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.

Pilus biogenesis and epithelial cell adherence of Neisseria gonorrhoeae pilC double knock-out mutants.

The phase-variable PilC proteins of pathogenic Neisseria species have recently been implicated in both assembly and cellular adherence functions of the type 4 pili of these pathogens. We describe here the cloning of full-length pilC1 and pilC2 genes and the complete sequencing of the pilC2 gene of Neisseria gonorrhoeae MS11. Sequential inactivation of both genes by gene replacement in piliated (P+) variants of N. gonorrhoeae MS11 led initially to a non-piliated (P-) phenotype; however, spontaneous P+ variants could be derived from some pilC1,2 double mutants which produced morphologically intact pili. Purified pili from pilC1,2 mutants revealed no detectable PilC protein. Instead, a novel protein about 70 kDa in size appeared in the pili preparations of P+ mutants; this protein exhibited no immunological cross-reactivity with PilC1 or PilC2. We propose that this novel factor replaces the function of PilC in pilus biogenesis. Using isogenic N. gonorrhoeae strains which produce identical PilE (pilin) proteins we demonstrate that pili associated with the 70 kDa protein do not confer gonococcal adherence to human epithelial cells, in contrast to pili assembled in the presence of PilC1 or PilC2.

Stable engraftment of human female genital mucous membrane xenografts on SCID mice.

We developed a model in which full-thickness human genital mucous membranes (fallopian tubes, endometrium) were heterotopically xenografted into the skin of severe combined immunodeficient (SCID) mice. The transplanted tissue retained its human phenotype for at least 4 weeks including the glandular epithelium, the lamina propria, and main parts of the grafted vessels. By using an occlusive chamber filled with covering phosphate-buffered saline we created a system that protected the moist human epithelial surface. This system will allow the study of the interaction of test substances, or of invasive, pathogenic microorganisms, with epithelial cells and other cellular components of the human genital mucosa under in vivo conditions.

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.

A new method for the 3-D in vitro growth of human RT112 bladder carcinoma cells using the alginate culture technique.

We studied the response to different in vitro culture conditions and the ability for polarization in three-dimensional (3-D) and two-dimensional (2-D) cell culture systems of the commonly used human bladder carcinoma cell line RT112. In the case of 2-D culture the cells were grown on glass or plastic coverslips, filter membranes, or bovine lens capsules. The alginate culture technique (ACT) was used to test the effects of 3-D cell culture on the polarization of the RT112 epithelial cells. Our studies show clear differences in the arrangement of the cells depending on the cultivation procedure. The RT112 cells cultured on glass and plastic supports were irregular and flattened in shape whereas the cells grown on filters or on lens capsules developed into 2-3 layers consisting of markedly polarized cells. However, ACT was superior to cell culture on either artificial supports or even on lens capsule. During the 3-D cultivation the transformed epithelial cells regenerated multicellular spheroids which maintained a tissue-like, geometrically well-ordered and highly prismatic organization. This ACT-induced morphogenesis is novel and distinct from that reported with conventional culture conditions. Microscopic investigations showed that RT112 cells grown in alginate were both much more tightly packed and more regularly organized compared to 2-D cultures. In addition, the spheroidal organized cells exhibited well developed cell-cell contacts, a distinct endoplasmatic reticulum, and a marked Golgi apparatus. In summary, ACT can be used for 3-D in vitro growth of the transformed human epithelial cell line RT112 that offers substantial advantages over conventional cell culture methods.

Defined tumor cell-host interactions are necessary for malignant growth.

Analyzing the different steps of malignant growth (primary tumor, metastasizing tumor cells, secondary tumor), one recognizes an intense interaction between normal and malignant cells. Tumor cells not only induce activities of normal cells, which normally are rarely activated, but also they exploit properties of normal cells for their own purposes. The major mechanisms and processes of this "parasitism" are described in more detail and the results are discussed. Tumors cannot grow beyond a certain size without a supply of blood and lymph vessels by the host (angiogenesis). Metastasizing tumor cells cannot leave the vessel (extravasate) in which they are transported without the cooperation of the respective endothelial cells of the host. An appropriate environment formed by the host tissues is essential for the settlement of tumor cells at secondary sites. Historically, these are a few examples that show intense cooperation between host and tumor. More are given in the present contribution.

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.

Cell interactions and motility in human lung tumor cell lines HS-24 and SB-3 under the influence of extracellular matrix components and proteinase inhibitors.

