Experimental infection of native human ureteral tissue with Neisseria gonorrhoeae: adhesion, invasion, intracellular fate, exocytosis, and passage through a stratified epithelium.

The exact mechanisms by which Neisseria gonorrhoeae invades the mucosal lining to cause local and disseminated infections are still not fully understood. The ability of gonococci to infect the human ureter and the mechanism of gonococcal infection in a stratified epithelium were investigated by using distal ureters excised from healthy adult kidney donors. In morphological terms, this tissue closely resembles parts of the urethral proximal epithelium, a site of natural gonococcal infection. Using piliated and nonpiliated variants of N. gonorrhoeae MS11, we demonstrated the importance of pili in the attachment of gonococci to native epithelial cells as well as their association with epithelial damage. By electron microscopy we elucidated the different mechanisms of colonization and invasion of a stratified epithelium, including adherence to surface cells, invasion and eventual release from infected cells, disintegration of intercellular connections followed by paracellular tissue infiltration, invasion of deeper cells, and initiation of cellular destruction and exfoliation resulting in thinning of the mucosa.

Isolating and maintaining highly polarized primary epithelial cells from normal human duodenum for growth as spheroid-like vesicles.

A method is described for the three-dimensional (3-D) in vitro culture of nontransformed gastrointestinal epithelial cells from the human duodenal mucosa. Biopsies obtained from human duodenum were finely minced. The tissue fragments were suspended in culture medium supplemented with 5% fetal calf serum and the appropriate antibiotics. The suspended mucosal fragments generated spheroid-like multicellular vesicles consisting of highly prismatic absorptive and goblet cells retaining most of the histological features of the tissue in vivo. We performed immunocytochemical studies to determine the origin of the vesicles using monoclonal antibodies against EP4. The histochemistry of the vesicles showed alkaline phosphatase activity. Ultrastructural studies revealed that these cells exhibit characteristics of normal duodenal cells in vivo: apical microvilli, glycocalyx, tight junctions and desmosomes, lateral membrane interdigitations, mucous droplets, and a well-developed Golgi apparatus. An overgrowth of the vesicles by fibroblasts was not seen during cultivation. In contrast with the two-dimensional cell cultures grown on artificial supports, the vesicle cells show organization similar to that of natural epithelia. The polarization and cytoarchitecture of normal gastrointestinal epithelial cells cultured as 3-D vesicles are comparable to those known for the native tissue. This study was undertaken to provide a morphological baseline for subsequent infection experiments.

[Therapeutic effect of argon plasma coagulation on small malignant tumors of the gastrointestinal tract]. / Therapeutischer Effekt der Argon Plasma Koagulation (APC) auf kleine Malignome des Gastrointestinums.

The use of argon plasma coagulation has proved to be an effective method of non-contact electrocoagulation with high efficiency in palliative treatment of gastrointestinal lesions. In this study there were 10 patients suffering from small oesophageal malignant tumors; in 5 patients no further tumor could be detected in biopsies during a period up to 34 months, in the other cases passage could kept open. These results suggest this treatment as an alternative therapy in selected patients.

Therapeutic effect of argon plasma coagulation on small malignant gastrointestinal tumors.

In endoscopy, argon plasma coagulation (APC) is a new principle of non-contact electrocoagulation and has proved to be a sufficient tool for palliative endoscopic treatment of gastrointestinal neoplasms, predominantly of the oesophagus and colorectum. In a study of 67 patients suffering from histologically confirmed and endosonographic T1-staged tumours of the gastrointestinum, 10 patients were selected for endoscopic APC treatment because of the impossibility of surgical therapy. Although the application was primarily of a palliative nature, in 9 of 10 cases of minor neoplasms, no further tumour could be detected in biopsies during the observation period (9.45 +/- 2.8 months). One patient was not cured locally. In none of the patients was any serious complication noticed during the outpatient follow-up. The effective results and lack of severe complications suggest this technique as an alternative therapy in selected patients with smaller gastrointestinal tumours.

Highly polarized primary urothelial cells from human ureter grown as spheroid-like vesicles.

