Ultrastructural findings in progressive macular hypomelanosis indicate decreased melanin production.

BACKGROUND: The pathogenesis of progressive macular hypomelanosis (PMH) is unknown. Recently, Westerhof et al. (Arch Dermatol 2004; 140: 210-214) hypothesized that Propionibacterium acnes produces a depigmenting factor that interferes with melanogenesis in the skin, resulting in hypopigmented spots. The purpose of the study is to gain an insight into the pathogenesis of PMH. MATERIALS AND METHODS: We took a biopsy of 2-mm diameter from normal and lesional skin in eight PMH patients. Using electron microscopy, we compared melanization of melanosomes, melanosome transfer and amount of epidermal melanin in normal and lesional skin. RESULT: Compared to non-lesional skin, we observed a decrease of epidermal melanin and less melanized melanosomes in lesional skin of all patients. When comparing normal and lesional skin of patients with skin type V and VI, we observed a difference in melanosome size and maturation and a switch of transferred melanosomes from single stage IV transferred melanosomes to aggregated stage I, II and III transferred melanosomes, as seen in healthy skin of skin type I to IV. CONCLUSION: Hypopigmentation in PMH seems to be the result of an altered melanogenesis based on a decrease in melanin formation and a change in the distribution of melanosomes. In lesional skin of PMH patients with skin type V and VI less melanized, aggregated melanosomes in stead of single, mature melanosomes are transferred from melanocytes to keratinocytes. This results in a decrease of epidermal melanin. Further investigations are needed to determine the precise role of Propionibacterium acnes in this alteration of melanogenesis.

Hypermelanocytic guttate and macular segmental hypomelanosis.

We report two sisters, 27 and 30 years of age, with a cutaneous pigmentary anomaly, which seems to be a new entity. At the age of 26 years the elder sister developed an asymptomatic and persistent rash consisting of discrete, grouped, round to oval, guttate and nummular, hypopigmented macules, 0.2-5 cm in diameter. The distribution of the lesions was unilateral. They were located on the right side of the thorax with a moderately sharp demarcation in the mid-line and ran in a segmental distribution over the right arm, hand and fingers. Microscopic examination of lesional skin scrapings was negative for fungi. Examination with Wood's light accentuated the lesions from the surrounding normal skin. The younger sister had experienced identical, mostly guttate, skin lesions for many years, which at examination were distributed on all extremities and buttocks, and to a lesser degree on the trunk, but here in a segmental distribution. Histological examination (Masson-Fontana staining) of lesional skin of both sisters was identical. A slightly thinned epidermis and a marked decrease in pigmentation of the epidermal basal layer was seen. Electron microscopic examination of lesional skin showed an overall linear increase of morphologically and cytologically normal melanocytes just above the epidermal basal membrane. At many places the density of melanocytes was so high that the keratinocytes were displaced from the basal layer. The melanocytic dendrites extended into the suprabasal layer. The keratinocytes of lesional skin showed a decreased number of melanosomes. It is paradoxical that a hypomelanotic macule shows a histological picture of an increase in normal functioning melanocytes. In all probability a deficient melanosome transfer is responsible for this unexpected phenomenon.

Misdiagnosis of Fabry disease: importance of biochemical confirmation of clinical or pathological suspicion.

Generalized angiokeratoma are associated with three lysosomal storage disorders, one of which is Fabry disease (alpha-galactosidase A deficiency). Treatment for Fabry disease with supplementation of recombinant enzyme is available in the European Union and subsequently physicians' awareness may rise. A patient who was erroneously diagnosed with Fabry disease is presented.

Cardiac pathology in neuronal ceroid lipofuscinoses--a clinicopathologic correlation in three patients.

We report the clinical details and the pathology of the heart at autopsy of three neuronal ceroid lipofuscinosis (NCL) patients. Two patients were diagnosed as classical juvenile NCL and one as a variant juvenile NCL (JNCL) with granular osmiophilic deposits (GRODs). Cardiac findings during life were retrospectively evaluated and included left ventricular hypertrophy with repolarization disturbances (ECG findings) in two patients with classical JNCL and severe bradycardia with periods of sinus arrest in one of them, severe supraventricular tachycardias during anaesthesia in the variant JNCL-patient. At autopsy myocardial and valvular storage of lipopigments, diagnostic for NCL, was observed histologically and confirmed ultrastructurally in all three cases. In two patients with JNCL the storage was associated with hypertrophy and dilation of both ventricles, degenerative myocardial changes, interstitial fibrosis and fatty replacement. Abundant accumulation and degeneration were seen in all components of the conduction system in three patients, which outreached at several places by far the storage of the adjacent myocardium. Our observations indicate a prominent involvement of the heart in NCL, with preference of storage for the conduction system of the heart.

