Liver sinusoidal endothelial cells in morphogenesis of pediatric autoimmune hepatitis. Ultrastructural characteristics - a novel report.

The pathogenesis of autoimmune hepatitis (AIH) is poorly understood. Up to now, little is known of the involvement of liver sinusoidal endothelial cells (LSECs), accounting for approximately 40% of nonparenchymal hepatic cells, in AIH morphogenesis in pediatric patients. The study objective was ultrastructural analysis of LSECs from pretreatment biopsies of 19 children, aged 4-17 years (14 girls), with clinically and histologically diagnosed AIH. Our study is the first to describe alterations in LSECs, from swelling to necrosis, demonstrating their important role in the morphogenesis and progression of pediatric AIH. Frequently damage to LSECs coexisted with significantly activated Kupffer cells, fibrogenesis and fibrosis, but not cirrhosis, accompanied by the appearance of transitional hepatic stellate cells. Interestingly, even though in half of the AIH children the sinusoidal vessels were found to undergo transformation of discontinuous into continuous endothelium showing features of defenestration, the true basement membrane did not form underneath. The fact that the basement membrane is not formed, even when LSECs are markedly damaged, may seem to indicate some regenerative capacities of these cells and lesion reversibility.

Ultrastructural Analysis of the Large Neuronal Perikarya in an Injured Dentate Nucleus Using an Experimental Model of Hyperthermia-Induced Convulsions: The First Qualitative and Quantitative Study.

Background: Febrile seizures are a common form of convulsions in childhood, with poorly known cellular mechanisms. The objective of this pioneering study was to provide qualitative and quantitative ultrastructural research on the large neuronal perikarya in the cerebellar dentate nucleus (DN), using an experimental model of hyperthermia-induced seizures (HSs), comparable to febrile seizures in children. Methods: The study used young male Wistar rats, divided into experimental and control groups. The HSs were evoked by a hyperthermic water bath at 45 °C for 4 min for four consecutive days. Specimens (1 mm3) collected from the DN were routinely processed for transmission electron microscopy studies. Results: The ultrastructure of the large neurons in the DN affected by hyperthermic stress showed variously pronounced lesions in the perikarya, including total cell disintegration. The most pronounced neuronal lesions exhibited specific morphological signs of aponecrosis, i.e., dark cell degeneration ('dark neurons'). In close vicinity to the 'dark neurons', the aponecrotic bodies were found. The findings of this qualitative ultrastructural study correspond with the results of the morphometric analysis of the neuronal perikarya. Conclusions: Our results may constitute interesting comparative material for similar submicroscopic observations on large DN neurons in HS morphogenesis and, in the future, may help to find potential treatment targets to prevent febrile seizures or reduce recurrent seizures in children.

The role of Kupffer cells in the morphogenesis of nonalcoholic steatohepatitis - ultrastructural findings. The first report in pediatric patients.

OBJECTIVE: Until now studies concerning the involvement of hepatic nonparenchymal cells (NPCs), particularly Kupffer cells/macrophages (KCs/MPs), in the pathogenesis of human nonalcoholic steatohepatitis (NASH) have been limited to adult patients; there are no similar reports referring to children. This study aimed to explore, based on ultrastructural analysis, the role of KCs/MPs in the morphogenesis of nonalcoholic steatohepatitis (NASH) in children. MATERIAL AND METHODS: Ultrastructural investigations of KCs were conducted on liver bioptates obtained from 10 children, aged 2-14 years, with clinicopathologically diagnosed NASH. Bioptatic material was fixed in solution of paraformaldehyde and glutaraldehyde in cacodylate buffer, routinely processed for transmission-electron microscopic analysis and examined using an Opton EM microscope. RESULTS: The current ultrastructural study revealed within the hepatic sinusoids the presence of numerous enlarged KCs with increased phagocytic activity, which reduced or blocked vascular lumen. Interestingly, the activated KCs not only contained primary and secondary lysosomes, altered mitochondria, and well-developed Golgi apparatus, but also absorbed fragments of erythrocytes. Such macrophages were frequently seen very close to the transformed hepatic stellate cells (T-HSCs) and progenitor/oval cells. Intensive fibrosis was observed in the vicinity of activated KCs/MPs. Bundles of collagen fibers were seen directly adhering to these cells and to other NPCs, especially T-HSCs. CONCLUSIONS: KCs are involved in the morphogenesis and development of pediatric NASH. Engulfment of erythrocytes by hepatic macrophages may lead to the accumulation of iron derived from hemoglobin in liver and play a role in triggering the generation of oxidative stress in the disease course.

