Histomorphometric analysis of liver biopsies of treated patients with Gaucher disease type 1

Gaucher disease (GD) is an autosomal recessive lysosomal disorder caused by a disturbance in the metabolism of glucocerebroside in the macrophages. Most of its manifestations - hepatosplenomegaly, anemia, thrombocytopenia, and bone pain - are amenable to a macrophage-target therapy such as enzyme replacement. However, there is increasing evidence that abnormalities of the liver persist despite the specific GD treatment. In this work, we adapted histomorphometry techniques to the study of hepatocytes in GD using liver tissue of treated patients, developing the first morphometrical method for canalicular quantification in immunohistochemistry-stained liver biopsies, and exploring histomorphometric characteristics of GD. This is the first histomorphometric technique developed for canalicular analysis on histological liver biopsy samples.

Cholestasis beyond the Neonatal and Infancy Periods / 대한소아소화기영양학회지

Cholestasis results from impairment in the excretion of bile, which may be due to mechanical obstruction of bile flow or impairment of excretion of bile components into the bile canaliculus. When present, cholestasis warrants prompt diagnosis and treatment. The differential diagnosis of cholestasis beyond the neonatal period is broad and includes congenital and acquired etiologies. It is imperative that the clinician differentiates between intrahepatic and extrahepatic origin of cholestasis. Treatment may be supportive or curative and depends on the etiology. Recent literature shows that optimal nutritional and medical support also plays an integral role in the management of pediatric patients with chronic cholestasis. This review will provide a broad overview of the pathophysiology, diagnostic approach, and management of cholestasis beyond the neonatal and infancy periods.

Prevalência da baixa densidade mineral óssea em mulheres na pós-menopausa tratadas de câncer de mama / Prevalence of low bone mineral density in postmenopausal breast cancer survivors

OBJETIVO: Avaliar a prevalência da baixa densidade mineral óssea (DMO) em mulheres na pós-menopausa tratadas de câncer de mama. MÉTODOS: Estudo de corte transversal que incluiu 115 mulheres tratadas de câncer de mama atendidas em Hospital Universitário do Sudeste do Brasil. Foram incluídas mulheres com amenorreia há 12 meses ou mais e 45 anos ou mais de idade, tratadas de câncer de mama e livres de doença há pelo menos 5 anos. A DMO foi mensurada pelos raios-X de dupla energia em coluna lombar (L1 a L4) e colo de fêmur. Considerou-se baixa DMO quando valores de T-score de coluna total e/ou colo de fêmur <-1,0 Score de Delphi (DP) (osteopenia e osteoporose). Por meio de entrevista, foram avaliados fatores de risco para baixa DMO. Na análise estatística, empregaram-se os testes do χ2 ou Exato de Fisher. RESULTADOS: A média de idade das pacientes foi 61,6±10,1 anos e o tempo de menopausa, 14,2±5,6 anos, com tempo médio de seguimento de 10,1±3,9 anos. Considerando coluna e colo de fêmur, 60% das mulheres tratadas de câncer de mama apresentavam baixa DMO. Avaliando os fatores de risco para baixa DMO, foi encontrada diferença significativa na distribuição percentual quanto à idade (maior porcentagem de mulheres com mais de 50 anos e baixa DMO), história pessoal de fratura prévia (11,6% com baixa DMO e nenhuma com DMO normal) e índice de massa corpórea. Maior frequência de obesidade foi observada entre mulheres com DMO normal (63%) quando comparadas àquelas com baixa DMO (26,1%; p<0,05). CONCLUSÃO: Mulheres na pós-menopausa tratadas de câncer de mama apresentaram elevada prevalência de baixa DMO (osteopenia e/ou osteoporose). .

