The role of the IL-8 signaling pathway in the infiltration of granulocytes into the livers of patients with alcoholic hepatitis.

BACKGROUND AND AIM: IL-8 (C-X-L motif chemokine ligase 8) and CXCR2 (C-X-C-motif chemokine receptor 2) are up regulated in alcoholic hepatitis (AH) liver biopsies. One of the consequences is the attraction and chemotactic neutrophilic infiltrate seen at the AH stage of alcoholic liver disease. MATERIALS AND METHODS: Human formalin-fixed, paraffin-embedded (FFPE) liver biopsies from patients who have AH were studied by (2.1) RNA sequencing, (2.2) PCR and (2.3) semi quantitation of specific proteins in biopsy sections using immunohistochemical measurements of antibody fluorescent intensity with morphometric technology. RESULTS: Immunohistochemistry of IL-8 showed that the expression was increased in the cytoplasm of the hepatocytes in AH liver biopsies compared to the controls. IL-8 and ubiquitin were co-localized in the MDBs. Numerous neutrophils were found throughout and satellitosis of neutrophils around MDBs was present. This suggested that IL-8 may be involved in MDB pathogenesis. RNA seq analysis revealed activation by IL-8 which included neutrophil chemotaxis by LIM domain kinase 2 (LIMK2) (17.5 fold increase) and G protein subunit alpha 15 (GNA15) (27.8 fold increase). CONCLUSIONS: The formation of MDBs by liver cells showed colocalization of ubiquitin and IL-8 in the MDBs. This suggested that IL-8 in these hepatocytes attracted the neutrophils to form satellitosis. This correlated with up regulation of the proteins downstream from the IL-8 pathways including LIMK2, GNG2 (guanine nucleotide binding proteins) and PIK3CB (phosphatidyl isitol-4, 5-biophosphate-3-kinase, catalytic subunit beta).

The mechanisms of Mallory-Denk body formation are similar to the formation of aggresomes in Alzheimer's disease and other neurodegenerative disorders.

There is a possibility that the aggresomes that form in the brain in neurodegenerative diseases like Alzheimer's disease (AD) and in the liver where aggresomes like Mallory-Denk Bodies (MDB) form, share mechanisms. MDBs can be prevented by feeding mice sadenosylmethionine (SAMe) or betaine. Possibly these proteins could prevent AD. We compared the literature on MDBs and AD pathogenesis, which include roles played by p62, ubiquitin UBB +1, HSPs70, 90, 104, FAT10, NEDD8, VCP/97, and the protein quality control mechanisms including the 26s proteasome, the IPOD and JUNQ and autophagosome pathways.

Upregulation of autophagy components in alcoholic hepatitis and nonalcoholic steatohepatitis.

There are many homeostatic mechanisms for coping with stress conditions in cells, including autophagy. In many studies autophagy, as an intracellular pathway which degrades misfolded and damaged protein, and Mallory-Denk Body (MDB) formation have been shown to be protective mechanisms against stress such as alcoholic hepatitis. Alcohol has a significant role in alteration of lipid homeostasis, sterol regulatory element-binding proteins (SREBPs) and peroxidase proliferator-activated receptors through AMP-activated protein kinase (AMPK)-dependent mechanism. AMPK is one of the kinases that regulate autophagy through the dephosphorylation of ATG1. Activation of ATG1 (ULK kinases family) activates ATG6. These two activated proteins relocate to the site of initial autophagosome and activate the other downstream components of autophagocytosis. Many other proteins regulate autophagocytosis at the gene level. CHOP (C/EBP homologous protein) is one of the most important parts of stress-inducible transcription that encodes a ubiquitous transcription factor. In this report we measure the upregulation of the gene that are involved in autophagocytosis in liver biopsies of alcoholic hepatitis and NASH. Electron microscopy was used to document the presence of autophagosomes in the liver cells. Expression of AMPK1, ATG1, ATG6 and CHOP in ASH were significantly (p value<0.05) upregulated in comparison to control. Electron microscopy findings of ASH confirmed the presence of autophagosomes, one of which contained a MDB, heretofore undescribed. Significant upregulations of AMPK-1, ATG-1, ATG-6, and CHOP, and uptrending of ATG-4, ATG-5, ATG-9, ATR, and ATM in ASH compared to normal control livers indicate active autophagocytosis in alcoholic hepatitis.

