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Using an indirect immunoperoxidase technique on frozen sections with the monoclonal antibody 96.5, we investigated the in situ distribution of melanotransferrin, a transferrin (Tf) and transferrin receptor (TfR) related glycoprotein, in human liver. Specimens included normal liver, liver in iron overload, hepatocellular carcinoma, angioma and foetal liver. On light microscopy, immunoreactivity was almost exclusively present on sinusoidal lining cells, apparently endothelial cells; the pattern was similar in normal and in iron-loaded liver. A gradient of more enhanced staining in acinar zone II and III was observed. The endothelial localization of the staining was supported by the positivity of the central vein endothelium and of the angiomas. Immunoelectron microscopy on three liver specimens showed positivity on sinusoidal endothelial cells but not on Ito and Kupffer cells. In addition, positivity on rough endoplasmic reticulum vesicles of some hepatocytes was also present. Four hepatocellular carcinomas showed an intense staining in tumour cells, 3 were weakly positive and 3 were negative. In the foetal livers, the central vein endothelium was positive from 21 weeks of gestation onward and additional positivity of zone III sinusoidal endothelial cells was present from 27 weeks on. The present results show that in the liver melanotransferrin has a localization different from Tf and the TfR. These latter molecules are predominantly localized in parenchymal cells. In addition, there does not appear to be a coordinate regulation secondary to iron storage, between melanotransferrin, Tf and the TfR. The observed gradient in the staining pattern in foetal and adult liver specimens further supports the heterogeneity of the endothelial cell population in the liver and suggests a developmental relationship between endothelial cells of sinusoids and central vein.

Whipple's disease is a multisystem disorder with protean manifestations and with poorly understood aetiopathogenesis. It is unclear how the immune system reacts, whether it functions normally or not, whether it protects the organism or is defeated in one way or another by the 'Whipple bacillus'. The purpose of our study was to assess humoral and cellular immunity at the level of the intestinal mucosa. This histochemical, immunocytochemical and electronmicroscopic study, based on 16 cases, has shown that the changes in components of the mucosal immune system in Whipple's disease are quite different from normal. The phagocytic capacity of the macrophages, assessed microscopically, is abnormal, the number of intra-epithelial lymphocytes is increased, the CD 4/CD 8 cell ratio is decreased and the IgM positive cells in the lamina propria outnumber the IgA positive cells. These changes may be inter-dependent.

The Nyctemera clatrathum complex (Lepidoptera: Arctiidae, Arctiinae, Callimorphini) is revised. It consists of seven species, occurring on New Guinea and surrounding islands, of which three are new to science: Nyctemera giloloensis sp. n., N. oninica sp. n. and N. dauila sp. n. Two taxa are synonymized: Leptosoma absurdum Swinhoe, 1892 syn. n. of Leptosoma clathratum Snellen van Vollenhoven, 1863 and Deilemera pratti Bethune-Baker, 1904 syn. n. of Nyctemera mesolychna Meyrick, 1889. Lectotypes are designated for Deilemera pratti Bethune-Baker, 1904 and Deilemera absurdum latimargo Rothschild 1915. Adults and genitalia of all species are figured and distribution maps are presented.

Extragenital malignant mixed mesodermal (müllerian) tumors (MMMT) are rare neoplasms, with but 24 well documented cases in the literature. Neuroendocrine differentiation in mixed müllerian neoplasms has been mentioned only anecdotally. We report on the clinical, pathological, and immunohistochemical features of a hitherto-undescribed extragenital MMMT with prominent neuroendocrine differentiation arising from the jejunal mesentery. This lesion was composed of a poorly differentiated epithelial component and a spindle cell component with heterologous (rhabdomyoblastic) differentiation. The bulk of the tumor consisted of small cell neuroendocrine carcinoma, which exhibited strong immunoreactivity for NSE, LEU-7, chromogranin A and synaptophysin. Electronmicroscopy confirmed the presence of neurosecretory dense-core granules. The primary mesenteric origin of the tumor was established at autopsy. Along with a brief review of previously reported extragenital MMMT some histogenetic concepts relevant to this case are discussed.

Aims: Synovial sarcoma with extensive calcification and ossification is a rare variant, the ultrastructural, cytogenetic and molecular analysis of which has not been reported previously. Methods and results: A large mass in the shoulder of a 20-year-old male patient led to a deformity of the chest wall, thus supporting the hypothesis that this is a slowly growing variant of synovial sarcoma. Nevertheless, the patient developed metastatic lung disease 7 months after resection. On histology, the monophasic spindle cell proliferation was in several areas obscured by the massive calcification and ossification. Immunohistochemistry showed keratin, epithelial membrane antigen, vimentin and CD99 expression. The cytogenetic analysis revealed a single t(X;18)(p11.2; q11.2), typical for synovial sarcoma. Additional fluorescence in-situ hybridization revealed SSX2 involvement. At the ultrastructural level, prominent needle-shaped intramitochondrial crystals were present, both in the cytoplasm and in the extracellular matrix. Conclusion: The presence of the t(X;18) with SSX2 involvement definitively characterizes this tumour as a variant of synovial sarcoma. In addition, the needle-like mitochondrial calcifications give a possible clue to the pathogenesis of the extensive metaplastic ossification and calcification.