PURPOSE: To evaluate the prevalence of low bone mineral density (BMD) in postmenopausal breast cancer survivors. METHODS: In this cross-sectional study, 115 breast cancer survivors, seeking healthcare at a University Hospital in Brazil, were evaluated. Eligibility criteria included women with amenorrhea ≥12 months and age ≥45 years, treated for breast cancer and metastasis-free for at least five years. BMD was measured by DEXA at the lumbar spine (L1-L4) and femoral neck. Low BMD was considered when total-spine and/or femoral-neck T-score values were <-1.0 Delphi Score (DP) (osteopenia and osteoporosis). The risk factors for low BMD were assessed by interview. Data were analyzed statistically by the χ2 test and Fisher's exact test. RESULTS: The mean age of breast cancer survivors was 61.6±10.1 years and time since menopause was 14.2±5.6 years, with a mean follow-up of 10.1±3.9 years. Considering spine and femoral neck, 60% of breast cancer survivors had low BMD. By evaluating the risk factors for low BMD, a significant difference was found in the percent distribution for age (higher % of women >50 years with low BMD), personal history of previous fracture (11.6% with low BMD versus 0% with normal BMD) and BMI. A higher frequency of obesity was observed among women with normal BMD (63%) compared to those with low BMD (26.1%) (p<0.05). CONCLUSION: Postmenopausal breast cancer survivors had a high prevalence of osteopenia and osteoporosis. .

Successful mouse hepatocyte culture with sandwich collagen gel formation / 대한외과학회지

PURPOSE: Primary mammalian hepatocytes largely retain their liver-specific functions when they are freshly derived from donors. However, long-term cultures of functional hepatocytes are difficult to establish. To increase the longevity and maintain the differentiated functions of hepatocytes in primary culture, cells can be cultured in a sandwich configuration of collagen. In such a configuration, hepatocytes can be cultured for longer periods compared with cultures on single layers of collagen. However, research regarding mouse hepatocytes in sandwich culture is lacking. METHODS: Primary mouse hepatocytes were sandwiched between two layers of collagen to maintain the stability of their liver-specific functions. After gelation, 2 mL of hepatocyte culture medium was applied. RESULTS: After 24 hours, 5, 10 days of culture, the collagen gel sandwich maintained the cellular border and numbers of bile canaliculi more efficiently than a single collagen coating in both high and low density culture dishes. Reverse transcription-polymerase chain reaction analysis of alpha-1-antitrypsin (AAT), hepatocyte nuclear factor 4 alpha (HNF4A), alphafetoprotein, albumin, tryptophan oxygenase (TO), the tyrosine aminotransferase gene, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase for mouse primary hepatocytes cultured on collagen coated dishes and collagen gels showed superior hepatocyte-related gene expression in cells grown using the collagen gel sandwich culture system. AAT, HNF4A, albumin, TO were found to be expressed in mouse hepatocytes cultured on collagen gels for 5 and 10 days. In contrast, mouse hepatocytes grown on collagen-coated dishes did not express these genes after 5 and 10 days of culture. CONCLUSION: The collagen gel sandwich method is suitable for primary culture system of adult mouse hepatocytes.

Curcumin Prevents Bile Canalicular Alterations in the Liver of Hamsters Infected with Opisthorchis viverrini

Opisthorchis viverrini infection causes inflammation and liver injury leading to periductal fibrosis. Little is known about the pathological alterations in bile canaliculi in opisthorchiasis. This study aimed to investigate bile canalicular alterations in O. viverrini-infected hamsters and to examine the chemopreventive effects of curcumin on such changes. Hamsters were infected with O. viverrini and one group of animals was fed with 1% dietary curcumin supplement. Animals were examined during the acute infection phase, days 21 and 30 post-infection (PI) and chronic infection phase (day 90 PI). Scanning electron microscopy revealed that in the infected group fed with a normal diet, bile canaliculi became slightly tortuous by 30 day PI and more tortuous at day 90 PI. Transmission electron microscopy showed a reduction in microvilli density of canaliculi starting at day 30 PI, with a marked loss of microvilli at day 90 PI. These ultrastructral changes were slightly seen at day 21 PI, which was similar to that found in infected animals fed with 1% curcumin-supplemented diet. Notably, curcumin treatment prevented the reduction of microvilli density, reduced the dilation of bile canaliculi, and decreased the tortuosity of the bile canaliculi relative to non-infected animals on a normal diet at days 30 and 90 PI. These results suggest that curcumin reduces alteration of bile canaliculi and may be a promising agent to prevent the onset of bile duct abnormalities induced by O. viverrini infection.