Ultrastructure of the mammary gland in lactating rats after local implantation of oestrogen.

Cannulae containing oestrogen were implanted in the mammary glands of rats on the first day after parturition and lactation assessed by daily measurements of the litter weight. Sham-implanted and intact lactating rats were used as controls. A clear-cut inhibition of the milk yield was observed in the oestrogen-implanted group. Mammary tissue was processed for fine structural study after 4, 8 or 10 days. The initial phase showed milk stasis and secretion of colostrum progressively involving the alveoli and culminating in regression of the mammary tissue. Disappearance of myofilaments from the myoepithelial cells was consistently observed. Secretion of colostrum and mammary regression could be the result of the stasis produced by an abnormal dynamic state of the myoepithelial cell elicited by the steroid.

Developmental studies on the rat vomeronasal organ: vascular pattern and neuroepithelial differentiation. I. Light microscopy.

The origin and the developmental sequence of the rat vomeronasal organ and its vascular supply are followed by means of India ink injection in serial sections of celloidin-embedded embryos from the eleventh day of gestation up to birth. The anlage of the vomeronasal organ has been established by the twelfth day of gestation (E 12). It appears as a shallow longitudinal impression of the medial wall of the nasal pit. At day E 14, it separates from the epithelium of the primary nasal cavity, forming a tube. The lumen of the organ remains continuous with the nasal cavity frontally, but ends blindly at the edge of the primary palate dorsally. From day E 16 to E 18 the lateral surface of the tubular vomeronasal organ invaginates toward the lumen forming a wide longitudinal furrow. The lumen is bordered by the developing neuroepithelium and receptor-free epithelium by this time. The vomeronasal organ receives a separate arterial blood supply arising from septal tributaries of the olfactory artery, a branch of the anterior cerebral artery from the earliest stage of development. Blood from the vomeronasal complex is collected in the vomeronasal vein lying in the longitudinal furrow next to the receptor-free epithelium. The typical vascular pattern of the vomeronasal organ is established by the eighteenth day of gestation. At this time, the first capillary loops appear within the neuroepithelium and the vomeronasal vein can already be seen to extend along the long axis of the organ.

Developmental studies on the rat vomeronasal organ: vascular pattern and neuroepithelial differentiation. II. Electron microscopy.

The present electron microscopical study demonstrates that the establishment of the typical vascular pattern of the rat vomeronasal organ by the eighteenth day of gestation is accompanied by neither complete maturation of the wall of the blood vessels nor of the vomeronasal neuroepithelium. In the newborn rat, however, the vasculature and the neuroepithelium of the vomeronasal organ present morphological elements suggesting functional capability at birth.

Changes in granule cells of the ferret olfactory bulb associated with imprinting on prey odours.

The maturation of the granule cells of the ferret olfactory bulb around the time of odour imprinting has been examined. Rapid Golgi impregnation studies revealed a temporal overshoot in the development of the spines on the external and internal dendrites of the granule cells. In contrast, the number of somatic spines decreased continuously. Electron microscopical examinations of the synaptic contacts in the external plexiform layer revealed that the time course of synapse and reciprocal synapse formation was similar to that of the formation of the spines on the external dendrites. The results were taken as evidence that both the Golgi and the electron microscopical investigations described the same developmental process of postnatal synaptic rearrangement.

[Computerized tomography of the elbow joint]. / Computertomographie des Ellbogengelenkes.