The Ultrastructure of Hepatic Stellate Cell-Macrophage Intercellular Crosstalk as a New Morphological Insight into Phenomenon of Fibrogenesis in Pediatric Autoimmune Hepatitis.

The aim of the study was the pioneering retrospective ultrastructural evaluation of respective forms of hepatic stellate cells (HSCs) and analysis of their crosstalk with other adjacent nonparenchymal cells (NPCs), especially Kupffer cells/macrophages (KCs/MPs), in pediatric autoimmune hepatitis (AIH). METHODS: Ultrastructural assessment of the HSC population and NPCs was performed in transmission electron microscopy (TEM) using pretreatment liver biopsies from 25 children (8 boys and 17 girls) aged 4-17 with clinic-pathologically diagnosed untreated AIH. RESULTS: Submicroscopic evaluation allowed easy identification of numerous HSCs in the form of transitory cells, i.e., T-HSCs, accompanied by signs of fibrosis. T-HSCs included cells with features of activation initiation (iHSCs) and activation perpetuation (pHSCs), indicating high HSC activation plasticity. The pHSCs were markedly elongated and mainly showed a distinct loss of lipid cytoplasmic material, expanded and dilated channels of granular endoplasmic reticulum, and linear bundles of microfilaments beneath the cell membrane. They were surrounded by usually mature collagen fibers. Frequently activated KCs/MPs adhered directly to T-HSCs. Between them, tight intercellular junctions were formed by means of point desmosomes. CONCLUSIONS: Our qualitative TEM observations indicate a key role of T-HSCs in liver fibrogenesis in pediatric AIH, with the essential involvement of activated KCs/MPs that directly adhere to them. Tight intercellular junctions, being the ultrastructural exponent of the specific cellular mechanisms of the crosstalk between NPCs, can play a vital role in hepatic collagen fibroplasia. A better understanding of HSC population morphology at the ultrastructural level in AIH seems important not only to improve the disease morphological diagnostics but to also provide new insights into therapeutic interventions for the phenomenon of liver fibrogenesis.

Chronic non-cholestatic liver disease is not associated with an increased fracture rate in children.

Chronic liver disease in adults is a risk factor of osteoporosis, but little is known about risk of fractures in children with non-cholestatic liver disease. The aim of this study was to investigate associations among the severity of liver fibrosis, bone mass and low-energy fractures in children. History of fractures, anthropometry, and bone mass and size were examined in 39 Caucasian children (25 boys, 14 girls) aged 7.1-18 years (mean 11.9 ± 3.1) with chronic hepatitis B and liver fibrosis evidenced by liver biopsy. Severity of liver fibrosis was based on histological classification according to the method of Batts and Ludwig (mild, 1-2 scores; advanced, 3 scores) and Ishak (1-3 and 4-5 scores, respectively). Bone mineral content (BMC), density (BMD) and body composition were determined in the total body and lumbar spine using dual energy X-ray absorptiometry. Seven subjects (4 girls, 3 boys; 18% of the sample) had low BMD in the total body and lumbar spine region (Z-scores below -2.0). No associations were found among BMC, BMD, bone size and the severity of liver fibrosis. Nine boys (36% of all boys) and one girl reported repeated fractures (forearm, wrist, tibia, ankle, humerus), showing trends similar to the prevalence in general population. Fractures were neither associated with lower BMD/BMC nor with scores of liver fibrosis. Deficits in BMD in children with chronic hepatitis B are not associated with the severity of liver fibrosis. This study suggests that non-cholestatic liver disease does not increase the risk of low-energy fractures during growth. From the practical perspective, however, children with chronic liver disease should be screened for history and clinical risk factors for fractures rather than referred to bone density testing.