Immunoelectron microscopy on material retrieved from paraffin: accurate sampling on the basis of stained paraffin sections.

As a rule, immunoelectron microscopy (immuno-EM) is performed on fresh material processed according to specialized methods (e.g., freezing or embedding in hydrophilic resins). Paraffin-embedded tissue has only occasionally been used as a source of material for immuno-EM; this was usually as a last resort, when no fresh material was available. The authors used archival formalin-fixed, paraffin-embedded basal cell carcinomas for studying the fine-structural distribution of the cell surface molecule CD44 and its variants, as well as some other antigens. The results demonstrate, firstly, that paraffin-embedded material is far more suitable for immuno-EM than frequently assumed and, secondly, that the use of paraffin-embedded material enables highly accurate sampling on the basis of immunohistochemically or conventionally stained light microscopic sections.

Intrauterine fetal death due to Farber disease: case report.

We report a case of Farber disease in a fetus who died in utero at a gestational age of 29 weeks. Macroscopic examination showed moderate postmortem changes in a microcephalic female fetus (46,XX) with mild internal hydrops, two vessels in the umbilical cord, and a moderately enlarged, relatively well-preserved spleen. Microscopic examination showed foamy cells in the spleen. Electron microscopic examination revealed the presence of Farber bodies within these foamy cells. Enzyme studies of the fetus were not possible because all tissues were formalin fixed. Lipids were extracted from formalin-fixed tissues and increased levels of ceramide and the presence of hydroxyceramide in tissue of the spleen, liver, and lung were found. Glucosylceramide was not increased excluding saposin-precursor-deficiency. Because of these findings, both parents were tested for acid ceramidase activity in their leukocytes. They both had markedly reduced enzyme activity consistent with heterozygosity for Farber disease. To the best of our knowledge, this is the first published case of Farber disease in Dutch nonconsanguineous parents.

Lung epithelial H292 cells induce differentiation of immature human HMC-1 mast cells by interleukin-6 and stem cell factor.

BACKGROUND: Immature mast cells migrate into tissues where they differentiate into mature mast cells under the influence of local factors. In the airways of asthmatics increased numbers of chronically activated mast cells are located nearby the airway epithelium. OBJECTIVE: The aim of this study was to evaluate whether and, if so, which products released by epithelial cells may affect mast cell proliferation and differentiation. METHODS: We performed in vitro studies using the human lung mucoepidermoid carcinoma-derived H292 cell line and the immature human mast cell line, HMC-1. Proliferation was assessed by 3H-thymidine incorporation. Differentiation of HMC-1 cells was inferred from tryptase production. RESULTS: Exposure of HMC-1 cells to medium conditioned for 48 h by H292 cells resulted in a reduction of proliferation with 65 +/- 4.9% (mean +/- SEM, n = 9) at day 5. Culturing HMC-1 cells for 8 days in the presence of H292-conditioned medium resulted in morphological changes indicative of differentiation, and in a 3.0 +/- 0.4-fold increase of tryptase production (P = 0.0039, n = 9). Conditioned medium from H292 cells that were stimulated by LPS also inhibited HMC-1 proliferation. Inhibitory antibodies against two mediators from H292 cells, interleukin-6 (IL-6) and stem cell factor (SCF), abolished the increase in HMC-1 tryptase production induced by H292-conditioned medium. Recombinant human (rh) IL-6, but not rhSCF, reduced HMC-1 proliferation with 44% and 13% at day 3 and 5, respectively. Surprisingly, rhIL-6 did not increase HMC-1 tryptase production significantly whereas incubation with rhSCF did (1.5 +/- 0.1-fold, P = 0.002, n = 10) although the increase was less than observed for conditioned medium. CONCLUSION: Epithelial-derived IL-6 and SCF are implicated in differentiation of HMC-1 cells but additional factors are not excluded. As activated primary bronchial epithelial cells also express IL-6 and SCF, it should be considered that these cells are involved in mast cell differentiation within the airways, particularly in diseases where epithelial cells are activated, such as asthma.

Biomaterial-associated persistence of Staphylococcus epidermidis in pericatheter macrophages.

