, but was accompanied by an enhanced level of autophagic and hydrolytic activity in which a cytosolic source of acid phosphatase became manifest in the extra-cisternal spaces. Normal and annexin-positive cells were found to occur in the younger nursing bees, whilst necrosis and an aberrant vacuolar type of apoptosis predominated in the older foraging bees. The relevance of these results to the classification of programmed cell death is discussed.