Alteration of Bile Acid Transporter Expression in Patients with Early Cholestasis Following Living Donor Liver Transplantation

BACKGROUND: Intrahepatic cholestasis can occur early after living donor liver transplantation (LDLT). We investigated the changes in the expressions of the bile acid transporters and the liver histology in the patients who suffered with early cholestasis (EC) following LDLT. METHODS: The histological differences between 15 graft livers with EC after LDLT and 5 graft livers with biliary stricture following LDLT were evaluated. The hepatic mRNA levels of the bile canaliculi transporters (BSEP, MRP2, MRP3, MDR1, MDR3, NTCP) in 40 (20 graft livers, 20 matched donor livers) liver biopsy tissues were analyzed by performing real-time reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: Microscopic examination revealed hepatocellular and/or bile canalicular cholestasis around acinar zone 3 in the livers of the patients with EC. In the livers with biliary stricture, the cholestasis was dominantly observed in the hepatocytic cytoplasm and in the bile ductules around the portal area rather than around acinar zone 3. The BSEP and MRP2 mRNA levels in the EC livers were significantly reduced by 44% and 23%, respectively (p=0.000), compared to the matched donor livers. The levels of MDR3 and NTCP mRNA in the EC livers increased by 738% (p=0.000) and 281% (p<0.01), respectively. The change of the expressions of the bile acid transporters in the patients with biliary stricture was less significant than that in the EC group. CONCLUSIONS: These results suggest that the altered expressions of the bile acid transporters may play a role in the pathogenesis of EC following LDLT.

Increased Microfilaments in Hepatocytes and Biliary Ductular Cells in Cholestatic Liver Diseases

To assess the extent of microfilaments in cholestatic liver diseases we examined the cytoplasmic microfilaments in intrahepatic and extrahepatic cholestasis in man by electron microscopy. Study subjects were two patients with drug-induced intrahepatic cholestasis, three patients with intrahepatic cholestasis due to viral hepatitis, four patients with extrahepatic cholestasis due to stones of the common bile duct and two patients with primary biliary cirrhosis. Two biopsied specimens from patients without clinical or histological evidence of liver disease served as noncholestatic controls. The microfilaments in hepatocytes and biliary ductular cells were significantly increased in cholestasis compared with those in non-cholestatic controls. Well developed bundles of microfilaments were noted around the pericanalicular ectoplasm and seemed to be parallel to plasma membrane of the hepatocytes in cholestasis. In cholestasis, there were increased bundles of microfilaments around the periluminal region, lateral cell wall, and nucleus of biliary ductular cells. Two patterns of microfilaments bundles (fine microfilamentous network and spindle-shaped dense or clusters of microfilaments) were associated with cholestasis. The clustered form of microfilaments also seemed to be clearly associated with intracytoplasmic vacuoles containing bile salts. In conclusion, the increase of microfilaments in hepatocytes and biliary ductular cells may be the consequence of various forms of cholestasis. Further studies are needed to clarify the functional significance of increased microfilaments in cholestasis.

Bile excretion of hepatocytes cultured in vitro / 中华外科杂志

<p><b>OBJECTIVE</b>To regain bile excretory function of hepatocytes cultured in vitro.</p><p><b>METHODS</b>Sandwich configuration was used to culture hepatocytes and the structure of bile canaliculi as well as the function of bile excretion was observed by immunocytochemistry and the test of FDA metabolism with a single collagen configuration as a control.</p><p><b>RESULTS</b>First, the formation of bile canaliculi was observed by immunocytochemistry. In sandwiched hepatocytes, the gradual development of bile canaliculi-like structures into an anastomotic network was observed. At 24 h after cell culture, the formation of bile canaliculi was observed. With time progressed, the bile canaliculi became more clear and the network was established at 120 h. In contrast, hepatocytes in single collagen configuration showed almost no network of bile canaliculi. Second, the ability for hepatocytes to internalize, metabolize and excrete compounds into bile was indicated by FDA metabolized in the hepatocytes. In sandwiched hepatocytes, the bile excretory function was shown at 96 h, but in single collagen system, no bile excretion was observed.</p><p><b>CONCLUSION</b>Cultured hepatocytes are able to regain bile excretion in a given certain condition. Sandwich-cultured hepatocytes can reestablish bile canalicular structure and regain bile excretory function.</p>