Single cells or small cell clusters, isolated from the rat lacrimal gland, were incubated on reconstituted basement membrane (matrigel) in a well-defined serum-free medium. During the first days of culture, cells reassociated and reorganized in structures resembling acini. These multicellular structures, maintained in culture for 2 weeks, consisted of well-polarized cuboidal cells surrounding a central lumen and exhibiting apically located microvilli. Myoepithelial cells were observed at the periphery of the acinar structures. Both in the native lacrimal and in the cultured aggregates, epithelial cells displayed strong immunoreactivity for cytokeratin 8, while myoepithelial cells were immunoreactive for vimentin and alpha-smooth muscle isoactin. These data indicate that the cultured aggregates closely mimic the in vivo architecture of lacrimal glands both by morphology and immunohistochemistry. We further demonstrated the presence of an intact androgen receptor and the ability of the cultured aggregates to respond to androgens with increased secretion of the secretory component. Comparable androgen responses were observed in lacrimal gland cultures of 5-week-old male and female rats. In conclusion, we report a morphologically and functionally differentiated culture system of primary rat lacrimal cells, in which androgen-regulated gene expression was observed. This culture model provides a unique experimental paradigm for studying the effects of hormones, cytokines, and growth factors on the morphogenesis, growth, and functional differentiation of lacrimal glands. (C) 1998 Academic Press.

The mechanism of bile ductular reaction and accompanying fibrogenesis depends on interactions of ductular cells with the matrix and growth factors. Heparan sulfate proteoglycans (HSPGs) are essential cofactors in cell-matrix adhesion processes, in cell-cell recognition systems, and in receptor-growth factor interactions. We used monoclonal antibodies specific for the cell surface HSPGs (syndecans, glypican), for matrix HSPG (perlecan), and for heparan sulfate carbohydrate (HS) to investigate their immunohistochemical expression in 20 specimens with chronic cholestatic liver disease and in five normal human liver specimens. Because activated hepatic stellate cells (HSC are a major source of fibrosis in the liver, we also examined HSPG expression in primary cultures of human activated HSC using immunocytochemistry and Western blotting and for syndecan-1 also Northern blotting. In comparison with bile ductular cells of normal liver, reactive ductules in chronic cholestasis were marked by an elevated expression of syndecan-1, surrounded by an increased perlecan expression. In acinar zone 1, large stimulated macrophages and HSC, present in increased numbers, were strongly positive for syndecan-3. Cultured HSC showed a membranous staining pattern for syndecan-1, syndecan-3, and heparan sulfate, and in addition intracellular staining for syndecan-2, -3, and 4. Perlecan immunoreactivity was detected as intercellular strings. Western blotting revealed positive bands with all antibodies and Northern blotting for syndecan-1 was also positive. These results show that cultured human HSC can synthesize all four syndecans, glypican, and perlecan. These data reveal changes in the expression of syndecan-1, syndecan-3, and perlecan in human chronic cholestatic liver disease, that may be important in the deposition of matrix components and activation of growth factors that support ductular reaction and accompanying fibrogenesis.

We report on the predominance of a special type of small histiocyte in the inflammatory infiltrate accompanying herpetic bullae. These histiocytes, which have previously been taken to be neutrophils, are freshly arrived cells with a hitherto unknown function. Until now, they have been found only in Sweet's syndrome and erythema nodosum where they form Miesscher's radial granulomas. Similar small histiocytes were found in half of those herpetic lesions with intact bullae, and in over two-thirds of ulcerated lesions in which these cells formed a palisade in the fibrinoid material covering the floor of the ulcerated vesicles. Small histiocytes, admixed with neutrophils, were in close proximity to virally infected keratinocytes. Immunohistochemical analysis confirmed their histiocytic nature. With the exception of ecthyma contagiosum (orf), similar small histiocytes were not found in other viral infections or in nonspecific ulcers of the skin. In cases of herpetic folliculitis, small histiocytes showed massive epidermotropism towards hair follicle epithelium. We conclude that cutaneous and oral herpetic infections represent yet another disease in which small, freshly arrived histiocytes occur. They may be involved in antigen presentation, or in killing of infected keratinocytes.

