Large-scale isolation of sinusoidal endothelial cells from pig and human liver.

OBJECTIVE: Hepatic in vitro studies, like those on hypoxia/reperfusion injury in liver transplants, demand large numbers of cultivated sinusoidal endothelial cells (SECs). In this article, we present and evaluate a new method for the isolation of SECs from porcine and human livers. METHODS: SECs were isolated employing a four-step collagenase perfusion. The sinusoidal character of the cells was validated by transmission and scanning electron microscopy, exclusion of Weibel-Palade bodies and factor VIII-related antigen, expression of scavenger receptor, and incorporation of latex beads. RESULTS: In 23 pigs, an average of 9 x 10(4) SECs were harvested from each liver. Cells were cultivated under standard conditions, as well as in multilayer cocultures of isolated SECs and hepatocytes in a "sandwich" configuration. Standard cultures showed an average of 90% SECs in primary cultures and 100% SECs after the first passage. The possibility of isolation of SECs from human livers was demonstrated in eight cases. CONCLUSION: With the four-step collagenase perfusion it is possible to easily isolate large numbers of viable and pure SECs from one organ. A further advantage is the possibility of isolating hepatocytes from the same organ.

[Scanning microscopy and immunohistochemical studies of an explanted phacic posterior chamber lens (ICL-M, STAAR)]. / Rasterelektronenmikroskopische und immunhistochemische Untersuchungen einer explantierten phaken Hinterkammerlinse (ICL-M, STAAR).

OBJECTIVE: This study was performed to find out if an explanted phacic collamer IOL (ICL/STAAR) showed structural or surface quality changes after an intraocular implantation. MATERIAL AND METHODS: We explanted an ICL 6 months after implantation and compared it to a new reference lens using scanning electron microscopy and immunohistochemical investigations. RESULTS: The explanted ICL showed no changes in comparison to a new ICL except for a coating with an unidentified material. Both the new and explanted ICL presented grooves on the surface and superficial holes of unknown origin measuring roughly 10 mu. Neither pigment nor macrophages could be found on the explanted lens. Although the lens material contained 0.1% collagen, the immunohistochemical staining for collagen was negative. CONCLUSIONS: The collagen content of the ICL could not be proven using immunohistochemistry. No changes were found between the new and the explanted lens, except for a coating of the explanted lens with an unidentified material.

LI-cadherin: a marker of gastric metaplasia and neoplasia.

BACKGROUND: Intestinal metaplasia is considered a risk factor for the development of gastric adenocarcinomas of the intestinal type and is found in approximately 20% of gastric biopsies. Conventional histology only detects advanced stages of intestinal metaplasia. AIMS: To study expression of the enterocyte specific adhesion molecule liver-intestinal (LI)-cadherin in intestinal metaplasia as well as in gastric cancer, and to evaluate its use as a diagnostic marker molecule. PATIENTS: Gastric biopsies (n=77) from 30 consecutive patients (n=30; aged 28-90 years) as well as surgically resected tissue samples (n=24) of all types of gastric carcinomas were analysed. METHODS: Single and double label immunofluorescence detection on cryosections of gastric biopsies; alkaline phosphatase antialkaline phosphatase method on paraffin embedded carcinoma tissue sections. RESULTS: Of 77 biopsies (from 30 patients), 12 (from 10 patients) stained positive for LI-cadherin. LI-cadherin staining correlated with the presence of intestinal metaplasia. Conventional histological diagnosis however failed to detect subtle gastric intestinal metaplasia (three of 10 patients). In contrast, only LI-cadherin and villin were positive in these cases whereas sucrase-isomaltase also failed to detect intestinal metaplasia in four of 10 patients. Well differentiated gastric carcinomas showed intense staining for LI-cadherin while undifferentiated carcinomas showed only weak diffuse cytoplasmic staining. CONCLUSIONS: To detect early metaplastic changes in the gastric mucosa, LI-cadherin has a sensitivity superior to sucrase-isomaltase and conventional histology and comparable with that of villin. Its specificity exceeds that of villin. Thus LI-cadherin represents a new, reliable, and powerful marker molecule for early detection of gastric intestinal metaplasia and well differentiated adenocarcinomas.

Liver transplantation after organ preservation with normothermic extracorporeal perfusion.