An essential pre-condition for the interpretation of computer tomographs of the elbow joint is a knowledge of the normal anatomy. The complexity of the joint requires a definite protocol for the examination, since only in this way is it possible to distinguish all anatomical structures and pathological processes. A clinical example is used to show that CT of the elbow joint is superior to all other radiographic methods for demonstrating osteochondritis dissecans. It is therefore an important additional method for demonstrating the elbow joint.

[CT of the upper ankle joint. Anatomy--pathology]. / CT des oberen Sprunggelenkes. Anatomie--Pathologie.

Diagnosis of ligament ruptures via the possibilities of conventional radiology is often insufficient. To verify CT efficiency, postmortem specimens were scanned in axial, coronal and sagittal planes and compared with anatomical findings of sections in identical planes. After the preliminary examinations we conducted a study on 32 patients. The examinations of the anterior talofibular ligament showed a correlation between CT and operative findings in 30 of 32 cases.

[Lectin histochemistry on the olfactory region and the vomeronasal organ or rats and golden hamsters]. / Lektinhistochemische Untersuchungen an der Regio olfactoria und am vomeronasalen Organ von Ratte und Goldhamster.

The present investigation describes the lectin-binding properties of the regio olfactoria (RO) and the vomeronasal organ (VNO) of the rat and golden hamster. Special attention is paid to the lectin-binding properties of the chemosensory epithelia as well as to the reactions of their specific glands. The following lectins were used: wheat germ agglutinin (WGA), horseshoe crab agglutinin (LPA), gorse agglutinin (UEA I), peanut agglutinin (PNA), soybean agglutinin (SBA), and horse gram agglutinin (DBA). Lectin-binding procedure was performed on paraffin sections of the RO and VNO using the peroxidase-antiperodixase method. Comparisons of the lectin-binding properties of the surface of the main olfactory epithelium (MOE) with that of the neuroepithelium (NE) of the VNO as estimated by the intensity of staining demonstrate that in both species differences exist between the lectin-binding properties, of the MOE and VNO-NE. Moreover, some reactions of the MOE and VNO-NE differ from species to species.

The effect of ifosfamide on tumor oxygenation at different temperatures.

In MX1 human breast cancer xenografts grown on the hind paw of thymusaplastic nude mice the effect of ifosfamide on tumor oxygenation, tumor pH and the concentration of lactic acid have been determined at mean tumor temperatures of 32 degrees C, 37 degrees C and 41 degrees C. For histological studies tumors were shock-frozen or fixed with formalin or glutaraldehyde. Treatment with Ifosfamide (250 mg/kg b.w.) reduced intratumoral laser Doppler flow, oxygenation and pH. This suggests that ifosfamide or its metabolites may have an effect on tumor vasculature.

Morphological studies on the rodent main and accessory olfactory systems: the regio olfactoria and vomeronasal organ.

The present study on the main olfactory system (MOS) and the accessory olfactory system (AOS) documents the functional morphology of the rodent olfactory region and that of the vomeronasal organ (VNO) using light and electron microscopical techniques. Special attention is given to the cytoarchitecture of the sensory epithelia, i. e. the olfactory epithelium (OE) of the regio olfactoria and the neuroepithelium of the VNO (VNO-NE). Both sensory epithelia consist of a pseudostratified columnar epithelium composed of three types of cells, i. e. receptor cells, supporting cells and progenitor cells. Even at the light microscopical level, however, distinctive morphological features can be distinguished which illustrate important differences between the two sensory epithelia. For example, the height of the respective epithelia differs considerably, the VNO-NE is approximately 170 microns tall and the OE is only about 90 microns. The receptors of the VNO-NE lack olfactory knobs which are typically found in the sensory cells of the OE. The perikarya of the receptor cells of the VNO-NE are very large when compared to those of the sensory cells of the OE. In contrast to the OE, blood vessels are found within the neuroepithelial layer of the VNO. The progenitor cells of the OE are located in a clearly distinguishable cell layer which is lacking in the rodent VNO-NE. The differences between the two epithelial layers become more obvious at the electron microscopical level. The olfactory knobs of the sensory cell dendrites of the OE reach the nasal cavity with numerous cilia. These olfactory hairs, on average 11 per knob, consist of a short proximal segment and a long and thin distal segment. This distal segment runs parallel to the epithelial surface and is embedded in the neuroepithelial mucosal layer. The dendrites of the receptor cells of the VNO-NE reach the lumen of the VNO with numerous branched microvilli which are also embedded in the mucous layer. Horizontal ultrathin sections through the apical portion of the OE reveal that each supporting cell completely envelopes several dendrites. This glia-like relationship is not found in the corresponding layer of the VNO-NE. The sensory cell perikarya of the OE contain only a few endoplasmatic reticulum (ER) profiles while the receptor cells of the VNO are characterized by an extensive smooth endoplasmatic reticulum (SER). In contrast to the fila olfactoria, numerous axons within the vomeronasal nerve show ellipsoidal varicosities without synaptic vesicles which may indicate the existence of at least two vomeronasal nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Treatment with the anti-tumor drugs, cis-platin and mafosfamide, does not affect the structure of prekinetochores in a human breast cancer cell line. An immunofluorescence study using human anti-centromere autoantibodies.