Focal nodular hyperplasia is a tumour like lesion, characterized by a central fibrous scar with irradiating fibrous septa that surround hyperplastic nodules and contain multiple bile ductules. The origin of the bile ductular structures is not clear. Recently, we found evidence for the existence of human counterparts of rat oval cells (potential stem cells) that have the ability of differentiating towards both bile duct cells and hepatocytes. These cells were found in regenerating human liver as well as in chronic cholestatic conditions. Because cholestatic features are seen in focal nodular hyperplasia, we initiated an immunohistochemical study on 23 surgical specimens using antibodies specific for cytokeratins 7 and 19 (bile duct type cytokeratins), OV6 (rat oval cell marker), chromogranin-A (shown to be positive in reactive bile ductules and human oval-like cells) and neural cell adhesion molecule--NCAM (shown to be positive in reactive bile ductules) to investigate whether 'undifferentiated progenitor cells' are also present in focal nodular hyperplasia. Electronmicroscopy was applied in five cases. Bile ductules invariably showed immunoreactivity for CK7 and 19, OV6, chromogranin-A and NCAM. In addition, small individual cells with an oval nucleus and a small rim of cytoplasm, in the vicinity of the septa, were immunoreactive for chromogranin-A, CK7 and 19 and OV6. These cells were hardly recognizable on routine light microscopy. Clusters of periseptal hepatocytes, seemingly in continuity with bile ductular structures, had a transitional phenotype: they stained positive for chromogranin-A, CK7 and OV6 and sometimes formed liver cell rosettes. The number of OV6-positive hepatocytes was greater than the number of chromogranin-A and CK7 positive hepatocytes. This indicates that, in human liver, OV-6 is not purely a marker of progenitor cells. Ultrastructurally, small immature cells, highly resembling rat oval cells, were recognized in the vicinity of septa. In addition, transitional cells displaying characteristics both of hepatocytes and bile duct cells were also present. These results confirm the presence of 'undifferentiated progenitor cells' in focal nodular hyperplasia and suggest that the ductular reaction in these lesions results, at least partly, from activation of these cells.

A typical case of alveolar soft-part sarcoma was examined using ultrastructural, immunohistochemical and cytogenetic methods. Immunohistochemical stains were performed on frozen sections and showed strong desmin expression with the three anti-desmin antibodies used. In addition, the tumour cells were weakly positive for vimentin and myosin. Neural markers were negative. Chromosomal analysis showed consistent involvement of 17q25--an abnormality which has been reported in another alveolar soft-part sarcoma. The histogenesis of alveolar soft-part sarcoma is still debatable but our findings support a myogenic origin. The finding of an apparently identical chromosomal abnormality in two of three thus far examined cases of alveolar soft-part sarcoma is of interest and must await further confirmation, but it may result in the identification of a chromosomal marker for this enigmatic tumour and thus pave the way for further molecular elucidation.

Summary. Background: Regulator of G-protein signaling (RGS) 2 negatively regulates Gs signaling by inhibiting the activation of adenylyl cyclase (AC). RGS2 mRNA contains four translation initiation sites, leading to four isoforms with different abilities to inhibit AC activity; the largest isoform is the most pronounced inhibitor. A role for RGS2 in platelets is not known. Objective: To describe a heterozygous RGS2 mutation (G23D) in three related patients, leading to Gs hypofunction in their platelets, and to study the mechanism behind the effect of the RGS2 mutation on platelet function and morphology. Methods: Gs signaling was studied ex vivo in platelets and in vitro in transfected cells. Translation initiation was evaluated in vitro, and the interaction of wild-type and G23D RGS2 with AC was unraveled via immunoprecipitation. Platelet granule content was analyzed with proteomics. Results: The mutation leads to reduced cAMP production after stimulation of Gs-coupled receptors. The largest RGS2 isoforms, with strong AC inhibitor activity, are enriched when the mutation is present, as compared with wild-type RGS2. Moreover, the mutation results in a stronger interaction of RGS2 with AC. G23D RGS2 carriers have enlarged, round platelets with abnormal α-granules. Proteomics of the platelet releasate revealed altered expression of some proteins involved in actin assembly, and carriers seemed to have a reduced platelet shape change. Conclusions: We present the first platelet Gs signaling defect caused by a heterozygous RGS2 variant that results in a unique mutational mechanism, such as the differential use of translation initiation sites resulting in different functional RGS2 isoforms.

A case of primary malignant rhabdoid tumour of the liver occurring in a 5-month-old girl is reported. Histologically the neoplasm presented the 'typical' features as described in malignant rhabdoid tumour arising in the kidney. In addition, some areas of the tumour showed a 'pseudoglandular' growth pattern. Immunohistochemistry revealed the neoplastic cells to be intensively positive for vimentin and for cytokeratin 19. Variable immunoreactivity for cytokeratin polypeptides 7, 8 and 19 was also detected. On electronmicroscopy the neoplastic cells contained bundles of intermediate filaments, tonofilaments and intercellular junctions. These findings are suggestive of a primitive epithelial differentiation.

