J Koudstaal

Nesidioblastosis and endocrine hyperplasia of the pancreas: a secondary phenomenon.

Diffuse endocrine cell proliferation (nesidioblastosis) and islet cell hyperplasia are considered causes of organic hyperinsulinism but have not been distinguished (by histometric or immunohistologic methods) from the normally variable pancreatic islet cell population during development and in adults. Therefore, in this study morphologic, immunohistologic (to detect insulin, glucagon, somatostatin, and pancreatic polypeptide), and morphometric features were evaluated in 1) normal pancreases (from fetal to adult; n = 49); 2) pancreases from patients with nesidioblastosis (n = 5); and 3) tumor-associated pancreases (TAP) from patients with insulin-producing islet cell tumors (n = 8). The study of normal postnatal development revealed that all features of fetal development remain present after birth and that the diagnosis of any diffuse endocrine disorder should therefore be based essentially on quantitative histometric parameters (total endocrine area, islet size distribution, distribution of each endocrine cell type). With these parameters endocrine cell hyperplasia was demonstrated in TAP from adults due to increased numbers of A and D cells. However, in the cases previously diagnosed as pathologic nesidioblastosis, all parameters were within the normal range. Thus, nesidioblastosis does not appear to be a pathologic entity. Careful re-examination of the pancreases, prompted by these data, revealed small islet cell tumors in three of these five cases. It is concluded that the endocrine pancreas can react rapidly, both morphologically and functionally, to changes in hormonal feedback, e.g., islet cell tumors. Therefore, the observation of a diffuse islet cell disorder in a patient with hyperinsulinism should not be considered an indication that an islet cell tumor is not present.

Zonal heterogeneity of rat hepatocytes in the in vivo uptake of 17 nm colloidal gold granules

SummaryThe in vivo uptake in hepatocytes of intravenously injected colloidal gold granules with a diameter of 17 nm or 79 nm and coated with bovine serum albumin or with polyvinyl-pyrrolidone was studied. Irrespective of coating only the 17 nm granules were taken up in hepatocytes. Perivenous hepatocytes did take up much more gold granules than periportal hepatocytes. The gold granules were found in lysosomes around bile canaliculi. Two hours after injection hepatocytes contained the maximal amount of granules. At least a portion of the granules was discharged into the bile. The observed zonal gradient in the uptake of 17 nm gold granules might be caused by the greater supply of granules to the perivenous hepatocytes as a combined result of the higher porosity of the endothelial lining and the smaller number of Kupffer cells with a low endocytic activity in this zone.

Proliferation rate of colonic mucosa in normal subjects and patients with colonic neoplasms: a refined immunohistochemical method.

An increased colonic epithelial proliferation rate and an increase of the cryptal proliferative zone are probable markers of increased susceptibility to colonic cancer. In this study an immunohistochemical method using 5-bromo-deoxyuridine (BrdUrd) to measure the proliferation rate of colonic mucosa in vitro was used. Fresh endoscopic colonic biopsy specimens were incubated with BrdUrd and then processed for immunohistochemistry using a monoclonal antibody. Essential procedures with respect to the equal distribution of nuclei stained with BrdUrd in the biopsy specimens proved to be the cutting of the specimens before incubation and the use of a microwave oven at the beginning of incubation. The use of the procedure of the running average showed that 12 length cut crypts are sufficient to determine reliably the proliferation rate, expressed as the labelling index (LI). This was determined in the biopsy specimens of 10 subjects without organic colonic disease, eight patients with adenomatous colonic polyps, and in six patients with (recent) colonic carcinoma. Mean LI in the controls was significantly lower than in patients with colonic polyps and in those with colon cancer. It is concluded that this method is promising for screening persons at risk for colon cancer and will be of great potential in performing dietary intervention studies in these subjects.

Influence of a highly purified senna extract on colonic epithelium

Background: Chronic use of sennoside laxatives often causes pseudomelanosis coli. A recent study suggested that pseudomelanosis coli is associated with an increased colorectal cancer risk. A single high dose of highly purified senna extract increased proliferation rate and reduced crypt length in the sigmoid colon compared to historical controls. Aims: To evaluate in a controlled study the effects of highly purified senna extract on cell proliferation and crypt length in the entire colon and on p53 and bcl-2 expression. Methods: Addition of a senna extract to colonic lavage was studied in 184 consecutive outpatients. From 32 randomised patients, 15 with sennosides (Sen), 17 without (NSen), biopsies were taken. Proliferative activity was studied in 4 areas of the colon, using 5-bromo-2′-deoxyuridine labelling and immunohistochemistry (labelling index, LI). Expression of p53 and bcl-2 in the sigmoid colon was determined immunohistochemically. Results: Crypts were shorter in Sen than in NSen in the transverse and sigmoid colon. LI was higher in Sen than in NSen in the entire colon. No difference in p53 expression was seen. Bcl-2 expression was higher in both groups when crypts were shorter and/or proliferation was increased. Conclusion: Sennosides induce acute massive cell loss probably by apoptosis, causing shorter crypts, and increased cell proliferation and inhibition of apoptosis to restore cellularity. These effects may reflect the mechanism for the suggested cancer-promoting effect of chronic sennoside use.