The goal of the present article was to determine whether a nuclear parameter, centromere structure of interphase cells, could serve as an indicator to assess cellular damage caused by anti-tumor drugs. These were cis-platin and mafosfamide, which are widely used for the management of solid tumors. To visualize the centromeres, we probed treated and untreated cells of a human breast cancer cell line, MX-1, with a human anti-centromere serum. The serum was obtained from a scleroderma patient and detects antigens associated with prekinetochores of the decondensed chromosomes. The DNA was simultaneously displayed by a specific fluorescent dye. The cells were grown on coverslips, incubated for 1 h in a drug-containing medium, transferred into a drug-free medium and observed 24 h later. Since the efficiency of many anti-tumor drugs increases with the temperature, two different temperatures, 37 and 42 degrees C, were used. The analysis revealed that the treatment did not visibly alter the labeling pattern. We conclude that chromosome structure remains largely intact and is not suitable for the cytological evaluation of the efficiency of anti-tumor drugs. This is in contrast with, for example, the microtubular cytoskeleton and mitochondria, which were extensively damaged under the conditions applied here (compare Wolf et al. 1995). Independent of the drug and the temperature selected, the nuclear lumen of mononucleated and multinucleated cells contained small fluorescent spots. Double dots corresponding to the sister centromeres in the G2 phase of the cell cycle were rare. In addition to the voluminous nuclei, some cells possessed micronuclei in the lateral cytoplasm and these were regularly labeled by the autoantibodies. A small subset of the mononucleated MX-1 cells had unusually large nuclei. It is reasonable to assume that they are polyploid. The fluorescent spots marking the prekinetochores were very large in these cells. This may indicate that the chromosomes remain associated after replication.

The vomeronasal organ of the New World monkey Saguinus fuscicollis (Callitrichidae). A light and transmission electron microscopic study.

The vomeronasal organ (VNO) of the New World monkey Saguinus fuscicollis (Callitrichidae) is located at the base of the most distal portion of the nasal septum opening into the nasal portion of the ductus nasopalatinus which also communicates with the oral cavity. The lumen of the VNO is limited medially and laterally by a neuroepithelium which is devoid of intraepithelially located blood vessels and composed of receptor, supporting and basal cells. Ultrastructural analysis of the VNO of Saguinus fuscicollis reveals morphological features which lead one to postulate the functional capacity of this organ in these primates. A possible mechanism through which scent marks may be incorporated into the VNO is discussed.

[The olfactory region of the bat Scotophilus heathi. Light and electron microscopic studies]. / Die Regio olfactoria der Fledermaus Scotophilus heathi. Licht- und elektronenmikroskopische Studien.