OBJECTIVE: To study normothermic extracorporeal liver perfusion (NELP) as a means to preserve livers for transplantation and to reverse warm ischemic injury. SUMMARY BACKGROUND DATA: The authors provide experimental evidence that successful transplantation after 4 hours of normothermic extracorporeal liver perfusion is possible and as reliable as 4 hours of cold preservation in University of Wisconsin solution. NELP preserves liver function completely and can reverse 60 minutes of warm ischemic injury in non-heart-beating donors. METHODS: Thirty-six German Landrace pigs received transplants in six groups. Group 1 animals received direct transplantation. Group 2 received transplants after 4 hours of cold preservation with University of Wisconsin solution and Group 3 animals after 4 hours of NELP. Group 4 animals sustained 1 hour of warm ischemia before transplantation. Group 5 animals received transplants after 1 hour of warm ischemia and 4 hours of cold preservation and Group 6 animals after 1 hour of warm ischemia and 4 hours of NELP. RESULTS: All animals receiving livers treated by NELP survived more than 7 days after the transplant (Groups 3 and 6). In contrast, all animals in Group 5 developed primary graft nonfunction within 24 hours after transplantation. CONCLUSION: The technique of NELP holds the potential to keep a mammalian liver outside the body completely functional, possibly for more than 4 hours. NELP can be used for liver preservation before transplantation or for the use of organs from non-heart-beating donors.

Cellular sources of enhanced brain-derived neurotrophic factor production in a mouse model of allergic inflammation.

The aim of this study was to investigate production and cellular sources of brain-derived neurotrophic factor (BDNF) production in allergic asthma. For this purpose a mouse model of chronic and severe ovalbumin (OVA)-induced airway inflammation was developed. Allergen-exposed mice developed elevated immunoglobulin E titers; airway inflammation with influx of lymphocytes, monocytes, and eosinophils; and airway hyperresponsiveness. In addition to an influx of inflammatory cells, interleukin (IL)-4 and IL-5 production were enhanced, macrophages showed morphologic signs of activation, and airway epithelium was thickened and displayed a goblet-cell hyperplasia with a marked mucus production. BDNF was detected using in situ hybridization and enzyme-linked immunosorbent assay. Constitutive expression of BDNF messenger RNA (mRNA) was observed in the respiratory epithelium of sensitized and nonsensitized mouse lungs. In addition, BDNF mRNA was detected in airway inflammatory infiltrations and bronchoalveolar lavage fluid (BALF) cells of OVA-sensitized and aerosol-challenged mice. Highest BDNF protein levels were detected in BALF after long-term allergen aerosol exposure. Analysis of BDNF production by isolated lymphocyte subsets revealed T but not B cells as a cellular source of BDNF. In addition, activated alveolar macrophages were identified as BDNF-positive cells. These data indicate that in allergic airway inflammation BDNF production is upregulated and immune cells serve as a source of BDNF.

Long-term results of total parathyroidectomy without autotransplantation in patients with and without renal failure.

