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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Matrix-derived serum markers in monitoring liver fibrosis in children with chronic hepatitis B treated with interferon alpha.

AIM: To evaluate prospectively 4 selected serum fibrosis markers (tenascin, hyaluronan, collagen VI, TIMP-1) before, during and 12 mo after IFN treatment of children with chronic hepatitis B. METHODS: Forty-seven consecutive patients with chronic hepatitis B (range 4-16 years, mean 8 years) underwent IFN treatment (3 MU tiw for 20 wk). Fibrosis stage and inflammation grade were assessed in a blinded fashion before and 12 mo after end of treatment. Serum fibrosis markers were determined using automated assays. RESULTS: IFN treatment improved histological inflammation but did not change fibrosis in the whole group or in subgroups. Only hyaluronan correlated significantly with histological fibrosis(r = 0.3383, P = 0.021). Basal fibrosis markers did not differ between responders (42.5%) and nonresponders(57.5%). During IFN treatment only serum tenascin decreased significantly in the whole group and in nonresponders. When pretreatment values were compared to values 12 mo after therapy, TIMP-1 increased in all patients and in nonresponders, and hyaluronan decreased in all patients and in responders. CONCLUSION: Tenascin reflects hepatic fibrogenesis and inflammation which decreases during IFN treatment of children with chronic hepatitis B. TIMP-1 correlates with nonresponse and hyaluronan with histological fibrosis.

Ultrastructure of Kupffer cells and hepatocytes in the Dubin-Johnson syndrome: a case report.

Ultrastructure of Kupffer cells and hepatocytes in liver bioptate was evaluated in a 17-year-old boy with Dubin-Johnson syndrome (DJS). The liver tissue obtained by needle biopsy was fixed in glutaraldehyde and paraformaldehyde and routinely processed for electron microscopic analysis. The ultrastructural examinations of liver bioptate revealed the accumulation of membrane-bound, electron-dense lysosomal granules within the cytoplasm of hepatocytes, characteristic of DJS. They were located mainly in the vicinity of the biliary pole, and preferentially in the centrilobular region that corresponded to the pigment deposits seen under light microscope. The presence of the granules was accompanied by dilated elements of the granular endoplasmic reticulum and paracrystalline mitochondrial inclusions as well as dilation of the bile canaliculi. The changes in hepatocytes co-existed with marked stimulation and enhanced phagocytic activity of Kupffer cells. This was manifested in the accumulation of pigment deposits within their cytoplasm that corresponded to those observed in hepatocytes. Hyperactive pericentral Kupffer cells which are involved in the response to pigmentary material originating from disintegrated hepatocytes may play an essential role in the development of DJS.

[Serum gamma-glutamyl transpeptidase activity in children with chronic hepatitis B]. / Aktywnosc gamma-glutamylotranspeptydazy w surowicy dzieci chorych na przewlekle zapalenie watroby typu B.

UNLABELLED: THE AIM OF THE STUDY was evaluation of serum activity of chosen enzymes (ALT, AST, GGT and ALP) in assessment of fibrosis degree in children with chronic hepatitis B. MATERIAL AND METHODS: We determined serum activity of liver enzymes in 47 children aged 4-16 with biopsy-verified chronic hepatitis B, prior to interferon alpha treatment. Fibrosis stage was assessed in a blinded fashion according to Batts and Ludwig. We defined advanced fibrosis as a score =3. Receiver operating characteristics (ROC) analysis was used to calculate the power of the assays to detect advanced liver fibrosis (AccuROC, Canada). RESULTS: Serum GGT activity of 19 IU/I had a sensitivity of 50% and a specificity of 90% (AUC=0.7324, p=0.0391) to predict advanced fibrosis. All other enzymes did not allow a useful prediction. CONCLUSION: GGT is suitable serum marker to predict advanced liver fibrosis in children with chronic hepatitis B.

Diagnostic accuracy of serum biochemical fibrosis markers in children with chronic hepatitis B evaluated by receiver operating characteristics analysis.

AIM: To investigate the diagnostic accuracy of potent serum biochemical fibrosis markers in children with chronic hepatitis B evaluated by receiver operating characteristics (ROC) analysis. METHODS: We determined the serum level of apolipoprotein A-I (APO A-I), haptoglobin (HPT) and a-2 macroglobulin (A2M) with an automatic nephelometer in 63 children (age range 4-17 years, mean 10 years) with biopsy-verified chronic HBeAg-positive hepatitis B. Fibrosis stage and inflammation grade were assessed in a blinded fashion according to Batts and Ludwig. We defined mild liver fibrosis as a score < or =2 and advanced fibrosis as a score equal to 3. ROC analysis was used to calculate the power of the assays to detect advanced liver fibrosis (AccuROC, Canada). RESULTS: Serum concentrations of APO A-I, HPT and A2M were not significantly different in patients with chronic hepatitis B compared to controls. However, APO A-I level of 1.19 ng/L had a sensitivity of 85.7% and a specificity of 60.7% (AUC = 0.7117, P = 0.035) to predict advanced fibrosis. All other serum biochemical markers and their combination did not allow a useful prediction. None of these markers was a good predictor of histologic inflammation. CONCLUSION: Apolipoprotein A-I may be a suitable serum marker to predict advanced liver fibrosis in children with chronic hepatitis B.