Ultrastructural Profile Combined with Immunohistochemistry of a Hepatic Progenitor Cell Line in Pediatric Autoimmune Hepatitis: New Insights into the Morphological Pattern of the Disease.

Considering that the heterogenic population of a hepatic progenitor cell line (HPCL) can play a vital role in autoimmune hepatitis (AIH), we decided to conduct pioneering retrospective evaluation of these cells in pediatric AIH by means of transmission electron microscopy (TEM). The aim of the study was to assess the ultrastructure of the HPCL in children with untreated AIH. Ultrastructural analysis of the HPCL population, preceded by immunohistochemical staining for cytokeratin 7 (CK7), was performed using pretreatment liver biopsies from 23 children with clinicopathologically diagnosed AIH. Immunohistochemical assessment for CK7 allowed detection of proliferating immature epithelial cells differentiating towards periportal and intralobular intermediate hepatocytes without marked formation of ductular reactions in AIH children. Using TEM, we distinguished three morphological types of HPCs: I-the most undifferentiated progenitor cells; III-intermediate hepatocyte-like cells; II-intermediate bile duct cells. Most frequent were the cells differentiating towards hepatocytes, most rare-those differentiating towards cholangiocytes. The results indicate that an HPCL may be an important source of hepatocyte regeneration. Ultrastructural analyses of the HPCL population, combined with immunohistochemistry for CK7, might be a useful tool to evaluate liver cell regeneration, including fibrogenesis, and may help better understand the morphological pattern of the disease, in pediatric AIH. Frequent appearance of an HPCL in the vicinity of fibrotic foci, often accompanied by hyperactive Kupffer cells and transitional hepatic stellate cells, may indicate their significant involvement in liver fibrogenesis.

Influence of Topiramate on the Synaptic Endings of the Temporal Lobe Neocortex in an Experimental Model of Hyperthermia-Induced Seizures: An Ultrastructural Study.

The objective of this pioneering study was to assess potentially neuroprotective properties of topiramate (TPM), a broad spectrum and newer-generation antiepileptic used against damage to synaptic endings of the temporal lobe neocortex in experimental hyperthermia-induced seizures (HS). TPM (80 mg/kg b.m.) was administered in young male Wistar rats with an intragastric tube before and immediately after HS. Specimens (1 mm3) collected from the neocortex, fixed via transcardial perfusion with paraformaldehyde and glutaraldehyde solution, were routinely processed for transmission-electron microscopic study, i.e., for descriptive and morphometric analysis. The ultrastructure of neocortical neuropil components affected by hyperthermic stress showed distinct swelling of pre and post-synaptic axodendritic and axospinal endings, including total disintegration. Mitochondria were markedly damaged in synaptic structures. Axoplasm of presynaptic boutons contained a decreased number of synaptic vesicles. Synaptic junctions showed active zone-shortening. Preventive administration of TPM before HS induction demonstrated neuroprotective effects against synaptic damage in approximately 1/4 of these structures. Interestingly, beneficial effects on synapsis morphology were more common in perivascular zones close to well-preserved capillaries. They were demonstrated by smaller swelling of both presynaptic and postsynaptic parts, well-preserved mitochondria, an increased number and regular distribution of synaptic vesicles within axoplasm, and a significantly increased synaptic active zones. However, topiramate used directly after HS was ineffective in the prevention of hyperthermia-evoked synaptic injury. Our findings support the hypothesis that topiramate applied before HS can protect some neocortical synapses via the vascular factor by enhancing blood-brain barrier components and improving the blood supply of gray matter in the temporal lobe, which may be significant in febrile seizure-prevention in children.

Aberrant distributions and relationships among E-cadherin, beta-catenin, and connexin 26 and 43 in endometrioid adenocarcinomas.

