Jochen Düllmann

Primary human hepatocytes on biodegradable poly(l-lactic acid) matrices: a promising model for improving transplantation efficiency with tissue engineering.

Liver transplantation is an established treatment for acute and chronic liver disease. However, because of the shortage of donor organs, it does not fulfill the needs of all patients. Hepatocyte transplantation is promising as an alternative method for the treatment of end‐stage liver disease and as bridging therapy until liver transplantation. Our group has been working on the optimization of matrix‐based hepatocyte transplantation. In order to increase cell survival after transplantation, freshly isolated human hepatocytes were seeded onto biodegradable poly(l‐lactic acid) (PLLA) polymer scaffolds and were cultured in a flow bioreactor. PLLA discs were seeded with human hepatocytes and exposed to a recirculated medium flow for 6 days. Human hepatocytes formed spheroidal aggregates with a liver‐like morphology and active metabolic function. Phase contrast microscopy showed increasing numbers of spheroids of increasing diameter during the culture period. Hematoxylin and eosin histology showed viable and intact hepatocytes inside the spheroids. Immunohistochemistry confirmed sustained hepatocyte function and a preserved hepatocyte‐specific cytoskeleton. Albumin, alpha‐1‐antitrypsin, and urea assays showed continued production during the culture period. Northern blot analysis demonstrated increasing albumin signals. Scanning electron micrographs showed hepatocyte spheroids with relatively smooth undulating surfaces and numerous microvilli. Transmission electron micrographs revealed intact hepatocytes and junctional complexes with coated pits and vesicles inside the spheroids. Therefore, we conclude that primary human hepatocytes, precultured in a flow bioreactor on a PLLA scaffold, reorganize to form morphologically intact liver neotissue, and this might offer an optimized method for hepatocyte transplantation because of the expected reduction of the initial cell loss, the high regenerative potential in vivo, and the preformed functional integrity. Liver Transpl 17:104–114, 2011.

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell

Many applications of hematopoietic gene therapy require selection for clones with active transgene expression. However, it was unclear whether the clonal progeny of a retrovirally transduced hematopoietic stem cell would be capable of maintaining transgene expression through serial repopulation and multilineage differentiation. Such investigations require simultaneous analyses of clonality, multilineage activity and transgene copy numbers. Using a mouse model, the present study demonstrates that a single hematopoietic stem cell expressing a marker gene from one or two insertions of a simple retroviral vector actively maintains multilineage transgene expression in the vast majority (80–99%) of bone marrow and peripheral blood cells. Gene expression persisted through serial transplantations for at least 97 weeks post gene transfer and was observed in the lymphoid (B, T and NK cells), myeloid (CD11b+, Gr-1+), erythroid (Ter119+, mature red blood cells) and megakaryocytic (as indicated by platelets) progeny. Therefore, a single immunoselection for hematopoietic stem cells expressing the transgene in vivo was sufficient to establish a completely chimeric hematopoiesis. These observations imply that the retroviral vectors used in this study contain cis-elements that mediate expression through massive clonal expansion and multilineage differentiation, provided the insertion occurred in genetic loci permissive for expression in hematopoietic stem cells.

Iron storage in macrophages and endothelial cells. histochemistry, ultrastructure, and clinical significance

Summaryl hour after i. v. infusion of colloidal iron in iron deficient subjects uniform phagosomal iron granules were observed in macrophages and endothelial cells of several organs. 7 to 10 days later transformation into ferritin could be visualized in macrophages only. Now, these cells showed diffuse iron staining of the cytoplasm due to dispersed ferritin molecules. Polymorphous lysosomes contained densely packed particles from still unchanged ferric hydroxide to paracristalline ferritin. The macrophageal iron was mobilizable in few days to several weeks. The uniform lysosomal iron granules of endothelial cells disappeared after 1 to 2 years. Endothelial iron siderosis without previous i.v. iron application was a frequent finding in pernicious anaemia and iron overload of diverse origin.Zusammenfassungl Stunde nach i.v. Infusion kolloidaler EisenprÄparate wurden gleichförmige phagosomale Eisengranula in Makrophagen und Endothelzellen verschiedener Organe gefunden. 7 bis 10 Tage spÄter konnte eine Umwandlung in Ferritin nur in Makrophagen sichtbar gemacht werden. Diese Zellen zeigten nun eine diffuse, durch fein verteiltes Ferritin bedingte EisenfÄrbung des Zytoplasma. Die polymorphen Lysosomen enthielten dicht gelagerte Eisenteilchen von z.T. noch unverÄndertem Ferrihydroxyd bis zu parakristallinem Ferritin. Das Makrophageneisen war in wenigen Tagen bis zu mehreren Wochen mobilisierbar. Die uniformen lysosomalen Eisengranula der Endothelzellen schwanden nach 1 bis 2 Jahren. Eine endotheliale Siderose ohne vorherige i.v. Eisengabe war ein hÄufiger Befund bei perniziöser AnÄmie und Eisenüberladung verschiedener Ätiologie.