Evidence for a migratory capability of rat Kupffer cells to portal tracts and hepatic lymph nodes

SummaryThe present study concerns the migratory ability of Kupffer cells in the rat. Phagocytic cells were labeled with colloidal carbon or gold, these markers being administered intravenously either into a tail vein, which resulted in generalized reticuloendothelial uptake, or in low dose into the portal vein, which produced uptake by Kupffer cells alone. Cells containing marker were observed in the portal tracts and in hepatic lymph nodes from 1 to 3 days after injection into the portal vein. The direct movement of single marker particles to the portal tracts could be excluded. Since injection of marker into the portal vein labeled Kupffer cells exclusively, whereas blood cells, splenic and bone marrow macrophages remained unlabeled, the labeled cells in the portal tracts and hepatic lymph nodes appeared to be former Kupffer cells migrating which had migrated to these sites.

Application of enzymehistochemical methods to isolated subcellular fractions and to sucrose-ficoll density gradients

To compare histochemical and biochemical determinations of enzyme activities, enzymehistochemical procedures are applied to sections of pellets of subcellular fractions. These investigations are of value to determine the subcellular localization of histochemically demonstrable enzyme activities and to test the homogeneity of an isolated fraction. In homogenating duckling liver a great part of the endothelial cells is not destructed and consequently is found in the nuclear fraction. Kupffer cell lysosomes land in the heavy mitochondrial fraction, whereas hepatocyte lysosomes are chiefly found in the light mitochondrial fraction. β-Glucuronidase activity shows a preferentially microsomal localization. Application of enzymehistochemical staining reactions to discontinuous gradients and comparison with biochemical data provides additional information about the validity of an enzymehistochemical reaction. In rat liver the tetrazolium reductases show a distinctly dual localization: activity in the mitochondrial band and in microsomal bands. As to their localization in different bands of the gradients non-specific esterases demonstrate a clear pH-dependency.SummaryTo compare histochemical and biochemical determinations of enzyme activities, enzymehistochemical procedures are applied to sections of pellets of subcellular fractions. These investigations are of value to determine the subcellular localization of histochemically demonstrable enzyme activities and to test the homogeneity of an isolated fraction. In homogenating duckling liver a great part of the endothelial cells is not destructed and consequently is found in the nuclear fraction. Kupffer cell lysosomes land in the heavy mitochondrial fraction, whereas hepatocyte lysosomes are chiefly found in the light mitochondrial fraction. β-Glucuronidase activity shows a preferentially microsomal localization. Application of enzymehistochemical staining reactions to discontinuous gradients and comparison with biochemical data provides additional information about the validity of an enzymehistochemical reaction. In rat liver the tetrazolium reductases show a distinctly dual localization: activity in the mitochondrial band and in microsomal bands. As to their localization in different bands of the gradients non-specific esterases demonstrate a clear pH-dependency.

Influence of fixation and buffer treatment on the release of enzymes from the plasma membrane.

Summary1. Pretreatment of frozen cryostat sections with formaldehyde or calcium ions inhibits diffusion of the plasma membrane enzymes 5′-nucleotidase, ATP-ase and alkaline phosphatase during incubation. 2. Treatment of fixed sections with different kinds of buffer at 37°C induces diffusion of enzyme activity from the plasma membrane to other sites of the section and into the incubation medium. This buffer influence depends on temperature: at 4°C only a slight diffusion occurs. Addition of phospholipase C, digitonin or taurocholate to the buffer opposes the buffer effect. 3. Pretreatment of frozen cryostat sections with a mixture of equal parts of chloroform and acetone gives a good fixation of the plasma membrane enzymes 5′-nucleotidase, ATP-ase, alkaline phosphatase and leucyl-β-naphthylamidase. During this treatment the different kinds of lipids present in the membrane are extracted equally. After this fixation buffer treatment does not cause a visible diffusion of enzyme activity in the section. Only a slight diffusion (1 till 7 percent) into the buffer solution takes place. 4. The mentioned treatments open up possibilities to get insight into the membrane anchorage of plasma membrane enzymes.