The optimal surgical procedure for severe renal secondary hyperparathyroidism (sHPT) is still a point of controversy. Total parathyroidectomy (PTX) without auto-transplantation was abandoned for fear of an adynamic bone condition; however, in the case of autotransplantation recurrent sHPT is frequent and promotes atherosclerosis. We studied 11 hemodialysis patients (age 59+/-12 years) on dialysis for 18 (12-30) years in whom total PTX was performed due to severe sHPT (group I; intact PTH: 1,240+/-230 pg/ml), and 5 patients (age 55+/-10 years) without renal insufficiency who inadvertently received total PTX during thyroid surgery (group II). After total PTX (group I, 26+/-18 [9-59] months; group II, 252+/-188 [22 480] months) both groups showed no measurable intact PTH levels. Calcium homeostasis was maintained by oral substitution with calcium (group I, calcium dialysate of 2.0 mmol/l), vitamin D and calcitriol (serum parameters in groups I and II: calcium 2.4 and 2.2 mmol/l; phosphate 1.8 and 1.1 mmol/l; 25(OH)-vitamin D(3) 21 and 34 ng/ml; 1,25(OH)(2)-vitamin D(3) 32 and 41 pg/ml, respectively). In group I, after total PTX there was a rapid and sustained improvement in bone pain with markedly enhanced physical activity and endurance. High turnover osteopathy markedly improved as indicated by declining levels of native osteocalcin (90+/-17 vs. 26+/-18 ng/ml), bone alkaline phosphatase (74+/-12 vs. 12+/-6 ng/ml), and carboxyterminal cross-linked telopeptide of type-I collagen (65+/-16 vs. 40+/-21 ng/ml) but increasing levels of carboxyterminal propeptide of type-I procollagen (120+/-36 vs. 148+/-41 ng/ml). Recalcification of bone was excellent as demonstrated by X-ray and confirmed by bone histology. Itching extravascular calcific deposits and calcifications of blood vessel and cardiac valves immediately stopped after total PTX. Moreover, 6 sHPT patients suffered from severe atherosclerotic lesions such as thoracic aortic aneurysm (n = 3) or abdominal aortic aneurysm (n = 3) which showed size progression before but not after total PTX when annually controlled by ultrasonography. In group II, even long after total PTX, there was no clinical, radiological, histological or biochemical evidence for low turnover osteopathy. In conclusion, our data indicate that substitution with vitamin D(3) metabolites and calcium can prevent deleterious bone effects of hypoparathyroidism in hemodialysis patients and in patients with normal kidney function and may compensate for the missing PTH action. Over this, a better survival rate is expected as a consequence of the beneficial effect of total PTX on the progression of atherosclerotic lesions. We suggest reconsideration of total PTX without autotransplantation in dialysis patients with severe sHPT who are not eligible for renal transplantation.

A human-SCID mouse model for allergic immune response bacterial superantigen enhances skin inflammation and suppresses IgE production.

Chronic skin colonization with Staphylococcus aureus is a well-known feature in atopic dermatitis. The aim of this study was to develop a human-SCID mouse model to analyze the possible role of bacterial superantigens in human allergic immune responses under in vivo conditions. SCID mice were reconstituted with peripheral blood mononuclear cells (between 2 and 9 x 10(7) cells per mouse) from atopic dermatitis patients sensitized to house dust mite allergen (Der p). Total and Der p specific antibody production required the following conditions: (i) injection of Der p; (ii) presence of CD14+ antigen-presenting cells; and (iii) IL-4 as shown by the inhibitory effect of human soluble IL-4 receptor on immunoglobulin E production. This model was used to study the immunomodulatory effects of the superantigen staphylococcal enterotoxin B in comparison with Der p. In intraperitoneally reconstituted human-SCID mice, topical treatment was ineffective in inducing skin inflammation. Therefore, additionally to intraperitoneal transfer, peripheral blood mononuclear cells from atopic donors were also injected intradermally. Such reconstituted SCID mice were then exposed via the skin to either Der p, staphylococcal enterotoxin B, or a combination of both. Maximal effects on epidermal inflammation and dermal T cell infiltration were obtained with staphylococcal enterotoxin B and Der p. Staphylococcal enterotoxin B alone was less effective and Der p only stimulated dermal T cell infiltration. These findings support the hypothesis that bacterial superantigens can act as trigger factors in allergic skin inflammation.

Comparison of hollow fibre membranes for hepatocyte immobilisation in bioreactors.

Various hollow fibre membranes of polyamide, cellulose and polypropylene were investigated as potential substrata for hepatocyte immobilisation in bioreactors for hybrid liver support systems. Membranes were subjected to a cytocompatibility test in which the attachment and morphology of primary hepatocytes were evaluated. The effect of coating with collagen and fibronectin was also studied. Adequate cell immobilisation was possible on polypropylene and polyamide membranes even without coating. The flattening process of the cells was dependent on the material and the coating. The incorporation of porous polypropylene hollow fibres in hybrid liver cell bioreactors and their specific permeability properties could also offer means for cell oxygenation, metabolite distribution and immuno-isolation purposes.

The relevance of murine animal models to study the development of allergic bronchial asthma.