The relation between copper tolerance and the sensitivity of plants with respect to the effect of copper on the plasmalemma of root cells was studied using plants from one copper sensitive and two copper tolerant populations of Silene cucubalus Wib. In each population, the external copper concentration needed to induce ion leakage (a measure of damage to the permeability barrier) was similar to the highest no-effect-concentration of copper for root growth in that population. At higher concentrations, the degree of root growth inhibition paralleled the rate of ion leakage, the degree of trypan blue staining (a measure of plasmalemma integrity) and the accumulation of lipid peroxidation products. The amount of copper taken up by the plants was inversely related to their level of copper tolerance. Compared to copper sensitive plants, copper tolerant plants showed no increased resistance to either the sulfhydryl reagent N-ethylmaleimide or the free radical-producing compound cumene hydroperoxide. These results indicate that damage to the permeability barrier of root cells constitutes the primary effect of copper toxicity in both sensitive and tolerant plants, and that copper tolerance is coupled to the ability of the plants to prevent such damage. This ability might depend on exclusion of copper by the root cell plasmalemma.

Malignant histiocytic neoplasms of dendritic cell lineage are rare and most are derived from the dendritic cells of the T-cell and B-cell areas of the lymph node, respectively. Only one case of a malignant histiocytic neoplasm of Langerhans' cells, the dendritic cell of the skin, has been reported. We report the occurrence of a true histiocytic neoplasm of Langerhans' cell type in a 23-year-old female patient. The diagnosis of this highly aggressive neoplasm is supported by histochemical, immunohistochemical, ultrastructural, and genotypic analysis—techniques which allow differentiation from malignant melanoma and large cell anaplastic lymphoma.

Background: Non-alcoholic-steatohepatitis (NASH) leading to fibrosis, end-stage cirrhosis and hepatocellular carcinoma is an increasing health problem in the Western world. Thus, the need for new therapeutic approaches is increasing. IKK2 plays a key role in the development of NASH by mediating inflammation and insulin resistance. Aim: Here the beneficial effects of a pharmacological IKK2 inhibitor (AS602868) on initial stages of NASH progression were tested. Methods: Mice were fed with a high sucrose diet (HSD) and daily-administered AS602868 and vehicle. The impact of AS602868 on NASH progression was studied using biochemical, histological and molecular markers. Results: AS602868 treatment prevented HSD-induced weight gain and visceral fat accumulation. In adipose tissue, AS602868-treated mice exhibited a lower degree of infiltrated macrophages along with reduced proinflammatory cytokine production. Further analysis demonstrated that AS602868 treatment efficiently inhibited nuclear factor (NF)-κB activation in liver non-parenchymal cells and as a consequence attenuated the inflammatory response in the liver. Accordingly, in HSD/AS602868 mice, liver and adipose tissue adiponectin levels remained at levels comparable with those of control chow-fed mice, while they were decreased in HSD/vehicle animals. Additionally, AS602868 improved lipid β-oxidation mediated by peroxisome proliferator-activated receptor (PPAR) α and PPARγ. Systemic pharmacological IKK2 inhibition by AS602868 treatment efficiently prevented liver steatosis and inflammation, and improved antioxidant response. All this contributed to attenuation of NASH progression as evidenced by lower hepatocyte apoptosis and early stages of liver fibrosis. Conclusion: The data demonstrate that AS602868-mediated IKK2 inhibition represents a new therapeutic approach to prevent dietary-induced NASH progression.

The mating behaviour and the factors affecting male mating success in individually marked free-ranging red squirrels (Sciurus vulgaris) were studied over a 4 year period with the aid of radio-telemetry. In the coniferous area 12 mating-chases were observed against 14 in the deciduous study area. A mean of 3.19 adult males took part in a mating-chase, one of which was the leading male that defended the oestrous female. The leading male accounted for 24 of the 29 observed matings, while in five cases other males were able to mate successfully. The leading male had in general the highest dominance rank, and the highest body weight of all competitors. It was normally an older male with large testes. The position of the leading male in a chase was stable in time and in space, although on two occasions it abandoned the female when she moved outside its home range. The relative success of three different mating strategies: the protective leader strategy, the persistent male strategy, and the sneaker male strateg...

Summary: We report a retrospective electron-microscopical study of liver biopsies and fibroblast cultures of 19 patients with congenital disorders of glycosylation (CDG) of different subtypes. A constant finding in liver biopsies of all CDG-I cases was that of abnormal lysosomal lamellar inclusions in the hepatocytes, which were not found in CDG-II. None of the patients showed significant abnormalities in their fibroblasts.