Epithelial cells growing in vitro are frequently non-polarized and lack histophysiological characteristics. Furthermore, the quality of two-dimensional cell layers is limited by the physico-chemical properties of the support. Therefore, for an in vitro system to reflect the normal epithelial physiology, it is necessary to maintain the inner and outer geometrical configuration of the cells. In order to avoid the disadvantages of two-dimensional cultures we have established an in vitro model that closely resembles the in vivo situation. Human ureteral epithelial cells (HUEC) were used to prepare multicellular vesicles which maintain a geometrically intact cell organization that is not achieved in conventional cultures. Light and electron microscopy investigations showed the morphology of the cells to be similar to that in situ. HUEC vesicles are more in vivo-like than two-dimensional cultures and therefore represent a suitable model for a variety of research purposes including studies on the pathogenesis of micro-organisms.

[Liver resection in hepatocellular carcinoma]. / Leberresektion beim Hepatozellulären Karzinom.

Hepatic resection offers the only chance of cure for patients with hepatocellular carcinoma. Preoperative tumor staging is based on radiologic procedures (ultrasound, CT- and MR-imaging). Compromised hepatic function will limit resectability, leaving only palliative procedures. Unfavorable prognostic tumor factors (diameter > 5 cm, multifocal, nonencapsulated, vascular invasion) should lead to a combination of adjuvant chemotherapy/chemoembolization and surgical intervention. In irresectable situations multimodality treatment protocols (combined i.v. chemotherapy, radio-immuno-therapy) play an increasing role.

Highly polarized primary epithelial cells from human nasopharynx grown as spheroid-like vesicles.

The value of experimental culture models using epithelial cells often depends on the degree of polarization and other critical features observed in natural tissues, including the formation of tight junctions, desmosomes and membrane interdigitations. However, growth of normal epithelial cells as monolayers on artificial supports also leads to partial loss of the original characteristics of epithelial cells, and the quality of the monolayer is strongly influenced by the physicochemical properties of the support. In addition, not all normal epithelial cell types are able to adhere and to grow well on artificial substrata. In order to circumvent the drawbacks of two-dimensional cultures we established an in vitro model that closely resembles the in vivo situation of the intact epithelium. Human epithelial cells from nasopharynx (HNPEC) were used to prepare multicellular epithelial vesicles consisting of both non-ciliated and ciliated mucosal cells. Electron microscopy investigations showed that the morphological appearance of the epithelial cells was similar to that in situ. HNPEC vesicle cultures maintain a geometrically intact organization of individual cells that is not achieved using conventional culture conditions. HNPEC vesicles are more in vivo-like than two-dimensional cultures and therefore represent a suitable model for a variety of research purposes including studies on the pathogenesis of invasive microorganisms.

New aspects of cellular thallium uptake: Tl+-Na+-2Cl(-)-cotransport is the central mechanism of ion uptake.

Cellular uptake mechanisms of 201Tl+ were studied in Ehrlich mouse ascites tumor cells. 201Tl+ passes the cell membrane of tumor cells using three transport systems: the ATPase, the Tl+-Na+-2Cl(-)-cotransport, and the Ca++-dependent ion channel. In the case of 201Tl+ the main route for entering the cells was the cotransport, its importance increasing with the age of the cells; in parallel, the ATPase activity was reduced. In contrast, the transport capacities of the ATPase and the cotransport were of the same magnitude in the case of 42K+ and 86Rb+. This change in ion distribution was not brought about by varying velocity relations but by changing the number of transport systems in the cell membrane. There was no relationship between transport rates and diameters of the ions. 201Tl+ distribution is proportional to that of K+ with a higher intracellular concentration of about 30%. Under physiological conditions the cotransport was reversible suggesting the ability to regulate steady state during varying extracellular ion concentrations. Cells and medium were two compartments, kinetically seen. Due to the significant difference of transport capacities between the three systems with the respective ions the term "potassium-thallium-analogy" may be misleading as it erroneously assumes identical uptake conditions.

Expression of polypeptide segments of the human complement component C3 in E. coli: genetic and immunological characterization of cDNA clones specific for the alpha-chain of C3.