Iron overload of the liver by trimethylhexanoylferrocene in rats.

Iron-deficient female Wistar rats were fed a diet, which contained 0.5% trimethylhexanoylferrocene, over a 56-week period. This dietary iron loading resulted in a progressive siderosis and enlargement of the liver with a maximum iron content of 947.0 +/- 148.0 mg (vs. 0.07 +/- 0.04 mg in iron deficiency) and a maximum organ weight of 39.4 +/- 6.6 g (vs. 6.9 +/- 1.4 g in iron-deficient control rats). Up to 43 weeks, whole liver iron rose by increase in iron concentration (max. 28.0 +/- 6.1 mg/g wet weight, w.w.) as well as by enlargement of the organ. Afterwards whole liver iron increased solely by ongoing hepatomegaly. At the commencement of iron loading, stainable iron was almost exclusively stored by hepatocytes equally throughout all areas of the liver lobule. Later, the distribution of iron-loaded hepatocytes became strikingly periportal, and, in addition, Kupffer cells as well as sinus-lining endothelia began to store iron. Animals with a liver iron concentration of more than 10.4 +/- 0.75 mg/g w.w. showed no further increase in ferritin and haemosiderin within hepatocytes. Iron-burdened Kupffer cells/macrophages, however, accumulated permanently, hereby forming intrasinusoidal and portal siderotic nodules and areas. First signs of liver damage such as necrosis of single hepatocytes and mild fibrosis began at a liver iron concentration of 14.7 +/- 1.4 mg/g w.w. With advancement of iron loading, focal necrosis of hepatocytes and iron-burdened macrophages took place, and significant perisinusoidal as well as portal fibrosis developed. Cirrhosis, however, the final stage of impairment in iron overload of the liver in humans, could not be induced in this animal model up to now.

Absence of macrophage and presence of plasmacellular iron storage in the terminal duodenum of patients with hereditary haemochromatosis

Biopsy specimens of the terminal duodenum obtained from 11 patients with hereditary haemochromatosis were examined by light and electron microscopy. Stainable iron was found in the lamina propria of the terminal duodenum in only 4 patients, all of whom were in an advanced stage of the disease. The iron was localized in the basal parts of the villi, sparing their tips, and between the crypts of Lieberkühn. The iron-storing cells could be identified as plasma cells, in which ferritin and haemosiderin were localized within lysosomes and ferritin molecules scattered in the cell sap. There was no storage of iron in macrophages. These observations demonstrate the impaired iron-storing capacity of macrophages in hereditary haemochromatosis, which may be related to the increased iron absorption in this iron storage disease.

[On the ultrastructure and cytochemistry of eosinophil-myelomonocytic leukemias].

In the bone marrow of two patients with acute leukemia 46% and 55% of atypical eosinophilie cells were found, respectively. Blood eosinophilia was absent. The N-AS-D-Cl-Esterase reaction of the granules was positive in the first case in 58%, and in the second case in 3% of the eosinophils, as well as the PAS-reaction in all cells of this series. The ultrastructure of the eosinophils reveals nuclear maturation up to hypersegmentation. The maturation of the granules, in part of abnormal size, is arrested at the primary stage. Typical secondary granules with cristalloid cores are lacking. Only in the first case a few, small, semicircular or circular profiles of lamellar substructure are seen in the granules. The cytoplasm of hypersegmentated eosinophils shows an abnormally high glycogen content. Besides the eosinophils, monocytic cells and their precursors proliferate in the bone marrow of the first patient. In the second patient myeloblasts, promyelocytes with Auer rods, and monocytic cells characterize further neoplastic cell populations. The elements of the monocytic series can be identified by their ultrastructural features, such as irregular configuration of the nuclei, bundles of cytoplasmatic microfilaments, and numerous small electron-dense lysosomal granules. In both cases the Alpha-Naphthyl-Acetate-Esterase reaction is weakly positive. The findings presented are summarized under the terms “eosinophil-monocytic leukemia” and “eosinophil-myelomonocytic leukemia” (collective term). Bei zwei Patienten mit akuter Leukämie wurden im Knochenmark 46% bzw. 55% atypische eosinophile Zellen gefunden. Eine Bluteosinophilie fehlte. Die N-AS-D-Cl-Esteraseaktivität der Granula war im ersten Fall in 58%, im zweiten in 3% der Eosinophilen, die PAS-Reaktion in allen Zellen dieser Reihe positiv. Ultrastrukturell zeigen die Eosinophilen auch bei Kernreifung bis zur Hypersegmentation fast ausschließlich z.T. abnorm große Primärgranula. Typische Sekundärgranula mit kristalloiden Interna fehlen in beiden Fällen. Lediglich im ersten Fall sind vereinzelt kleine, hakenoder ringförmige lamelläre Strukturen in den Granula nachweisbar. Das Cytoplasma kleiner, übersegmentierter Eosinophiler weist einen abnorm großen Glykogengehalt auf. Neben den Eosinophilen proliferieren im Knochenmark im ersten Fall ausschließlich monocytoide Zellen und deren Vorstufen, im zweiten Myeloblasten, Promyelocyten mit Auer-Stäbchen und monocytoide Zellen. Die Elemente der monocytären Reihe lassen sich aufgrund feinstruktureller Merkmale wie unregelmäßig konfigurierter und gelappter Kerne, Bündel cytoplasmatischer Mikrofilamente und zahlreicher, kleiner, elektronendichter lysosomaler Granula identifizieren. In beiden Fällen ist die Alpha-Naphthyl-Acetat-Esterase-Reaktion nur schwach ausgeprägt. Die vorgelegten Befunde werden unter den Begriffen „eosinophil-monocytäre” und „eosinophil-myelomonocytäre Leukämie” (Sammelbegriff zusammengefaßt.