Nasal carriage of Staphylococcus aureus represents a risk factor for subsequent invasive infections and interpatient transmission of strains. No physiological in vitro model of nasal epithelial cells is available to study both patient- and bacteria-related characteristics and their interaction, leading to adherence and colonization. Starting with tissues from human nasal polyps, a confluent, squamous, nonkeratinized epithelium in collagen-coated 96-well microtiter plates was obtained after 14 d. This in vitro cell-layer was characterized histologically, ultrastructurally, and immunohistochemically and showed features that were indistinguishable from those observed in the squamous nonkeratinized epithelium found in the posterior part of the vestibulum nasi. Adherence experiments were performed with four different 3H-thymidine-labeled Staphylococcus aureus strains. The effect of bacterial inoculum size, temperature of incubation, and incubation medium were studied. The adherence results were found to be reproducible, reliable and sensitive, allowing detection of small quantitative differences in adherence between the Staphylococcus aureus strains. There was no significant difference in adherence at 23 degrees C and 37 degrees C, nor between the incubation medium M199 and phosphate-buffered saline. Plastic adherence could be reduced and standardized with use of siliconized tips and a constant bacterial inoculum volume of 100 microl/well. This physiological and reliable in vitro cell-culture model offers a unique opportunity to study Staphylococcus aureus adherence to squamous, nonkeratinized nasal epithelial cells and both patient and bacterial characteristics involved in this interaction.

A 57-year-old female patient presented with painless obstructive jaundice and mild mesogastric pain; she was in good general condition on admission. Abdominal ultrasonography revealed diffuse tumoral invasion of the liver, suggesting diffuse metastases. A liver biopsy showed a tumour with a trabecular growth pattern, composed of uniform relatively small cells, very suggestive of an endocrine carcinoma. Additional immunohistochemical stains, however, did not show any endocrine differentiation, but showed positivity for both hepatocyte-type cytokeratins (cytokeratin 8 and 18) and bile duct-type cytokeratins (cytokeratin 7 and 19). In addition, parathyroid hormone-related peptide, shown to be a good marker for cholangiocarcinoma, was immunoreactive. Electron microscopy revealed tumour cells with an intermediate phenotype: the cells clearly showed hepatocyte features on one hand and bile duct cell features on the other hand. Nine days after admission, the patient died due to liver failure and hepatic encephalopathy. Autopsy excluded another primary tumour site. Overall, this tumour was a primary liver tumour with an intermediate phenotype and with a very rapid clinical course. The intermediate (between hepatocyte and bile duct cell) phenotype suggests an immature progenitor cell origin, which is concordant with a rapid clinical course. This type of tumour has not been described previously and provides additional evidence for the existence of progenitor cells in human liver.

Benign peripheral nerve tumors with a mesenchymal component are rare and are represented principally by the neuromuscular choristoma. We describe an 11-month-old male infant who presented with a mass arising from the left brachial plexus. The lesion was bound firmly to the involved nerves and consisted histologically and ultrastructurally of bundles of well-differentiated smooth muscle cells intermingled with striated muscle fibers and unmyelinated nerve fibers. In the 13 previously published cases of neuromuscular choristoma, no smooth muscle component was observed. This unique peripheral nerve choristoma with not only striated but also smooth muscle closely resembled neuromuscular choristoma and was considered a variant of it.

At endoscopy, the duodenum in Whipple's disease frequently appears abnormal and some clinical features such as gastrointestinal blood loss and anaemia suggest epithelial damage. However, the intestinal epithelial cells themselves appear to be normal on light and electron microscopy. The aims of this study were to analyse in detail the cytological changes in epithelial cells over time and in response to therapy in biopsies obtained from 20 patients, to investigate the functional repercussion on digestive enzymes such as lactase, and to assess the expression by the epithelial cells of MHC antigens. Cytological changes were minimal at both the light- and the electron-microscopic level and MHC class I expression was preserved. However, changes indicative of functional deficits were demonstrated. Lactase and MHC class II expression were reduced or even absent. Antibiotic therapy resulted in normalization within 3-6 months. These findings are consistent with the clinical evolution and are of interest with regard to the importance of the immune response in aetiopathogenesis.

The macroscopic and light microscopic structure of the vesicular and bulbourethral glands was investigated in male pigs, which were castrated early in life and examined at slaughter weight. The vesicular glands showed a constant morphological pattern while in the bulbourethral glands an individual variation was observed in the number of mucous acini and the amount of interstitial tissue. In spite of this variation in the normal structure, the latter gland as well as the vesicular gland showed reliable changes after treatment with diethylstilboestrol.