The third component of complement C3 and its fragments have a central role in a variety of host defense mechanisms. The identification of functionally relevant C3 domains is important because of the marked functional versatility of the C3 molecule. Several human C3 cDNA clones from a human liver cDNA library were isolated and characterized. A bacterial expression vector system was used to express cDNA clones that were identified by an immunological screening procedure. The C3 cDNA clones produced in E. coli the hybrid proteins consisting of cro-beta-galactosidase and polypeptide segments of human C3, as revealed by Western blotting with antisera to human C3. The C3 moiety of the hybrid proteins had a m.w. of up to 46.000. Polyclonal antibodies against the C3 segments expressed by one of the C3 cDNA clones (ReC3-1) have been raised in mice and rabbit, and in addition, a monoclonal antibody was produced. The antisera and the monoclonal antibody reacted in Western blotting analysis selectively with the alpha-chain, but not the beta-chain of human C3. Restriction mapping of the different cDNA clones was performed, and revealed that the different clones were partially overlapping. The ReC3-1 cDNA clone included a 0.7 kb noncoding region at the 3' terminal end of the C3 cDNA. One of the restriction sites (Hind III) identified in the ReC3-1 cDNA clone was not present in the recently published sequence of human C3 cDNA. This difference in nucleotide sequence provides direct evidence for C3 polymorphism at the DNA level. The combination of immunologic procedures with recombinant DNA methodology should facilitate additional analysis of the structure-function relationship of the C3 molecule.

[Chemiluminescence measurement in AIDS, lymphadenopathy and hemophilia patients]. / Chemilumineszenzmessung bei AIDS, Lymphadenopathie- und Hämophiliepatienten.

In order to reveal the activity of polymorphonuclear neutrophil leukocytes (PMNL) representing the first step of defence against infections, measurements of chemiluminescence (CL) were performed in patients suffering from acquired immune deficiency syndrome (AIDS), lymphadenopathy, or hemophilia. In comparison with healthy controls, AIDS patients revealed significant reduction (about 50 per cent) of phagocytic, i.e. CL activity of neutrophils, which had been induced by Zymosan. Only part of the patients suffering from lymphadenopathy answered with decreased granulocyte activity on the application of Zymosan. If concanavalin A was used as stimulant of metabolic activity of PMNL-independently of phagocytosis-again AIDS and some of the lymphadenopathy patients showed a markedly reduced neutrophil response. In conclusion it should be stated that there is some evidence for at least two defects of cellular immunity associated with AIDS and to some extent, with AIDS-endangered homosexuals suffering from lymphadenopathy: first the defect of PMNL to answer to concanavalin A with increased metabolic activity, and secondly the defect of PMNL to start phagocytosis induced by Zymosan with a subsequent release of oxygen radicals which are measurable as chemiluminescence. The appraisal of granulocyte activity by means of measurements of chemiluminescence might become an additional criterion for AIDS diagnostics.

Uptake mechanism of interstitially injected 99mTc-labeled antimony trisulphide colloid in the popliteal lymph node of rabbits.

The right popliteal lymph node was studied in rabbits by well counting, histoautoradiography and electron microscopy, after interstitial injection of 99mTc-labeled antimony trisulphide (Sb2S3) colloid into the right hind pad. The highest radioactivity concentration (96.8%/g) was measured 6 hr following injection. At 24 hr, the concentration had dropped to nearly half of the maximum (51.5%/g). At each time, only a single or a few lymph node sectors were found to contain 99mTc. Initially, the radioactivity distribution pattern in the draining lymph node was stripy. Beginning at 15 min p.i., there was a progressive change from stripy to focal radioactivity distribution pattern. Until 6 hr after injection, the bulk of radioactivity was trapped by macrophages in the lumen and wall of the lymph node sinus system, predominantly in medullary sinuses. Surprisingly, at 24 hr the majority of labeled cells were eosinophilic polymorphonuclear leukocytes located in medullary sinuses and medullary cords. Up to 24 hr p.i., no accumulation of radioactivity could be detected in the cortical and paracortical lymph node parenchyma. In conclusion, interstitially injected 99mTc-Sb2S3 colloid is not homogeneously but sectorially distributed in the draining lymph node. Moreover, both macrophages and eosinophilic polymorphonuclear leukocytes are involved in the filtration process.

Axillary lymph node groups--the center in lymphatic drainage from the truncal skin in man. Clinical significance for management of malignant melanoma.

In order to identify regional lymph node drainage groups of primary malignant melanomas resp. suspicious pigmented tumors on truncal skin, lymphoscintigraphy using 99mTc-Sb2S3 colloid was performed in 50 patients of either sex prior to operative treatment. The lymphatic drainage pattern proved to be practically unpredictable by conventional anatomic guidelines based on the thesis of "lymphatic watersheds". In 96 per cent of the patients examined radiocolloid uptake was to be found in one or both of the axillary lymph node groups, either solely or combined with inguinal, supraclavicular, posterior cervical or parasternal node-bearing areas. In conclusion, axillary lymph node drainage groups are the center in lymphatic drainage from the truncal skin in man.