Bronchial asthma (BA) develops on the basis of a genetic predisposition and involves a characteristic sequence of changes in immune functions. In the immunopathogenesis, several phases can be distinguished: the initial stage is defined as the development of allergic sensitization. This step is dependent on: (i) T cell activation; (ii) IL-4 production; (ii) IgE synthesis; and (iv) mediator release by effector cells. The second phase of allergic inflammation as a consequence of the T cell dependent sensitization is characterized by IL-5 production and eosinophil activation and recruitment. Airway mucosa remodelling is the consequence of chronic inflammatory processes and represents the final stage of BA. In this article animal models will be discussed with regard to their relevance for these different phases in development of chronic allergic BA.

[Leiomyosarcoma of the pulmonary artery]. / Leiomyosarkom der Pulmonalarterie.

In a 58-year-old man, a leiomyosarcoma was found to be the cause of unclear thoracal pain on the right side. The initial diagnose was an embolus of the pulmonary artery. The MRI showed a suspicious area similar to an intravasal tumor.

Improved hepatocyte in vitro maintenance in a culture model with woven multicompartment capillary systems: electron microscopy studies.

Primary pig hepatocytes form a tissuelike structure in an in vitro culture model that has provision for three-dimensional cell orientation, cell aggregation, decentralized cell perfusion with low metabolite gradients, integral oxygenation, and nonparenchymal cell coculture. Scanning electron microscopy (SEM) has shown that hepatocytes spontaneously form aggregates in a three-dimensional structure between and on the surface of artificial capillaries. Transmission electron microscopy (TEM) has shown that after 7 weeks of in vitro perfusion, the cell ultrastructure remains similar to that of the parenchyma in vivo. Golgi complexes, active membrane processes, reorganization of cell junctions, and bile canaliculi-like intercellular spaces were demonstrated.

[High-resolution magnetic resonance tomography (HR-MRT) of the pleura and thoracic wall: normal findings and pathological changes]. / Hochauflösende Magnetresonanztomographie (HR-MRT) von Pleura und Thoraxwand: Normalbefund und pathologische Veränderungen.

AIM: To determine the value of high-resolution MRI in pleural and chest wall diseases, the normal and pathologic costal pleura and adjacent chest wall between paravertebral and the axillary region were examined with contrast enhanced high-resolution T1-weighted MRI images using a surface coil. MATERIAL AND METHODS: Normal anatomy was evaluated in 5 healthy volunteers and a normal specimen of the thoracic wall, and correlation was made with corresponding HR-CT and histologic sections. CT-proved focal and diffuse changes of the pleura and the chest wall in 36 patients underwent HR-MRI, and visual comparison of MRI and CT was done retrospectively. RESULTS: Especially sagittal T1-weighted HR-MRI images allowed accurate delineation of the peripleural fat layer (PFL) and the innermost intercostal muscle (IIM), which served as landmarks of the intact inner chest wall. PFL and IIM were well delineated in 3/4 patients with tuberculous pleuritis, and in all 7 patients with non-specific pleuritis, as opposed to impairment of the PFL and/or the IIM, which was detected in 15/18 malignancies as a pattern of malignant chest wall involvement. In one case of tuberculous pleural empyema with edema of the inner chest wall HR-MRI produced false positive diagnosis of malignant disease. CONCLUSION: HR-MRI images improved non-invasive evaluation of pleural and chest wall diseases, and allowed for differentiation of benign and malignant changes.

Liver-intestine cadherin: molecular cloning and characterization of a novel Ca(2+)-dependent cell adhesion molecule expressed in liver and intestine.

A novel member of the cadherin family of cell adhesion molecules has been characterized by cloning from rat liver, sequencing of the corresponding cDNA, and functional analysis after heterologous expression in nonadhesive S2 cells. cDNA clones were isolated using a polyclonal antibody inhibiting Ca(2+)-dependent intercellular adhesion of hepatoma cells. As inferred from the deduced amino acid sequence, the novel molecule has homologies with E-, P-, and N-cadherins, but differs from these classical cadherins in four characteristics. Its extracellular domain is composed of five homologous repeated domains instead of four characteristic for the classical cadherins. Four of the five domains are characterized by the sequence motifs DXNDN and DXD or modifications thereof representing putative Ca(2+)-binding sites of classical cadherins. In its NH2-terminal region, this cadherin lacks both the precursor segment and the endogenous protease cleavage site RXKR found in classical cadherins. In the extracellular EC1 domain, the novel cadherin contains an AAL sequence in place of the HAV sequence motif representing the common cell adhesion recognition sequence of E-, P-, and N-cadherin. In contrast to the conserved cytoplasmic domain of classical cadherins with a length of 150-160 amino acid residues, that of the novel cadherin has only 18 amino acids. Examination of transfected S2 cells showed that despite these structural differences, this cadherin mediates intercellular adhesion in a Ca(2+)-dependent manner. The novel cadherin is solely expressed in liver and intestine and was, hence, assigned the name LI-cadherin. In these tissues, LI-cadherin is localized to the basolateral domain of hepatocytes and enterocytes. These results suggest that LI-cadherin represents a new cadherin subtype and may have a role in the morphological organization of liver and intestine.