Quantitation of proliferation-associated markers Ag-NOR and Ki-67 does not contribute to the prediction of lymph node metastases in squamous cell carcinoma of the vulva

A key prognostic parameter for vulvar carcinoma is the presence of lymph node metastases. Determination of proliferation markers has been suggested as a method to predict lymph node metastases in several tumor types. If this were true in vulvar carcinomas, reduced surgical therapy for patients with low-risk vulvar carcinoma could be considered. The authors analyzed whether the proliferation-associated markers silver nucleolar organizer region (Ag-NOR) and Ki-67 are predictors for inguinofemoral lymph node metastases in women with vulvar carcinoma. The authors also analyzed whether these proliferation markers are interrelated. Data were obtained from samples of 145 patients with T1/T2 squamous cell carcinoma of the vulva who were treated with vulvectomy and bilateral lymphadenectomy. None of these patients received preoperative therapy, and the invasion depth of the tumors was more than 1 mm. The median age was 71 years. The group consisted of 67 patients with differentiation grade 1, 64 with grade 2, and 18 with grade 3; 22% (15 of 67) of the patients with grade 1, 45% (29 of 64) with grade 2, and 43% (six of 14) with grade 3 had lymph node metastases. Formalin-fixed, paraffin-embedded sections were stained for proliferation markers Ag-NOR and MIB-1 (an equivalent of Ki-67 for fixed material). Both parameters were scored at the tumor stroma interface. Ag-NOR number and areas were quantified by interactive image analysis and Ki-67 index was scored microscopically with a grid. No relation was found between Ki-67 or Ag-NOR and lymph node metastases. A relation was found between Ki-67 and mitotic index (MI), but not between Ag-NOR and MI or Ki-67 index. Therefore, it is questionable whether Ag-NOR is, indeed, a marker for proliferation. The authors conclude that quantitation of Ki-67 and Ag-NOR does not contribute to the prediction of inguinofemoral lymph node metastases in squamous cell carcinoma of the vulva.

Gastrointestinal toxicity of chemotherapy and the influence of hyperalimentation.

The effect of combination chemotherapy on human small intestinal morphology and disaccharidase activities and their relation with clinical and chemical (fecal wet weight and K‐excretion) parameters for gastrointestinal toxicity were evaluated in patients with disseminated malignant melanoma receiving enteral normoalimentation (NA). Also evaluated were the supposed protective effects on gastrointestinal toxicity of enteral hyperalimentation (HA) with an elemental diet. After chemotherapy, a comparable decrease in villus height, total mucosal height, and mitotic index was found in jejuna biopsy specimens of both groups. However, in the NA group, the crypt depth decreased (in contrast to the HA group), whereas the disaccharidase activities in the HA group deteriorated to lower values than in the NA group. The authors found no correlation between disaccharidase levels and mucosal morphology, nor was there a correlation between these variables, fecal parameters and clinical diarrhea, suggesting that diarrhea occurring after chemotherapy was not due to loss of mucosal tissue or decrease in enzyme activities. A protective effect of HA with an elemental diet on gastrointestinal toxicity could not be established.

Bone Histomorphometry in Children with Newly Diagnosed Acute Lymphoblastic Leukemia

The objective of this study was to obtain insight into bone formation and resorption in children with newly diagnosed untreated acute lymphoblastic leukemia (ALL). In 23 consecutive children with ALL, a bone biopsy was taken from the crista iliaca posterior under ketamine anesthesia, together with the diagnostic marrow aspiration, before any treatment was given. Histomorphometric assessment was done of bone volume, bone area, trabecular thickness, osteoid volume, osteoid area, osteoid width, number of osteoblasts, erosion area, and number of osteoclasts. Data were analyzed in age groups under and over 10 y and compared with biopsies from 15 children, obtained during the work-up for other malignancies (only patients without bone marrow involvement were included). In ALL patients, bone volume and trabecular thickness were decreased in children <10 y. In patients >10 y, these parameters were not significantly different from the controls; bone densitometry showed no significant loss of bone in patients >10 y as well. Numbers of osteoblasts and osteoid surface occupied with osteoblasts were reduced in both age groups, as was the number of resorbing osteoclasts. No indications of osteomalacia were found. Childhood ALL results in a reduced number of both osteoblasts and osteoclasts, with a subsequent reduced osteoid formation and reduced bone resorption. This leads to a reduced bone volume and trabecular thickness, especially in younger children. In adolescents, the disturbance of bone (re)modeling is less serious, probably because of the strong stimulus on bone formation of sex hormones. The skeletal impairment at diagnosis is potentially reversible.