Implementación de un ensayo de ELISA para el diagnóstico de la fascioliasis equina / Implementation of an ELISA technique in the diagnosis of equine fascioliasis

And indirect enzyme linked immunosorbent assy was developed for the diagnosis of fascioliasis in naturally infected equines as a screening test with secretory and excretory antigens of adult flukes, in order to support the diagnosis of the disease. Fascioliasis in an economically important disease and the trematode Fasciola hepatica infects a wide variety of mammals and occasionally can infect man. The standarization was performed with negative sera (collected in the XII Region of Chile) and positive sera checked by post mortem examination. The evaluation was done in 226 sera samples: 115 negative, and 70 positive with confirmed fascioliasis, 19 from animals with other pathologies and 22 from apparently healthy animals. The ELISA presented a 85.7 percent of sensibility and a 97.4 percent of specificity

Histological study of extrahepatic part of human biliary tree with emphasis on peribiliary glands

Extrahepatic part of human biliary tree was lined with simple tall columnar epithelium. Supranuclear cytoplasm of the cell was darkly stained. Histochemistry of the epithelium revealed that it was mucus secreting. Below the epithelium there was a thick compact layer of connective tissue. Outside the compact layer there was a loose layer of areolar tissue in which smooth muscle, blood vessels and nerves were found. Glandular acini were present within and outside the compact layer of intrapancreatic bile duct [IPBD] and common bile duct [CBD] whereas these were present only within the compact layer of bile duct of ampulla of Vater [BDAV]. Statistical significance of quantitative difference between glandular acini of BDAV with other regions revealed that the difference was significant [P < 0.01] with IPBD and non significant [P > 0.05] with CBD. Statistical significance of quantitative difference between glandular acini of IPBD with other regions revealed that the difference was significant [P < 0.01 and P < 0.05] with BDAV and CBD respectively. Statistical significance of quantitative difference between glandular acini of CBD with other regions also revealed that difference was significant [P<0.05] with IPBD whereas it was not significant with BDAV [P>0.05]. Intramural and extramural glandular acini were present in IPBD and CBD whereas BDAV contained only intramural glands. The difference between intramural and extramural glandular acini was statistically significant [P < 0.02] in IPBD. The difference between intramural and extramural glandular acini of CBD was also statistically highly significant [P<0.001]. Glandular acini as demonstrated by different stains were 100% mucous type in BDAV and IPBD whereas it was mixed type in CBD. The acini of CBD were serous, mucous and mixed type. The difference between serous and mucous, mucous and mixed glandular acini was statistically highly significant [P<0.001]. The difference between serous and mixed glandular acini was statistically not significant [P>0.05]. Ratio of serous, mucous and mixed glandular acini was 1:22:1 respectively

A Study on Intrasplenic Transplantation of Embryonic Stem Cells

PURPOSE: The authors evaluated the morphological and proliferative properties of embryonic stem (ES) cells following intrasplenic transplantation. The results were compared with those obtained following intrasplenic transplantation of cultured hepatocytes. METHODS: ES cells of blastocysts were collected from superovulated Sprague Dawley rats. Hepatocytes were collected from the liver of 7-week old rats by perfusion of collagenase. The ES cells and hepatocytes were cultured for 6 days and transplanted into the rat spleen. The properties of the ES cells and cultured hepatocytes following transplantation were investigated by morphological methods. RESULTS: ES cells in the culture proliferated faster than hepatocytes, and differentiated to various shaped cells. Following transplantation, ES cells were distributed near the periarterial lymphatic sheath. Cultured hepatocytes gathered chiefly around the trabeculae. On PCNA stain of transplanted ES cells, positive cells appeared on day 7 and became distinct on days 10 and 14. Transplanted hepatocytes showed no PCNA positive cells on day 14. On electron microscopic examination, ES cells differentiated to hepatocyte-like structures on day 10, and became functioning hepatocytes on day 14. Transplanted hepatocytes formed bile canaliculi on day 10, although development of organelles was insufficient on day 14. CONCLUSION: ES cells proliferated faster than cultured hepatocytes. Intrasplenic ES cells proliferated at the germinal center and hepatocytes around the trabecula. ES cells differentiated to cells that had the function of hepatocytes.