An ultrastructural morphologic, histochemical, and morphometric study was made on the canalicular structures in rat liver after experimental bile duct ligation (BDL). Four different canalicular structures are distinguished, which are proved by morphometry to correspond to the different types observed during perinatal maturation. Canaliculus type 4, corresponding to the excretory pole of a normal adult hepatocyte, is characterized by a lumen filled with regular, slender microvilli. Canaliculus type 3, corresponding to the canalicular structure described in cholestasis, has a wide lumen devoid of microvilli and is found 2–3 days after BDL. A transitional canalicular structure between type 3 and 4, partly provided with microvilli, is found from the first day after BDL. After 3 days of BDL an increasing number of other canalicular types is observed. Canaliculi type 2, showing an irregular lumen with irregular microvillus-like projections, and canaliculi type 1, representing intracytoplasmic invaginations of two adjacent cell membranes, are found together with canaliculi type 3 and type 3–4. After incubation for alkaline phosphatase, the enzyme activity is detected in canaliculi type 1, 3–4, and type 4. The striking analogy of the sequential changes in canalicular structures after BDL and during perinatal maturation leads to the following interpretation: (1) Canaliculi type 1 and 2, more frequently observed in longstanding cholestasis correspond to newly formed excretory poles of the hepatocytes. (2) Canaliculus type 3, or the so-called cholestatic canalicular type, reflects an inactive secretory function. Morphometrically this canalicular type shows a very low S/V relation which may be partly responsible for this secretory inactivity. (3) Canaliculus type 3–4 reflects a disturbed secretory function after BDL and a beginning secretory activity during perinatal maturation.

The nopaline Ti-plasmid pTiC58 of Agrobacterium tumefaciens C58 was characterized by restriction endonuclease analysis. Fragments generated by HindIII (52), HpaI (17), SmaI (37), KpnI (20), BamHI (>50), EcoRI (>50), and XbaI (9) were arranged into a circular map corresponding to a genome size of 132 × 106 D. The map was established by analysis of large overlapping plasmid segments of pTiC58 derived from a partial HindIII digest and cloned in the vector pBR322. Taken together, the segments present in the 33 hybrid plasmids obtained cover the entire pTiC58 DNA except for 2% located between map coordinates 22 and 24.7 T. The restriction map of the pTiC58 region containing the T-DNA is presented in greater detail.

In spite of recent electromyographic experiments and myotomy on the pylorus, a detailed morphological study of that region is still lacking. In this study the pyloric part of the abomasum and the first 15 cm. of the duodenum have been investigated macro- and microscopically in 20 veal calves and 20 adult cattle. The circular muscle layer of the terminal part of the pars pylorica thickens into a proximal and a distal sphincter. Two U-shaped muscle loops, one on the visceral and the other on the parietal surface, are fixed by their base to the sphincter pylori. Together with longitudinal fibers of the abomasum and the duodenum, they may form a dilator pylori. A fibrous septum, which separates the muscular coat of the stomach from that of the duodenum is absent. Some longitudinal fibers of the abomasum are continuous with those of the duodenum. The loose mucosal folds in the pars pylorica and also the torus pyloricus may act as a mucosal plug in closing the pylorus. These findings are discussed in view of possible functional implications.

The expression of transferrin receptors (TfR's) has been investigated in eight liver biopsy specimens (four from patients without demonstrable iron and four from patients with iron storage due to primary hemochromatosis (HC)) using immunoelectron microscopy to demonstrate TfR's by the simultaneous application of two specific monoclonal antibodies (OKT9 and B3/25) to tissue chopper sections. In the four specimens without iron overload, hepatocytes, but not sinusoidal lining cells, stained positively and immunoreactivity was mainly localized in the cytoplasm. Positively stained cisternae of the endoplasmic reticulum indicated synthesis of the TfR. The presence of TfR's on segments and coated invaginations of the sinusoidal membrane and in small, but otherwise unidentified vesicles in the cytoplasm is compatible with endo-/exocytotic transport and recycling of TfR's as demonstrated by biochemical studies. Occasional positively stained material in canalicular lumina together with positively stained canalicular microvilli and pericanalicular vesicles suggest that transcellular transport may be an additional pathway for TfR's. In three biopsies showing severe iron overload due to HC, TfR immunoreactivity was completely absent. The remaining specimen showing HC, exhibited relatively mild iron overload and showed only a few positively stained hepatocytes. This supports the previously reported disappearance of hepatic TfR expression in HC when iron overload is severe.

This study of 185 vertebral columns of male and female goats describes the gross variation in numbers of segmental vertebrae and the occurrence of a variety of transitional vertebrae. The variations in number and gross anatomical organization of transitional vertebrae are discussed and a hypothesis presented to explain their occurrence.

Extract: Histochemical staining of alkaline phosphatase was performed on liver tissue of three groups of Wistar rats: (group 1) 44 fetuses between 14 days gestational age and birth; (group 2) 76 neonatal rats between 0 and 10 days of age and; (group 3) 44 neonatal rats between 0 hr and 10 days of age, born to dams subjected to bile duct ligation 2 days before delivery. In tissue from animals of group 1 a progressive evolution was observed in the pattern of “canalicular‘’ staining and in the degree of positive reaction obtained. In tissue from animals of group 2 a striking deviation from the usual staining pattern observed at birth was seen from the 12th hr on, returning gradually to “normal‘’ by the 10th day. This change resembled different cholestatic conditions seen in tissue of adult rats. From the 6th hr until the 2nd day a slight accumulation of histochemically detectable conjugated bilirubin was observed. In animals from group 3 a similarly “abnormal‘’ alkaline phosphatase picture was observed at birth (0 hr), whereas no bilirubin could be visualized. The findings seen in animals of group 2 suggest the occurrence of a cholestatic phenomenon in early neonatal life of normal rats, presumably caused by temporary immaturity of the liver secretory function, hepatocytes or bile ducts, or both. The results found in animals of group 3 indicate that the sequence of changes observed in the neonatal period can be accelerated by enforcing a secretory load upon the fetal liver. Speculation: A secretory immaturity of the liver cell seen in neonatal rat liver tissue could also exist in human neonatal liver, and may be an important factor in different conditions of human neonatal jaundice. One could further speculate whether the so-called “inspissated bile syndrome” observed in young children with severe hemolysis might not be partly due to a similar immaturity of the excretory mechanisms of the liver cell.