Biomaterial surfaces may be modified to reduce bacterial adhesion. The susceptibility in mice to Staphylococcus epidermidis infection in tissue surrounding the commonly used catheter materials-silicon elastomer (SE), polyamide (PA), and their surface-modified polyvinylpyrrolidone (PVP)-grafted derivatives, SE-PVP and PA-PVP, respectively-was assessed. Abscesses developed around SE-PVP. Around SE, PA, and PA-PVP catheters, no signs of infection were observed, although mice carrying PA-PVP developed septicemia after 14-21 days. S. epidermidis was cultured from the tissue surrounding PA-PVP segments. Cells around PA-PVP segments containing large numbers of bacteria were identified as macrophages by use of immunohistochemistry and electron microscopy. This persistence of intracellular bacteria was also observed around SE-PVP, SE, and PA catheters, although to a lesser extent. The cytokine profiles around the 4 materials were different. Implanted biomaterial induces an inflammatory response favorable to the persistence of S. epidermidis. Intracellular persistence of bacteria inside macrophages may be a pivotal process in the pathogenesis of biomaterial-associated infection.

Gangliocytic paraganglioma of the appendix.

AIMS: A case of gangliocytic paraganglioma is reported in the appendix which, to the best of our knowledge, is the first case at this particular site to be described in modern literature. METHODS AND RESULTS: A 47-year-old man with signs and symptoms of acute appendicitis underwent appendectomy. In the resected specimen a tumour with a diameter of 9 mm was found, which microscopically consisted of three different cell types: (a) epithelioid cells lying in a trabecular pattern and in formations reminiscent of 'Zellballen' as seen in paragangliomas (b) spindle cells and (c) ganglion-like cells. A diagnosis of 'gangliocytic paraganglioma' was made and confirmed by immunohistochemical and ultrastructural examination. CONCLUSION: Gangliocytic paragangliomas are rare tumours of uncertain histogenesis. More than 40 years ago a tumour in the appendix with features similar to our case was described by Masson as 'neuro-carcinoïde'. Concerning its origin, Masson, as well as other authors describing gangliocytic paragangliomas decades later, referred to the endodermal-neuroecto dermal complexes found by Van Campenhout. It is felt that the current finding of a gangliocytic paraganglioma in the appendix supports the hypothesis that gangliocytic paragangliomas arise from these embryonal structures.

Extracellular matrix of the human aortic media: an ultrastructural histochemical and immunohistochemical study of the adult aortic media.

Aortic distensability is the key to normal aortic function and relates to the lamellar unit in the media. However, the organization of the extracellular matrix components in these lamellar units, which are largely responsible for the distensability, is insufficiently known, especially in the human. We therefore performed a detailed ultrastructural analysis of these components. Thoracic aortas of 56 individuals (age 45-74 years), none of whom suffered from aortic disease, were studied by immunoelectron microscopy of elastin, collagen types I, III, IV, V, and VI, fibronectin, and fibrillin-1, and by ultrastructural histochemistry of proteoglycans, which were further characterized by enzymatic digestion. The elastic lamellae were closely associated with thick collagen fibers containing types I, III, and V collagen. Between these collagen fibers, numerous complex, circumferentially oriented streaks of elastin protruded from the lamellae. In contrast to what is usually reported in the aortas of experimental animals, the smooth muscle cells preferentially adhered to these ill-defined streaks rather than directly to the solid lamellae. Fibrillin-1- and type VI collagen-containing bundles of microfibrils (oxytalan fibers) were also involved in the smooth muscle cell-elastin contact. The smooth muscle cells were invested by basal lamina-like layers connecting them to each other as well as to the oxytalan fibers. Unexpectedly, these layers were abundantly labeled by anti-fibronectin, whereas type IV collagen, a specific basement membrane component, was mainly found in larger, flocculent deposits. The proteoglycans present were collagen-associated dermatan sulfate proteoglycan, cell-associated heparan sulfate proteoglycan, and interstitial chondroitin sulfate proteoglycan. Our observations demonstrate that the extracellular matrix in the human aorta is extremely complex and therefore differs from most descriptions based on experimental animals. They serve as reference for future studies on aortic diseases, such as aneurysmas and dissections.

The expression of CD44 glycoprotein adhesion molecules in basal cell carcinomas is related to growth pattern and invasiveness.