During carcinogenesis, loss of intracellular cohesion is observed among cancer cells with altered expression of such adhesion molecules as E-cadherin and beta-catenin, and aberrant expression and cellular location of intercellular gap junction proteins-connexins. The aim of this study was to evaluate immunohistochemically the expression and relationship between E-cadherin and beta-catenin, and the connexins Cx26 and Cx43 in 86 endometrioid adenocarcinomas. The aberrant cytoplasmic translocation of the studied proteins was a predominant finding, whereas only a minority of cases showed normal, nuclear beta-catenin labeling or membranous distribution of the remaining molecules. E-cadherin was positively and significantly associated with beta-catenin (P=0.001, r=0.366), as was Cx26 with Cx43 (P<0.001, r=0.719), E-cadherin with Cx26 (P<0.001, r=0.413), and E-cadherin and Cx43 (P<0.001, r=0.434) in all cancers. A subgroup of endometrioid adenocarcinomas (FIGO IB+II) exclusively showed a positive significant association between the expression of beta-catenin and Cx26 (P=0.038, r=0.339). In addition, there were significantly more beta-catenin-positive carcinomas among superficially spreading cancers (FIGO IA) than among deeper invading neoplasms (FIGO IB+II) (P=0.056). The altered location of the studied proteins indicates impairment of their physiological functions. In particular, normal membranous distribution of E-cadherin and connexins is lost and replaced by abnormal cytoplasmic accumulation in most cancers, and thus intercellular ties are expected to be weakened and loosened as a consequence. In contrast, the lack of relationship between beta-catenin and connexins, E-cadherin seems to be closely associated with the expression of Cx26 and Cx43 in endometrioid adenocarcinomas.

Eosinophilic Esophagitis in Children in North-Eastern Poland.

BACKGROUND: An increase in the incidence of eosinophilic esophagitis worldwide is being observed in children. The aim of the study was to analyze the incidence, clinical manifestations, biochemical markers and endoscopic features of children with eosinophilic esophagitis in comparison to patients with non-eosinophilic esophagitis. METHODS: This single-center retrospective study included newly diagnosed children with eosinophilic (EoE) and non-eosinophilic (non-EoE) esophagitis based on endoscopic and histopathological results between January 2013 and December 2018. RESULT: Among 433 of enrolled children with esophagitis, 36 (8.31%) were diagnosed with EoE (median age of 10 years). Male predominance and an increased percentage of allergy cases in the EoE group were noticed. Dysphagia was the only symptom that significantly differentiated both groups (p = 0.006). Endoscopic findings with relevant relationships with EoE included linear fissuring, decreased vascular pattern, trachealization and whitish exudates. No significant difference in the prevalence of other reported diseases between groups was observed. CONCLUSION: The results of EoE analysis in children from North-Eastern Poland did not differ from reports from other countries. The reported symptoms were not specific for EoE, and only dysphagia and some endoscopic lesions were helpful to differentiate children with EoE from non-EoE.

Hypoxia related growth factors and p53 in preoperative sera from patients with colorectal cancer--evaluation of the prognostic significance of these agents.

BACKGROUND: Insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) belong to a group of hypoxia related proteins. IGF-I induces expression of VEGF and decomposes wild type p53 in cancer cell lines. The goal of our study was to evaluate serum IGF-I, VEGF and p53 with respect to overall and disease free survival of patients with colorectal cancer (CRC) patients compared with healthy volunteers. METHODS: Preoperative blood samples from 125 patients with CRC and 16 healthy volunteers were examined using ELISA for serum IGF-I, p53 and VEGF concentrations. RESULTS: Concentrations of p53 and VEGF were significantly higher in CRC patients than in controls (p<0.0006 and p<0.0001, respectively). IGF-I was not statistically different between both groups. Serum IGF-I showed negative correlation with p53 in CRC patients (p<0.04, r=-0.193). IGF-I and VEGF showed negative correlation in poorly differentiated cancers (G3) (p<0.03, r=-0.339). Patients with VEGF concentrations that were above average for the cancer population survived for a shorter period of time (p=0.065 in evaluation of overall survival and 0.071 in estimation of disease-free survival during a 3-year follow-up) compared with patients with serum VEGF lower than the highest values seen in controls. CONCLUSIONS: Comparisons between serum IGF-I and p53 appear to confirm the metabolism of p53 by IGF-I. Serum VEGF showed prognostic significance in our study. Serum concentrations of IGF-I and VEGF did not show positive correlation, as expected due to IGF-I induction of VEGF in malignant colon cell lines.