The present investigation reports light and electron microscopical aspects of the main olfactory epithelium (MOE) of the insectivorous bat Scotophilus heathi. Serial frontal sections of the nose and associated structures reveal: 1) that the MOE is located on most of the ethmoturbinals and on the proximal upper portion of the nasal septum; and 2) that the vomeronasal organ is absent in this species. The ultrastructure of the MOE of Scotophilus heathi is similar to that observed in other vertebrates. Moreover, we did not observe significant morphological differences between the MOE of male and female animals. Nevertheless, the supra-nuclear region of the supporting cells of males and females shows a different amount and distribution of "lysosome-like" cell organelles at the two times of the year investigated.

Electron microscopic observations of the lymphatic vessels of the mammalian testis.

This paper is a report on preliminary investigations into the morphology of elastic fibers surrounding the lymphatic vessels of the testis, and it includes a description of the three-dimensional architecture of the lymph vessels and elastic fibers of the mammalian testis obtained by indirect injection of glutaraldehyde and Mercox. Different types of filaments and blind ends were observed. The filaments repeatedly divide and fuse to form the reticular networks. These networks provide a specific microenvironment for the lymphatic vascular system under different physiological and pathological conditions.

The morphology of xenotransplanted human breast carcinoma MX-1 growing in nude mice. A light and transmission electron microscopic study.

The present investigation is concerned with the morphological features of the human breast carcinoma MX-1, transplanted subcutaneously into nude mice. Three weeks after transplantation the tumor tissue is clearly distinct from the dermis. Solid tumor cell groups are separated incompletely by thin connective tissue septa, giving rise to a lobular appearance. The tumor cells are characterized by very irregularly formed nuclei with three or more nucleoli. The cytoplasm of these cells displays some lysosomes, the cisternae of the rough endoplasmic reticulum, mitochondria and a variable number of ribosomes. The Golgi fields are frequently observed, particularly near the nucleus. The cells are connected to each other by desmosomes, which also persist during mitotic activity. Ductular formations can occasionally be seen. The ultrastructure of the blood vessels discloses the morphological features necessary for the regulation of blood flow. Capillaries present a sinusoidal aspect with distended and narrow lumina. Interruptions of the endothelial wall, however, were not observed. This morphological appearance was found in all the MX-1 tumors investigated, reflecting the stable growth of this tumor cell line in nude mice.

DNA-containing cytoplasmic bridges in a human breast cancer cell line, MX-1: morphological markers of a highly mobile cell type?

We have used a CREST anti-centromere serum and a DNA-specific fluorescent dye to study the composition of extended cytoplasmic bridges between interphase cells of a human carcinoma cell line, MX-1, grown on coverslips. Under natural conditions, approximately 8% of the cells possessed cytoplasmic bridges up to 60 microns long. Elongated extensions from the cell surface were also observed and were interpreted as severed cytoplasmic bridges. The bridges were extremely slender throughout most of their lengths and could not be properly resolved by phase-contrast microscopy. Staining with a DNA-specific fluorescent dye revealed, however, the presence of a thin DNA thread. This finding strongly suggests that the bridges arise during mitosis through faults in chromosome segregation. The bridges persist and most probably elongate, when the cells separate from one another after completion of mitosis. Some bridges showed also highly fluorescent DNA masses, which were detected by a CREST anti-centromere serum. Thus, a subset of the cytoplasmic bridges contained centromeres. The DNA-containing bridges between carcinoma cells in culture signal continuous rearrangements of the karyotype at a relatively high rate. The presence of extended cytoplasmic bridges between the cells could be a morphological marker for highly mobile tumor cell types and has, therefore, diagnostic value.

The cell coat of the developing olfactory epithelium in the chick.

During development of the olfactory epithelium in the chick embryo, the cell coat is revealed by treatment with Ruthenium red. On day 4 of incubation the developing sensory epithelium displays a thicker apical and basal cell coat than the neighbouring head ectoderm. The lateral cell coat is of equal thickness in both epithelia. The apical cell coat of the olfactory epithelium increases in thickness from day 4 to day 19 of embryonic life, finally attaining a thickness of about 55 nm.