Comparison of four methods for mass hepatocyte isolation from pig and human livers.

Using the pig liver, parameters for large scale hepatocyte isolation were studied in order to develop a technique suitable for human organs. These investigations led to a 5-step modification of the original 2-step method. Four groups were compared. A nonenzymatic EDTA perfusion technique has been shown to be inconvenient for mass cell isolation. The enzymatic 2-step perfusion, using 0.08% collagenase and 20-kg pigs, resulted in a mean hepatocyte viability of 61 +/- 1.9%, with a mean yield of 67 +/- 6.5% wet weight of the organ. The enzymatic 5-step method resulted in a mean hepatocyte viability of 74 +/- 1.7% with a mean yield of 80 +/- 1.8% wet weight. Five-step portal venous perfusion in combination with arterial perfusion resulted in 76 +/- 2.6% viability with a yield of 82 +/- 6.1%. The results were dependent on collagenase concentration and weight of the donors, improving with decreasing body weight. The 5-step method with combined arterial and portal vein perfusion developed for pig liver was used for mass human liver cell isolation with a minimum viability of 57% and a minimum yield of 58% wet weight.

Hepatocyte culture between woven capillary networks: a microscopy study.

A multi-compartment capillary membrane culture model with independently perfused three-dimensionally woven capillaries was developed for immobilization of hepatocytes in bioreactors. This enables spatial restructuring of cells and enhanced mass transfer performance with more efficient oxygenation and metabolite exchange. Seeding density defines cell behaviour in this model. With low densities cells attach to the membranes and flatten. Increasing density leads to spontaneous formation of aggregates which are immobilized between the capillaries.

Inhaled nitric oxide in advanced paraquat intoxication.

No effective treatment is available for adult respiratory distress syndrome, pulmonary hypertension and progressive lung fibrosis in severe paraquat poisoning. A potentially beneficial effect of nitric oxide inhalation on the mean pulmonary artery pressure and gas exchange in a subject with advanced paraquat intoxication is reported. Eight days after the suicidal ingestion of an unknown dose of paraquat, a 52-year-old female had a PaO2 < or = 50 mm Hg despite ventilation with an FiO2 of 1 and a positive end-expiratory pressure of 14 to 18 cm H2O. After administration of 25 ppm nitric oxide, PaO2 increased and the mean pulmonary artery pressure and the right-to-left shunt decreased. Discontinuation of nitric oxide resulted in rapid reversal. Ventilatory function was stabilized for three days during nitric oxide inhalation but the patient developed massive pleural effusions and died on d 11 during an interruption of nitric oxide therapy. The response of serious paraquat intoxications to nitric oxide therapy may merit further study. A remarkable post-mortem finding was extensive myonecrosis supporting prolonged muscular retention of paraquat with toxic myopathy or neuromyopathy as a late manifestation of paraquat toxicity.

Hepatocyte aggregate culture technique for bioreactors in hybrid liver support systems.

Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates. The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures. This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems.

A case of rhinoscleroma cured with ciprofloxacin.

The diagnosis of rhinoscleroma was confirmed in a 17-year-old female patient from Tehran, Iran, suffering from a roundish tumour of the nose. Prior treatment with streptomycin and tetracycline had been unsuccessful. A three-month course of high-dose oral ciprofloxacin (750 mg b.i.d.) led to prompt cessation of the growth of the granuloma which was removed later by plastic surgery. Although serology alone appeared to have little value for the specific diagnosis of rhinoscleroma, a significant increase of IgG antibodies during treatment with ciprofloxacin confirmed infection by Klebsiella rhinoscleromatis in this case.