Utilidad del ultrasonido endoscópico durante una difícil canulación de la vía biliar / Utility of endoscopic ultrasound during a difficult biliary tract catheterization

La pancreatocolangiografía retrógrada endoscópica es actualmente uno de los métodos más eficaces en el diagnóstico y tratamiento de la coledocolitiasis; sin embargo no es factible realizarla en todos los pacientes, presentándose situaciones de difícil canulación en el 2-3 por ciento de los casos. El ultrasonido endoscópico es un procedimiento mínimamente invasivo con un bajo índice de morbilidad, el cual posee una alta eficacia en la evaluación de la anatomía topográfica y en el diagnóstico de patología biliar extrahepática. A continuación se presenta un caso de canulación difícil por variante anatómica de la papila mayor en el cual el USE demostró la localización y relaciones anatómicas del conducto colédoco, permitiendo el drenaje endoscópico efectivo de la vía biliar

Aumento transitorio de fosfatasa alcalina sérica post trasplante hepático en un paciente pediátrico / Transient high alkaline phosphatase activity in serum of a pediatric patient after liver transplantation

En este estudio se presenta el caso de un paciente pediátrico que luego de ser sometido a un transplante hepático con donante vivo relacionado, presentó un cuadro de hiperfosfatasemia alcalina sérica. Se pone énfasis en la importancia de la utilización de la técnica de isoelectroenfoque para determinar el origen tisular del hallazgo mencionado

Morphological Changes of Cultured Rat Hepatocytes Induced by Phalloidin and Cytochalasin D / 대한해부학회지

The microfilaments of hepatocyte are distributed throughout the vicinity of cell membranes, especially numerous around the region of bile canaliculus, and provide the maintenance of cell shape, cellular wall tension, canalicular motility, the secretion for bile, etc. To evaluate the relationship between the microfilament and alteration of cell shape, we examined the morphological changes of cultured rat hepatocytes, following treatments with phalloidin or cytochalasin D with fluorescent and electron microscopes. 1. In the fluorescent micrographs, actin microfilament was distributed near the plasma membrane and bile canaliculus. 2. Both drugs, phalloidin or cytochalasin D, produce the cytoplasmic protrusions from the surface. Their shapes were pedunculated with narrow neck or bulged with broad base, respectively. 3. In the phalloidin treated group, cytoplasmic protrusion was seperated from the internal cytoplasm by microfila-ments networks at the narrow base. In contrast, in the cytochalasin D treated group, cytoplasm was bulged with broad base and kept in direct continuity with the canalicular ectoplasm. 4. Pericanalicular ectoplasm of phalloidin treated group was widened and accumulated with microfilaments. But, bile canaliculus of cytochalasin D treated group was markedly dilated and devoid of microvilli, and the ectoplasm was almost disappeared. Considering above results, dysfunction of microfilaments leads to the structural changes and inhibition of bile secretion of hepatocytes.