We compared the paracortical area in 4 cases of dermato-pathic lymphadenitis (DL) with the same area in 11 cases of various other reactive conditions of the lymph node by immuno- and enzymehis-tochemical techniques. In addition, electron microscopy was performed on threee cases of DL. The paracortical nodules in DL proved to be composed of a variable number of dendritic, OKT6+ OKIa+ ATP-ase+ cells, admixed with helper T-lymphocytes. All other lymph nodes studied lacked dendritic OKT6+ cells, whereas OKIa positivity was found in the cortical (follicular centers and mantle zones) and paracortical area (lymphocytes and scattered dendritic cells). Short incubation for ATPase revealed a paracortical, pericellular staining pattern in cases of DL, whereas in all other cases this staining pattern was observed only after long incubation times. On electron microscopy, three types of dendritic cells were found in DL, namely interdigitating reticulum cells (IDRC), Langerhans cells (LC) and macrophages. Intermediate forms between IDRC and LC, containing a few Birbeck granules and a well developed rough endoplasmic reticulum, were found. It is suggested that immunoreactivity for the monoclonal antibody 0KT6 is restricted to cases of DL, and is due to the appearance of dendritic cells that have LC-characteristics. These cells either arrive from the skin along afferent lymph vessels, or are the result of a local transformation process of IDRC that acquire LC-characteristics, i.e. OKT6 immunoreactivity and Birbeck granules.

This paper reports the case of a child in which the clinical and laboratory data indicate a progressive intrahepatic cholestasis of the type described as Byler's disease. The histological and histochemical findings suggest an intrahepatic cholestasis. Electron microscopy reveals interruptions of the bile canalicular membrane, which have been described as characteristic of this disease. A striking feature in the present case is the remarkable increase of microfilamentous structures in the pericanalicular ectoplasm and in the hepatocytic cytoplasm. The findings suggest a primary disturbance in bile acid secretion as the casue of cholestasis, entailing a hypertrophy of pericanalicular microfilaments which supposedly play a role in the final step of biliary secretion.

Eighteen liver biopsy specimens from patients with hepatitis B surface antigen (HBsAg) positive chronic aggressive hepatitis were studied by electron microscopy. All cases were selected on the basis of positive liver cell membrane fluorescence for HBsAg on immunohistochemical investigation. Striking changes in the morphology of the liver cell membrane were observed in nearly all cases. Furthermore, a dual aspect of hepatitis B core antigen (HBcAg) is described. HBcAg particles may occur as either 'naked' or 'cloudy' particles surrounded by semi electron dense material. The nature of the 'cloud' remains to be identified.

HLA-DR-positive dendritic cells present at the dermo-epidermal junction in skin disorders associated with the deposition of immunoglobulins and/or complement factors in the skin biopsy are investigated ultrastructurally. They demonstrate long, slender cytoplasmic processes, an indented nucleus, and strikingly dilated cisternae of the endoplasmic reticulum. HLA-DR antigen is present on the plasma membrane and within some of the dilated cisternae. These findings further support our previously formulated hypothesis that these cells represent a functionally active stage of antigen-presenting cells.

Various fixation and staining procedures for the demonstration of surface and cytoplasmic antigens have been described. An immunostaining procedure was sought that would allow the demonstration of these antigens, especially in small human tissue samples at the ultrastructural level. A modification and adaptation of the technique of Eldred, Zucker, Karten, and Yazula (J Histochem Cytochem 31:285, 1983) was applied on several varieties of human tissue, including liver, skin, and lymphoid tissue, using monoclonal and polyclonal antibodies in an indirect peroxidase procedure. In this way a reliable and generally applicable procedure was developed that satisfied the following demands: Use of a universal fixative that allows preservation of the antigenicity of various antigens; Adequate penetration of the tissue by the immunological reagents; Optimal preservation of subcellular structures; and Possibility to store the tissue samples for considerable periods of time.