Ultrastructural features of astrocytes in the cortex of the hippocampal gyrus and in the neocortex of the temporal lobe in an experimental model of febrile seizures and with the use of topiramate.

The objective of the current study was ultrastructural assessment of astroglia in specimens of the hippocampal cortex and neocortex of the temporal lobe in our own experimental model of febrile seizures (FS) in rats, as well as the analysis of the influence of a structurally novel broad spectrum anticonvulsant, topiramate (TPM), upon these cells in the CNS regions studied. The current study was inspired by some interesting literature reports on the in vitro investigation into the biological effects of TPM in primary cultures of rat cortical astrocytes and by the lack of data concerning astroglial morphology in vivo in an experimental model with this antiepileptic. In the FS group, the most pronounced changes in the study cell population referred to protoplasmic astroglia and were observed in approximately 3/4 of these cells. The abnormalities were similarly expressed in the two CNS regions studied, in terms of both quantity and quality. They were characterized by considerable swelling and degenerative changes, both in astrocytic perikarya and their processes. Changes were visible in the elements of the granular endoplasmic reticulum and mitochondria, which had a condensed configuration. In the group receiving topiramate directly after the induction of FS, submicroscopic changes in protoplasmic astrocytes were similarly expressed as in the FS group. However, in the group receiving the drug prior to the induction of FS its protective action was observed on the morphology of approximately 1/3 of the population of the protoplasmic astroglial cells. The remaining protoplasmic astrocytes still showed features of considerable or moderately pronounced injury. The beneficial effect of TPM on the ultrastructure of part of the population of the protoplasmic astroglia in the group in which the drug was applied prior to the induction of FS can be explained, among others, by a protective effect of the blood-brain barrier enhanced by the drug administration, as indicated by our earlier findings.

Effects of topiramate on the ultrastructure of synaptic endings in the hippocampal CA1 and CA3 sectors in the rat experimental model of febrile seizures: the first electron microscopy report.

The present study aimed at exploring a potentially neuroprotective effect of topiramate (TPM), one of the most commonly used newer-generation, broad-spectrum, antiepileptic drugs against ultrastructural damage of hippocampal synaptic endings in the experimental model of febrile seizures (FS). The study used male young Wistar rats aged 22-30 days, divided into three experimental groups and the control group. Brain maturity in such animals corresponds to that of 1- or 2-year-old children. Hyperthermic stress was evoked by placing animals in a 45°C water bath for four consecutive days. TPM at a dose of 80 mg/kg b.m. was administered with an intragastric tube before and immediately after FS. Specimens (1 mm3) collected from the hippocampal CA1 and CA3 sectors, fixed via transcardial perfusion with a solution of paraformaldehyde and glutaraldehyde, were routinely processed for transmission-electron microscopic analysis. Advanced ultrastructural changes induced by hyperthermic stress were manifested by distinct swelling of hippocampal pre- and post-synaptic axodendritic and axospinal endings, including their vacuolization and disintegration. The axoplasm of the presynaptic boutons contained a markedly decreased number of synaptic vesicles and their abnormal accumulation in the active synaptic region. The synaptic junctions showed a dilated synaptic cleft and a decreased synaptic active zone. TPM used directly after FS was ineffective in the prevention of hyperthermia-induced injury of synaptic endings in hippocampal CA1 and CA3 sectors. However, "prophylactic" administration of TPM, prior to FS induction, demonstrated a neuroprotective effect against synaptic damage in approximately 25% of the synaptic endings in the hippocampal sectors, more frequently located in perivascular zones. It was manifested by smaller oedema of both presynaptic and postsynaptic parts, containing well-preserved mitochondria, increased number and regular distribution of synaptic vesicles within the axoplasm, and increased synaptic active zone. Our current and previous findings suggest that TPM administered "prophylactically", before FS, could exert a favourable effect on some synapses, indirectly, via the vascular factor, i.e. protecting blood-brain barrier components and through better blood supply of the hippocampal CA1 and CA3 sectors, which may have practical implications.