Basal cell carcinomas (BCCs) of the skin exhibit a wide range of histological growth patterns as well as a highly variable rate of invasiveness. A large body of experimental and clinical studies supports a role for the CD44 glycoprotein family in the latter process. In the present study, we explored the distribution and the level of expression of pan-CD44, CD44v3, CD44v5 and CD44v6 in BCCs. The use of paraffin sections, combined with an antigen retrieval procedure, yielded far more detailed data than would have been possible with frozen sections. On average, the level of expression of the four CD44 isoforms studied appeared to differ relatively little. However, tumours or tumour areas consisting of thin tumour cell strands showed a significantly stronger expression of all four isoforms than those consisting of solid tumour cell groups. Furthermore, the highest CD44 expression was frequently observed in the smallest tumour cell strands in the tumour periphery. In these strands, the label seemed to be located not only at the tumour cell-tumour cell interface, as in other tumour areas, but also on the tumour cell surfaces facing the stroma. We are presently assessing the exact localization of CD44 at the cellular level by immunoelectron microscopy. In most cases, different growth patterns with significantly different levels of CD44 expression were found side by side within individual tumours. CD44 expression is therefore not a static tumour cell characteristic but is correlated with tumour architecture and tumour-stroma interaction.

Infantile fibre type disproportion, myofibrillar lysis and cardiomyopathy: a disorder in three unrelated Dutch families.

An apparently new cardioskeletal myopathy is reported in three unrelated families. Five infants were affected by rapidly progressive generalized muscle weakness, with onset shortly after birth, and dilated cardiomyopathy. All had generalized tremor (clonus) starting in the first week of life. The disease was lethal in all cases between 4 and 6 months. Muscle biopsy, performed in four of the five patients, showed a light microscopic pattern of small type I and normal-sized type II fibres. By electron microscopy small fibres were affected by myofibrillar disruption and swelling of organelles. Findings in blood and urine suggested a disturbance in energy metabolism but an extensive search for respiratory chain disorders and disorders of mitochondrial fatty acid oxidation in frozen muscle and cultured fibroblasts was negative. The findings support a new progressive autosomal recessive infantile cardioskeletal myopathy in which type I muscle fibres are preferentially affected.

Bacterial translocation from the biliary tract to blood and lymph in rats with obstructive jaundice.

BACKGROUND: The disruption of the hepatocyte tight junctions observed in biliary obstruction suggests altered permeability of the blood-bile barrier. In this study the role of biliary obstruction and increased biliary pressure on the translocation of bacteria from biliary tract to bloodstream and lymphatic system were evaluated. MATERIALS AND METHODS: Rats underwent distal bile duct ligation (BDL, n = 33) for two weeks or a sham celiotomy (n = 21). Seventeen of the 33 BDL rats underwent subsequent biliary decompression by a choledochojejunostomy (CJ). Two weeks after the final operation, a laparotomy was performed again and the CBD, the thoracic duct, and the caval vein were canulated. Next, a suspension containing 10(8) Escherichia coli/ml was retrogradely infused in the CBD for 5 min at 5 or 20 cm H2O above the secretory biliary pressure. RESULTS: A higher biliary infusion pressure resulted in a significant increase of cfu E.coli per milliliter of blood in all the three groups (Sham, BDL, CJ). BDL rats showed significantly more bacterial translocation to the bloodstream than the shams. After biliary decompression, translocation normalized to the control levels. At 5 cm H2O infusion pressure only one lymph culture was positive (CJ group). At 20 cm H2O overpressure, nine lymph cultures were E.coli positive (P = 0.03). These were found mainly in groups with a nonobstructed bile duct (Sham and CJ 40% vs BDL 10%). CONCLUSION: Translocation of bacteria from biliary tract to bloodstream increased at higher intrabiliary pressures. Longstanding bile duct obstruction was an independent determinant for cholangiovenous reflux. Bacterial translocation to the lymphatic system did not parallel translocation to the bloodstream, although in the nonobstructed biliary tract, increased bacterial translocation to the lymphatic system was pressure related.

Effects of Ursodeoxycholate and cholate feeding on liver disease in FVB mice with a disrupted mdr2 P-glycoprotein gene.