DNA content of leukaemic cells in non-Hodgkin's lymphomas of low-grade malignancy

ZusammenfassungBei 17 Patienten mit leukämischen Non-Hodgkin-Lymphomen niedriger Malignität entsprechend der Kieler Klassifikation [13] wurde der DNA-Gehalt der Blutzellen flußzytometrisch bestimmt und zu zytologischen Befunden in Beziehung gesetzt. Die Histogramme von acht Patienten mit chronisch lymphatischer Leukämie (CLL), zwei Patienten mit Prolymphozyten-Leukämie (PL), zwei Patienten mit Haarzell-Leukämie (HCL) und einem Patienten mit Immunozytom (IC) zeigen unimodale DNA-Verteilungskurven, die einem diploiden DNA-Gehalt der Zellen entsprechen dürften. Bimodale Histogramme wurden in einem Fall von CLL (Rai IV) mit hochgradiger Leukozytose, in einem Fall von Sézary-Syndrom mit kleinen und einigen großen Lutzner Zellen und in einem Fall von IC mit „prolymphozytoider“ Transformation beobachtet. Bei der CLL (Rai IV) entsprechen die zwei Gipfel des Histogramms einer großen Population wahrscheinlich diploider Zellen (88%) und einer kleinen von G2-Zellen (3%), während 9% der Lymphozyten in DNA-Synthesephase angetroffen wurden. Die großen Lutzner Zellen des Patienten mit Sézary-Syndrom machen 4% der Blutzellen aus und konnten als hypotetraploid (3,5c) identifiziert werden, während die kleinen Lutzner Zellen (44%) diploid sind. Das transformierte 1C, das sich aus einem lymphozytischen Typ mit kleinen, kristalloide Immunglobulineinschlüsse enthaltenden Zellen entwickelt hatte, war durch 84% tetraploider Zellen im Blut charakterisiert. Ihre großen und eingebuchteten Kerne zeigen eine relativ dichte Chromatinstruktur und große Nukleolen, die im Semidünn- und Feinschnitt besser als im Blutausstrich zu erkennen sind. Da Zellen in DNA-Synthesephase bei den Patienten mit Sézary-Syndrom und transformiertem IC fehlen, werden die hyperploiden Elemente als nicht im Blut proliferierend angesehen. Die tetraploiden „prolymphozytoiden“ Zellen des Patienten mit IC werden als ein Zeichen der Malignitätssteigerung des Non-Hodgkin-Lymphoms interpretiert.SummaryThe DNA content of blood cells from 17 patients with leukaemic non-Hodgkins lymphomas of low-grade malignancy according to the Kiel classification [13] was determined by a flow-cytometric assay and compared with cytological findings. The histograms from eight patients with chronic lymphocytic leukaemia (CLL), two patients with prolymphocytic leukaemia (PL), two patients with hairy cell leukaemia (HCL), and one patient with immunocytoma (IC) showed unimodal DNA distributions falling in with a diploid DNA content (2c) of the cells. Bimodal histograms were found in one case of CLL (Rai IV) with marked leukocytosis, in one case of Sézary syndrome with small and some large Lutzner cells, and in one case of IC with “prolymphocytoid” transformation. In the case of the CLL (Rai IV) the two peaks of the histogram represent a large population of probably diploid cells (88%) and a small one of G2 cells (3%), while 9% of the lymphocytes were found in the DNA synthesis phase. Large Lutzner cells of the patient with Sézary syndrome comprising 4% of the blood cells could be identified as hypotetraploid (3,5 c), whereas small Lutzner cells (44% of all cells) were recorded at 2c. The transformed IC which evolved from a lymphocytic ] type with small, crystalloid immunoglobulin bearing cells was characterized by 84% tetraploid (4c) cells in the blood. Their large and indented nuclei exhibited a relatively dense chromatin pattern and large nucleoli that were easily recognized in semithin and ultrathin sections but not in blood films. Since cells in DNA synthesis phase were absent, the hyperploid elements in the case of the Sé-zary syndrome and the transformed IC had to be regarded as non-proliferating in blood. The tetraploid “prolymphocytoid” cells of the patient with IC can be interpreted to reflect increased malignancy.