Liver pathology in children with newly diagnosed celiac disease.

AIM OF THE STUDY: To evaluate the prevalence and the type of liver pathology in children at the time of diagnosis of celiac disease (CD). MATERIAL AND METHODS: Data from newly diagnosed children with CD hospitalized in the university hospital were retrospectively reviewed. Liver pathology was defined as elevated alanine transaminase (ALT) and/or gamma-glutamyl transpeptidase (GGT) serum activity and/or pathological changes of the organ in ultrasound. RESULTS: Liver pathology was detected in 17 of 149 children (11.4%). Ten patients (6.7%) had an elevated ALT serum activity, whereas no child had an elevated GGT activity. Pathological changes of liver in ultrasound (mainly enlargement or steatosis of the organ) were found in 12 patients (8.1%), of whom 5 children (3.4%) had simultaneously elevated ALT serum activity. Children with liver pathology had lower iron (Fe) (p = 0.02) and folic acid (p = 0.01) concentrations compared to the rest of the patients. There were no statistically significant differences between liver pathology existence and age, sex, serum immunoglobulin A anti-tissue transglutaminase type 2 antibodies (IgA anti-TG2), ferritin, vitamin B12, or vitamin D concentrations. Moreover, a positive correlation between IgA anti-TG2 concentration and ALT serum activity was found (p < 0.01, R = 0.29). CONCLUSIONS: Liver pathology is present at diagnosis in a significant proportion of children with CD in the form of hypertransaminasemia and pathological changes of the organ in ultrasound. There is a correlation between IgA anti-TG2 concentration and ALT serum activity.

The combination of fecal calprotectin with ESR, CRP and albumin discriminates more accurately children with Crohn's disease.

PURPOSE: Increased fecal calprotectin is a sensitive marker of various types of intestinal inflammation. We investigated correlations between high fecal calprotectin concentration and serum inflammatory markers in children with different intestinal diseases with diarrhea with/without blood and/or abdominal pain, to test whether the combination of these markers can differentiate potential patients with inflammatory bowel disease. MATERIALS/METHODS: The study included 128 children with high fecal calprotectin concentration (>150ug/g) and symptoms suggesting bowel disorders, hospitalized in the years 2013- 2015. Twenty-six (20%) patients were diagnosed with Crohn's disease, 55 (43%) with ulcerative colitis, 32 (25%) with intestinal infection and 15 (12%) with food protein induced proctocolitis. RESULTS: Significantly increased inflammatory markers were detected in children with inflammatory bowel disease, with a correlation between calprotectin and erythrocyte sedimentation rate - ESR (R = 0.53), mean corpuscular volume - MCV (R=-0.64), red blood cell distribution width (R = 0.56), albumin (R = -0.52), hemoglobin (R = -0.53) only in Crohn's disease patients. To discriminate Crohn's disease patients from patients with intestinal infection and patients with food protein induced proctocolitis, AUC analysis was performed. It revealed that considering ESR, CRP and albumin as additional markers to fecal calprotectin significantly improved diagnostic performance (AUC 0.917, p = 0.038). CONCLUSIONS: In children with abdominal pain and/or diarrhea, increased ESR, CRP and decreased albumin combined with a high fecal calprotectin level yields additional diagnostic value in screening potential patients with Crohn's disease. As far as differentiation of ulcerative colitis is concerned, low additional diagnostic value was found when high fecal calprotectin was combined with albumin.

Ultrastructure of the blood-brain barrier of the gyrus hippocampal cortex in an experimental model of febrile seizures and with the use of a new generation antiepileptic drug--topiramate.