Ultrastructural Changes of the Cultured Hepatocytes in Microfilamentous Dysfunction induced by Drugs / 대한해부학회지

To examine the role of actin microfilaments which are located at beneath the plasma membrane, we observed the ultrastructural changes of rat hepatocyte induced by alteration of the microfilamentous integrity. The isolated hepatocytes from Sprague-Dawley were cultured in the L-15 medium containing phalloidin (agent that cause polymerization of actin) or cytochalasin D (agent that cause depolymerization of actin) for 30 min, 1 hour, 2 hours, 4 hours, 10 hours and 20 hours, respectively. The results observed with scanning and transmission electron microscope were as follows. 1. Following the alteration of actin microfilaments, bile canaliculi were dilated and devoid of microvilli. In phalloidin treated group, the thickening of microfilamentous ectoplasm was more marked than that of cytochalasin D treated group. Whereas, the dilation of bile canaliculi was more marked in cytochalasin D group. 2. Both drugs, phalloidin or cytochalasin D, produced the alteration of cell shape to form cytoplasmic protrusions at the cell surface. In the phalloidin treated group, protrusions were pedunculated, and the microfilament networks were accumulated at the narrow neck region. 3. In cytochalasin D treated group, no microfilament barrier was seen at the broad base of protrusion which exhibit direct continuity with the internal cytoplasm. 4. Single hepatocyte tend to recover their structural integrity as those in vivo. The new bile canaliculus was sealed off at the intercellular space by tight junctions, and intercellular contacts were established by the junctional complexes. The results demonstrated that excessive accumulation or depletion of microfilaments induced by phalloidin or cytochalasin D altered the cell shape different, respectively. The microfilaments of ectoplasm play an important role in the maintenance of the structural integrity of cultured hepatocytes.

The Effects of Cytochalasin B and Colchicine on Fine Structure of Bile Canaliculi and Hepatocytes in Mouse Liver / 대한해부학회지

Bile canaliculi is closely related to the cytoskeleton; actin filament web, microtubules and cytokeratin intermediate filaments. To understand how cytoskeletal alteration affects bile canalicular structure, the investigators injected cytochalasin B and colchicine into mice intraperitoneally to inhibit the polymerization of actin filaments and microtubules respectively, and observed the structural changes of bile canaliculi and hepatocytes with transmission and scanning electron microscopes. Bile canaliculi were dilatated and microvilli were decreased in number and length after injection of cytochalasin B and colchicine. Some bile canaliculi branched irregularly after colchicine treatment. Actin filament web in the canalicular ectoplasm was disrupted leaving granular zone after cytochalasin B treatment, but was intact after colchicine treatment. Intermediate filament bundles located at angles to the canalicular membrane appeared after colchicine treatment. Intercellular junctions delimiting bile canaliculi were intact after colchicine treatment, however were disrupted after cytochalsin B treatment. Focal junctions resembling desmosome were formed between microvilli after colchicine treatment. In both cytochalasin B and colchicine treated groups, lumen of rough endoplasmic reticulum were dilated, Golgi apparatus became prominent, and lipid droplets were appeared in the cytoplasm. These results suggest that both intact actin filaments and microtubules are necessary to keep the structural integrity of bile canaliculi.

New look on resection of segment VII of the human liver

Sao reconhecidos tres subsegmentos hepaticos (IXb, IXc e IXd), dos quais os dois ultimos devem ser bem identificados na resseccao do segmento VII. As relacoes anatomicas , os limites, os pediculos portais, o pediculo normal e as veias hepaticas tem grande importancia cirurgica. O eixo portal transverso, a identificacao dos pediculos e a posicao das fissuras devem ser conhecidas, principalmente nas segmentectomias parciais superiores. Ultra-sonografia e disseccao cuidadosa sao os recursos disponiveis para resolver dificuldades emergentes de variacoes anatomicas.

Uma contribuição ao estudo do acometimento pancreático na síndrome de imunodeficiência adquirida / A contribution to the study of pancreatic involvement in acquired immunodeficiency syndrome

Com o objetivo de avaliar a incidência, as principais etiologias e as formas clínicas do acomentimento pancreático na SIDA, o autor estudou 280 pacientes, todos necropsiados, selecionando 82 casos com alguma forma de anormalidade pancreática...Após análise estatística dos dados, o autor concluiu que: 1 - o acomentimento pancreático na SIDA é menos frequente em nosso meio (quando comparado com a literatura mundial), 2 - seu comportamento é subclínico e completamente atípico em relação às formas clássicas da literatura, 3 - a tuberculose se destacou como a maior causa de pancreatopatia