The human NSCLC cell lines HS-24 (squamous cell carcinoma) and SB-3 (metastasis derived from an adenocarcinoma) were investigated in respect to cell interactions, motility and invasive properties. HS-24 revealed high self adhesion capacity. Testing the interactions with collagens type I/III or IV, laminin and fibronectin by adhesion, non directional motility and haptotaxis assays, tight interactions and stimulation, particularly with collagen type I/III, was detected. Proteinase inhibitors (E64, Stefin A or leupeptin) revealed a slightly negative influence. Invasion in vitro of lung explants was reduced by leupeptin in a dose dependent manner and slightly increased by plasmin. SB-3 cells revealed low self adhesion. As judged from interaction with fibronectin, these cells have low integrin receptor concentrations and thus reduced adhesiveness to extracellular matrix. Collagen type I/III was inhibitory for undirectional motility and not permissive for haptotaxis. Therefore, it may play a restrictive role during the spread in vivo of these cells. Colonization of lung explants was low and was not influenced by cathepsin B proteinase inhibitors. The results emphasize a particular role of collagens for primary site tumor and metastasis development.

Influences of various substrata on morphology, motility and invasiveness of rat tumour cells. II. Studies on the highly metastatic variant BSp 73 ASML.

In a previous paper we studied the influences of the basal lamina and its components on the invasive but nonmetastatic variant BSp 73 AS of a rat pancreatic adenocarcinoma. In continuation of this publication we used the in vivo strongly metastasizing but hardly invasive variant BSp 73 ASML. On plastic or glass supports the low passaged ASML-cells grew separately, spread very scarcely and remained passive. Compared to these standard culture conditions, the behaviour of the tumour cells could be modified by biomaterials like collagen type I/III and type IV, fibronectin, laminin, matrigel, lens capsule and basal lamina. On these substrata (compared to plastic supports) the ASML-cells showed increased spreading, filopodial outgrowth and secretion of membrane vesicles. Only signs of motility as well as penetration of the biomatrices could be observed. Interestingly, highly passaged ASML-cells displayed an increased tendency to spread even on plastic. Under dynamic conditions these cells even failed to adhere. Obviously the biomatrices in vitro as well as long-term passaging are able to lead the ASML-cells partly to reactivate the behaviour that is characteristic of malignant tumour cells. Additionally, there is some evidence that the ASML-cells in vitro can strongly respond to chemotactic stimuli by exhibiting both flattening and translocative motility.

Influences of various substrata on morphology, motility, and invasiveness of rat tumour cells. I. Studies on the nonmetastatic variant BSp 73 AS.

BSp 73 AS is a highly invasive but scarcely metastatic cell line from a rat pancreatic adenocarcinoma. In vitro the AS-cells flatten and move actively and were originally able to form monolayers. Our highly passaged AS-cells had lost this ability and formed only two-dimensional clusters and small aggregates on plastic or glass supports. The in vitro behaviour of the As-cells, however, could be dramatically modified by biomatrices like collagen type I/III (vitrogen), Iaminin, fibronectin, basement membrane matrigel, lens capsule and bovine corneal endothelial basal lamina. On these substrata the AS-cells in most cases showed both faster and stronger attachment as well as spreading. Furthermore, degradation, alteration and penetration of the materials could be observed as well as filopodial outgrowth, secretion of membrane vesicles, modification of the cell shape and the forming of monolayers. Shaking the culture dishes decreased the attachment of the AS-cells to laminin, fibronectin and matrigel. Generally, the nonmetastatic AS-cells exhibited a rather aggressive activity in vitro. It could also be demonstrated that not only the cells alter the substratum, but also the cell phenotype is influenced by the extracellular components with which they were confronted.

An in vitro model study of BSp73 rat tumour cell invasion into endothelial monolayer.

In order to study the process of invasion in more detail we developed an in vitro model of the vessel wall. Rat tumour cells derived from an adenocarcinoma of the pancreas, BSp73 AS--of high invasive but low metastatic capacity--and BSp73 ASML--not invasive but highly metastatic--were compared for their mode of invasion into confluent monolayers of endothelial cells. Corneal as well as vascular endothelial cells were plated alternatively onto the basal lamina-like bovine lens capsule that was mounted in a combi-ring dish or reconstituted extracellular matrix (Basement Membrane Matrigel) as substrata. The endothelial monolayers were confronted with AS- and ASML-tumour cells. The interaction of the various cell types was followed by scanning and transmission electron microscopy. The invasive cell type AS was able to force the endothelial cells to retract and subsequently undermined the endothelial cell layer. In the noninvasive cell population ASML most cells remained in the typical roundish morphology and did not interact with the endothelial cell layers. Only a very minor fraction of ASML populations was able to attach to and also invade into the endothelial cell monolayer. It could be shown that AS-cells individually and as small groups penetrated the endothelial cell layer. The results of transmission and scanning electron microscopy suggest that endothelial cell retraction and underlapping of adjacent endothelial cells by tumour cells play an important role in invasion and extravasation through blood vessels. Against all expectations, the nonmetastasizing tumour cell variant (AS-cells) exhibited a dramatic invasive behaviour whereas the highly metastatic ASML-variant mostly retained its spherical shape and showed invasive activity only in exceptional cases.