The ultrastructure of the blood-brain barrier (BBB) of the gyrus hippocampal cortex in an experimental model of febrile seizures in rats and the effect of a new generation antiepileptic drug, topiramate, on the morphological status of this barrier were investigated. Advanced changes indicating a substantial increase in BBB permeability were observed in the animals with induced febrile seizures (FS), with approximately 2/3 of capillaries and perivascular astroglial processes being affected. Almost total occlusion of the capillary lumen was frequently seen, caused by damaged endothelial lining and by external pressure from markedly swollen perivascular astrocytic processes. Mitochondrial changes predominated among the abnormalities found in endoplasmic organelles of endothelial cells. Lesions in the BBB coexisted with damage to pyramidal neurons, mainly with features of aponecrosis ("dark neurons"). The study on topiramate seems to demonstrate its protective action on the BBB components of the ammonal cortex in the group receiving the drug as prevention, i.e. against febrile seizures. It was found to prevent marked BBB damage in over half of the capillaries. However, the application of topiramate directly after FS induction had no distinct beneficial effect on the structural BBB components.

Ultrastructural characteristics of the respective forms of hepatic stellate cells in chronic hepatitis B as an example of high fibroblastic cell plasticity. The first assessment in children.

PURPOSE: Activation of hepatic stellate cells (HSCs), mainly responsible for extracellular matrix synthesis, is assumed to be central event in the process of liver fibrogenesis. The major objective of the research was to analyze the ultrastructural profile of activated HSCs in children with chronic hepatitis B (chB), with respect to fibrosis intensity. MATERIALS/METHODS: Ultrastructural investigations of HSCs were conducted on liver bioptates from 70 children with clinicopathologically diagnosed chB before antiviral treatment. Biopsy material, fixed in paraformaldehyde and glutaraldehyde solution, was routinely processed for electron-microscopic analysis. RESULTS: In children with intensive liver fibrosis (S-2 and S-3), the ultrastructural picture showed almost total replacement of quiescent HSCs (Q-HSCs) by activated, i.e. transitional HSCs (T-HSCs). Among T-HSCs, two types of cells were distinguished: cells exhibiting initiation of HSC activation (Ti-HSCs), never before described in chB, that were frequently accompanied by activated Kupffer cells, and cells with features of perpetuation of activation (Tp-HSCs). Tp-HSCs were elongated and characterized by substantial loss of cytoplasmic lipid material; they contained an increased number of cytoskeletal components, extremely dilated channels of granular endoplasmic reticulum and activated Golgi apparatus, which indicated their marked involvement in intensive synthesis of extracellular matrix proteins. Many collagen fibers were found to adhere directly to Tp-HSCs. CONCLUSIONS: The current study showed T-HSCs to be an important link between Q-HSCs and myofibroblastic HSCs (Mf-HSCs). Transformation of HSCs into new morphological variations (Ti-HSCs; Tp-HSCs and Mf-HSCs), observed along with growing fibrosis, indicates their high plasticity and a key role in fibrogenesis in pediatric chB.

Ultrastructure of oval cells in children with chronic hepatitis B, with special emphasis on the stage of liver fibrosis: the first pediatric study.

AIM: To investigate the ultrastructure of oval cells in children with chronic hepatitis B, with special emphasis on their location in areas of collagen fibroplasia. METHODS: Morphological investigations were conducted on biopsy material obtained from 40 children, aged 3-16 years with chronic hepatitis B. The stage of fibrosis was assessed histologically using the arbitrary semiquantitative numerical scoring system proposed by Ishak et al. The material for ultrastructural investigation was fixed in glutaraldehyde and paraformaldehyde and processed for transmission-electron microscopic analysis. RESULTS: Ultrastructural examination of biopsy specimens obtained from children with chronic hepatitis B showed the presence of two types of oval cells, the hepatic progenitor cells and intermediate hepatic-like cells. These cells were present in the parenchyma and were seen most commonly in areas of intense periportal fibrosis (at least stage 2 according to Ishak et al) and in the vicinity of the limiting plate of the lobule. The activated nonparenchymal hepatic cells, i.e. transformed hepatic stellate cells and Kupffer cells were seen in close proximity to the intermediate hepatic-like cells. CONCLUSION: We found a distinct relationship between the prevalence of oval cells (hepatic progenitor cells and intermediate hepatocyte-like cells) and fibrosis stage in pediatric patients with chronic hepatitis B.

Serum hyaluronan and laminin level in children with chronic hepatitis B during long-term lamivudine treatment.