BACKGROUND & AIMS: The mouse mdr2 gene encodes a P-glycoprotein expressed in the hepatocanalicular membrane. Inactivation of this gene causes lack of biliary phospholipid and cholesterol secretion and non-suppurative cholangitis. The aim of this study was to investigate the role of bile salt hydrophobicity in induction of liver pathology in mdr2 (-/-) mice. METHODS: Mice (+/+) wild type or (-/-) knockout for the mdr2 gene were fed with either purified control diet or this diet supplemented with cholate (0.1%) or ursodeoxycholate (0.5%) for 3, 6, or 22 weeks after weaning. Liver histology was semiquantitatively scored. RESULTS: Each mouse fed bile acid became the major constituent of the bile salt pool. The cholate diet during 22 weeks induced only very mild liver pathology in (+/+) mice. By contrast, lever histology had already deteriorated after 3 weeks in the (-/-) mice and caused pronounced inflammatory nonsuppurative cholangitis and fibrosis in the 75% of mice that survived. Dietary ursodeoxycholate had no effect on histology in (+/+) mice but improved liver pathology significantly in (-/-) mice compared with purified control diet; the decrease of ductular proliferation and portal inflammation was most prominent after 22 weeks. CONCLUSIONS: The cholangiolitis and its sequelae in the mdr2 knockout mice depend on bile salt hydrophobicity.

Depletion of mitochondrial deoxyribonucleic acid in a family with fatal neonatal liver disease.

We describe a family in which three children of consanguineous parents died of hepatic failure before the age of 3 months. The first child had clinical symptoms of liver disease with hypoglycemia that were evident at birth. The second child was healthy and has normal development. The third child had severe liver dysfunction noted a few days after birth. Liver failure also developed in the fourth child soon after birth. Recently a mitochondrial disorder was considered as a possible cause. Deficiency of respiratory chain enzymes that contain polypeptides encoded by mitochondrial DNA (mtDNA) and depletion of mtDNA were found in the liver of the fourth child, but mitochondrial abnormalities were absent in muscle of the third child. The similarities in clinical presentation suggest that liver-specific depletion of mtDNA was the cause of the hepatic failure in all three children. We conclude that liver dysfunction with onset in the perinatal period can be caused by depletion of mtDNA.

Multiple labeling in electron microscopy: its application in cardiovascular research.

The heart is a muscular pump kept together by a network of extracellular matrix components. An increase in collagens, as in chronic congestive heart failure (CHF), is thought to have a negative effect on cardiac compliance and, thus, on the clinical condition. Conventional electron microscopy allows for the study of cellular and extracellular components and scanning electron microscopy (SEM) can put these structures in three-dimensional perspective. However, in order to study extracellular matrix components in relation to cells, immunoelectron microscopy is superior. We have used this technique in our studies on heart failure. Heart specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in sodium cacodylate buffer, dehydrated by the method of progressive lowering of temperature and embedded in LR Gold plastic. Immunolabeling could be achieved with different sized gold-conjugated secondary antibodies or protein-A gold conjugates. Depending on the objective, ultra small gold (USG) conjugates or a regular probe size can be used. Labeling efficiency could be increased by bridging antibodies. The double and triple staining procedures were based on single staining methods using one- and two-face labeling. The choice of antibodies and gold conjugates depended on the objectives. Immunoelectron microscopy, using multiple labeling, allowed a detailed study of the organization of the extracellular matrix and its relationship with cardiac myocytes. This may prove to be a useful tool for the study of chronic heart failure.

Mice with homozygous disruption of the mdr2 P-glycoprotein gene. A novel animal model for studies of nonsuppurative inflammatory cholangitis and hepatocarcinogenesis.

The mouse mdr2 gene (and its human homologue MDR3, also called MDR2) encodes a P-glycoprotein that is present in high concentration in the bile canalicular membrane of hepatocytes. The 129/OlaHsd mice with a homozygous disruption of the mdr2 gene (-/- mice) lack this P-glycoprotein in the canalicular membrane. These mice are unable to secrete phospholipids into bile, showing an essential role for the mdr2 P-glycoprotein in the transport of phosphatidylcholine across the canalicular membrane. The complete absence of phospholipids from bile leads to a hepatic disease, which becomes manifest shortly after birth and shows progression to an end stage in the course of 3 months. The liver pathology is that of a nonsuppurative inflammatory cholangitis with portal inflammation and ductular proliferation, consistent with toxic injury of the biliary system from bile salts unaccompanied by phospholipids. Thus, the mdr2 (-/-) mice can serve as an animal model for studying mechanisms and potential interventions in nonsuppurative inflammatory cholangitis (in a generic sense) in human disease, be it congenital or acquired. When the mice are 4 to 6 months of age, preneoplastic lesions develop in the liver, progressing to metastatic liver cancer in the terminal phase. The mdr2 (-/-) mice therefore also provide a tumor progression model of value for the study of hepatic carcinogenesis. Interestingly, also in this regard, the model mimicks human disease, because chronic inflammation of the biliary system in humans may similarly carry increased cancer risk.