Summary We report a case of Histiocytosis X (HX) associated with a high bone marrow and peripheral blood monocytosis in a 70 yr-old man. The histiocytic clusters in the skin showed the characteristics of epidermal Langerhans cells (LC): positivity for OKT 6 , OKla 1 , ATP-ase and presence of Birbeck granules. Histiocytes in all other locations showed posivity for OKM 1 , OKla 1 , acid P-ase and absence of Birbeck granules. The circulating monocytes were also negative for OKT 6 and ATP-ase and lacked Birbeck granules From these data we assume that the proliferation of monocytes in the bone marrow was the primary event, resulting in the release of large numbers of circulating monocytes and histiocytic proliferation in various tissues. These histiocytes acquired differing characteristics according to the microenvironment in which they underwent their final maturation. In the skin they differentiated into cells presenting the morphological, enzyme-and immunohistochemical characteristics of LC

Bilirubin UDP-glucuronyltransferase and UDP-xylosyltransferase activity could already be demonstrated in rat liver from day 19 of fetal life onwards (4 days before birth). Bilirubin-glucuronide was present in hepatocytes of 21-day-old fetal liver strongly suggesting that the enzyme detected in vitro was really active in vivo. This further supports the theory that secretion from the cell is also immature. The known deficiency of UDP-glucose dehydrogenase in fetal and neonatal rat liver could lead to decreased intracellular concentrations of UDP-glucuronic acid and possibly to increased concentrations of UDP-glucose. However, no glucosides were present in the fetal hepatocytes.

In order to complement previous electron microscopic studies on the differentiation of the biliary pole of the rat hepatocyte during perinatal maturation, a morphometric analysis was undertaken of the bile canaliculi in fetal and adult rat liver. Four morphologic canalicular types were found and morphometrically characterized using a surface/volume relation (s/v index) as key parameter. The surface/volume relation of the canaliculus in fetal and postnatal maturation as well as in the adult rat liver supports the previously formulated hypothesis that these different types correspond to successive maturation stages of the biliary pole of the hepaocyte. Furthermore, the s/v index of type 3 canaliculus supports the concept that this canalicular type represents a canalicular structure inefficient for secretion.

The differentiation of the biliary pole of the hepatocyte was studied during development. Four successive types can be distinguished morphologically. Type I, found at 16 days of fetal life, is formed by an intracytoplasmic invagination of the cell membrane. At 19–20 days, most canaliculi correspond to type 2, formed by sequestration of cytoplasmic fragments. In the perinatal period most canaliculi are of type 3, characterized by a wide and regular lumen, without microvilli. From the 2nd day of life on, an increasing number of canaliculi acquire microvilli and correspond to type 4. Histochemical results after incubation for alkaline phosphatase further support the proposed maturation scheme. The morphologic and histochemical characteristics of type 3 canaliculi suggest the existence of a cholestatic phenomenon of the neonatal liver.

An electron microscopical investigation is carried out on the proximal kidney tubules of rats submitted to experimental bile duct ligation. Twelve hours after obstruction, a strong increase of pinocytotic organelles in the subapical cytoplasm of the tubule cells, presumably due to an active transport of bile acids, is observed. These organelles stain positively after incubation for alkaline phosphatase. After 2 days of obstruction, the cytosomes mostly stain positively for acid as well as for alkaline phosphatase. It is suggested, therefore, that pinocytotic vacuoles have fused with cytosomes. In the same period and in the same cells, large focal cytoplasmic degenerations or cytosegresomes arise, apparently due to an injurious effect of bile acids. With longer duration of the bile duct obstruction (10–25 days) the cytoplasm of the tubule cells contains very heterogeneous cytosomes partly filled with an amorphous and dense material comparable to bile pigment-stained cytoplasmic compounds described in cholestatic human liver tissue. Exocytosis of cytosomes into the tubular lumen also occurs in later periods. The formation of cytosegresomes and the exocytosis of cytosomes point to an active defence of the cell toward an injurious agent. The present findings point to a striking analogy between cholestatic liver cells and renal cholestasis.

Three cases of Kikuchi's lymphadenitis were studied immunohistochemically on paraffin-embedded material in order to compare the plasmacytoid monocyte aggregates occurring in this disorder with plasmacytoid monocyte clusters in nonspecific reactive lymphadenitis. One of the cases was also analyzed on frozen material. In Kikuchi's lymphadenitis, plasmacytoid monocytes expressed the CD38 and LN1 antigens in addition to their monocyte/macrophage antigens found in nonspecific reactive lymph nodes. These plasmacytoid monocyte aggregates contained large numbers of Mac387+ lysozyme+ macrophages, scattered HLADR+ dendritic cells, and high numbers of CD8+ T-lymphocytes. Collections of T-immunoblasts or areas of necrosis were observed within these aggregates. The authors speculate that the histologic feature of Kikuchi's lymphadenitis is the morphologic substrate of a T-cell-mediated cytotoxic immune reaction occurring in plasmacytoid monocyte aggregates and resulting in plasmacytoid monocyte damage and necrosis.