BACKGROUND/AIMS: The aim was to assess the effect of long-term lamivudine treatment on liver fibrosis by direct assessment of histological scores and by indirect assessment of serum biomarkers in children with chronic hepatitis B (chB). METHODOLOGY: The observation was carried out on 31 children with biopsy proven chB who were nonresponders to previous IFNalpha therapy. The serum concentration of hyaluronan and laminin were measured before and up to 24 months of therapy. ROC analysis was used to calculate the power of the assays to detect advanced liver fibrosis (score > 2 according to Batts & Ludwig). RESULTS: Serum hyaluronan and laminin level were significantly higher in children with chB compared to controls. There was a significant correlation between serum hyaluronan level and the stage of liver fibrosis. The ability of serum hyaluronan to differentiate children with advanced fibrosis from those with mild fibrosis was significant (AUC = 0.7767). Laminin did not allow a useful prediction. Two-year lamivudine treatment did not improve histological fibrosis but it caused significant decrease of serum hyaluronan level. CONCLUSIONS: Hyaluronan is a better fibrosis marker than laminin to diagnose children with advanced liver fibrosis. The significant decrease of hyaluronan level during therapy suggests antifibrotic effect of lamivudine in children with chB.

Glassy droplet inclusions within the cytoplasm of Kupffer cells: A novel ultrastructural feature for the diagnosis of pediatric autoimmune hepatitis.

Since Kupffer cells/macrophages (KCs/MPs) may be involved in the pathogenesis of autoimmune hepatitis (AIH), this pioneer study was undertaken to evaluate KCs/MPs in pediatric AIH in transmission-electron microscope. METHODS: Ultrastructural analyses were performed using liver biopsies from 14 children with clinicopathologically diagnosed AIH. RESULTS: In all AIH children, ultrastructural findings revealed changes in the cells lining sinusoidal vessels, especially KCs/MPs and endothelial cells. KCs/MPs showed increased phagocytic activity and damaged mitochondria, frequently with accompanying intense fibrosis. In 10/14 AIH patients, the cytoplasm of sinusoidal KCs/MPs contained characteristic glassy droplet inclusions. They were round, sharply circumscribed, and contained homogeneous material and distinct translucent fields. Their ultrastructure was identical with the Russel bodies of plasma cells, which were also found in liver biopsies in the same patients. CONCLUSION: Ultrastructural identification of characteristic cytoplasmic droplets with glassy appearance in KCs/MPs, never before described in AIH, provides a useful novel morphological feature in the diagnosis of this disease.

Ultrastructural Characteristics of Rat Hepatic Oval Cells and Their Intercellular Contacts in the Model of Biliary Fibrosis: New Insights into Experimental Liver Fibrogenesis.

PURPOSE: Recently, it has been emphasized that hepatic progenitor/oval cells (HPCs) are significantly involved in liver fibrogenesis. We evaluated the multipotential population of HPCs by transmission electron microscope (TEM), including relations with adherent hepatic nonparenchymal cells (NPCs) in rats with biliary fibrosis induced by bile duct ligation (BDL). METHODS: The study used 6-week-old Wistar Crl: WI(Han) rats after BDL for 1, 6, and 8 weeks. RESULTS: Current ultrastructural analysis showed considerable proliferation of HPCs in experimental intensive biliary fibrosis. HPCs formed proliferating bile ductules and were scattered in periportal connective tissue. We distinguished 4 main types of HPCs: 0, I, II (bile duct-like cells; most common), and III (hepatocyte-like cells). We observed, very seldom presented in literature, cellular interactions between HPCs and adjacent NPCs, especially commonly found transitional hepatic stellate cells (T-HSCs) and Kupffer cells/macrophages. We showed the phenomenon of penetration of the basement membrane of proliferating bile ductules by cytoplasmic processes sent by T-HSCs and the formation of direct cell-cell contact with ductular epithelial cells related to HPCs. CONCLUSIONS: HPC proliferation induced by BDL evidently promotes portal fibrogenesis. Better understanding of the complex cellular interactions between HPCs and adjacent NPCs, especially T-HSCs, may help develop antifibrotic therapies in the future.