Quantification of ventilation characteristics of a helmet.

Despite the augmented safety offered by wearing a cyclist crash helmet, many cyclists still refuse to wear one because of the thermal discomfort that comes along with wearing it. In this paper, a method is described that quantifies the ventilation characteristics of a helmet using tracer gas experiments. A Data-Based Mechanistic model was applied to provide a physically meaningful description of the dominant internal dynamics of mass transfer in the imperfectly mixed fluid under the helmet. By using a physical mass balance, the local ventilation efficiency could be described by using a single input-single output system. Using this approach, ventilation efficiency ranging from 0.06 volume refreshments per second (s(-1)) at the side of the helmet to 0.22s(-1) at the rear ventilation opening were found on the investigated helmet. The zones at the side were poorly ventilated. The influence of the angle of inclination on ventilation efficiency was dependent on the position between head and helmet. General comfort of the helmet can be improved by increasing the ventilation efficiency of fresh air at the problem zones.

Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers.

Entamoeba (E.) histolytica trophozoites initiate amebiasis through invasion into the enteric mucosa. It was our aim to understand the molecular interactions between amebic trophozoites and enterocytes during the early steps of invasion. Trophozoites of E. histolytica strain HM1:IMSS were seeded on the apical side of enteric T84 cell layers, which were established on filters in two-compartment culture chambers. Cocultures were analyzed for paracellular permeability by measurement of transepithelial electrical resistance (TER) and for the tight junction proteins ZO-1, ZO-2, occludin, and cingulin by immunocytochemistry and immunoprecipitation. On direct contact with the apical side of the enteric cells, trophozoites caused an increase in paracellular permeability as evidenced by a decrease of TER associated with an increase in [(3)H]mannitol flux. Immunoprecipitation of cocultures revealed dephosphorylation of ZO-2, loss of ZO-1 from ZO-2, and degradation of ZO-1 but less so of ZO-2 and none of occludin or E-cadherin. In conclusion, trophozoite-associated increase in paracellular permeability of enteric cell layers is ascribed to disturbance of the molecular organization of tight junction proteins.

Mechanisms of downregulation of transfected E-cadherin cDNA during formation of invasive tumors in syngeneic mice.

Loss of E-cadherin expression has been observed both in experimental tumors and in human cancers and is related to invasiveness and poor differentiation. The E-cadherin-negative mouse mesenchymal tumor cell line MO4 was transfected with several plasmids expressing mouse E-cadherin cDNA. These plasmids differed from each other by the extent of E-cadherin-specific 3' untranslated region (UTR) sequences and by the use of different constitutive promoters. Transfectants were isolated that expressed functional E-cadherin in a homogeneous way. In syngeneic mice, such MO4-Ecad transfectants invariably produced malignant fibrosarcoma-like tumors, which were completely E-cadherin-negative at the protein level. Northern blotting revealed that E-cadherin mRNA expression was downregulated in some but not all MO4-Ecad tumors. Downregulation was caused by mRNA instability triggered by particular 3' UTR sequences. This in vivo downregulation of E-cadherin in malignant MO4-Ecad tumors turned out to be reversible and is likely to be mediated by host factors to be further identified.

Bacterium-assisted invasion of Entamoeba histolytica through human enteric epithelia in two-compartment chambers.

Entamoeba histolytica trophozoites initiate amebiasis by invasion into the enteric mucosa. It is the aim of our experiments to understand how bacteria and leukocytes act during amebic invasion through enteric cell layers. Cocultures were established in two-compartment chambers and studied by measurement of transepithelial electrical resistance (TER) and by histological examination. Trophozoites caused a decrease in TER that was followed by formation of holes in the enteric cell layer and transfilter migration of trophozoites. Phagocytosed bacteria activated trophozoites that opened the intracellular junctions and provided access for the invasion of bacteria. Leukocytes had no effect on the different steps of invasion of the trophozoites through the human enteric cell layers. We conclude that trophozoites, eventually assisted by enteric bacteria, disrupt enterocytic tight junctions before they open the enteric cell layer and invade through it.

Morphotypic plasticity in vitro and in nude mice of epithelial mouse mammary cells (NMuMG) displaying an epithelioid (e) or a fibroblastic (f) morphotype in culture.

Transition from an epithelioid (e-) to a fibroblastic (f-) morphotype marks invasiveness in clinical and experimental cancer. To understand better the factors influencing such transitions, we have subcloned and manipulated mouse mammary gland (NMuMG) cell cultures and compared the invasive phenotype of multiple subclones in vitro and in vivo. Cell lines with an e-morphotype expressed E-cadherin homogeneously and were not invasive in vitro. Cells with an f-morphotype were E-cadherin-negative and became fully invasive in vitro upon expression of the ras oncogene. Invasive tumors were produced in node mice after subcutaneous injection of e-type or f-type cells. These tumors showed cystic, glandular and undifferentiated structures. Tumors from f-type cells were E-cadherin-negative whereas e-type tumors stained heterogeneously in immunohistochemical preparations. Our observations demonstrate the impact of the micro-ecosystem on the invasive phenotype, with in vivo downregulation of E-cadherin and stimulation of the e- to f-morphotype transition.

Contact-dependent transfer of the galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture.

In a study to investigate early interactions of Entamoeba histolytica with epithelial cell monolayers, we found that a monoclonal antibody (MAb), CD6, against an ameba surface antigen recognized the lateral surface of epithelial cells after coculture with trophozoites. Display of the CD6 antigen on the epithelial cells necessitated contact with active trophozoites. It was found neither at 4 degrees C, nor with prefixed trophozoites, nor with trophozoite-conditioned media, nor when a filter prevented direct contact. Monolayers exposed to amebic sonicates or detergent lysates showed random immunostaining. Access to the antigenic site was limited, as immunostaining occurred predominantly with subconfluent monolayers. CD6 epithelial cell binding was first observed after 5 min of coculture; trophozoite-mediated target cell lysis was not detected until 15 min following coculture. Epithelial cell immunostaining occurred with some other ameba-specific antibodies but not with MAbs raised against the 170-kDa subunit of the galactose-N-acetylgalactosamine (Gal/GalNAc)-specific lectin. The CD6 MAb as well as some other ameba-specific antibodies immunoprecipitated from trophozoite lysates the same bands as the MAbs against the cysteine-rich domain of the 170-kDa subunit of the Gal/GalNAc-specific lectin. Why the latter MAbs failed to stain epithelial cells in the vicinity of attached trophozoites is presently unknown. We concluded that E. histolytica trophozoites transferred the intact amebic Gal/GalNAc-specific lectin or a portion of it to the lateral surface of epithelial cells. This juxtacrine transfer preceded killing of target cells.

Transition from the noninvasive to the invasive phenotype and loss of alpha-catenin in human colon cancer cells.

Loss of epithelioid organization in carcinoma cell lines has been related to invasiveness and poor differentiation of tumors. We investigated the invasion in vitro of various human colon cancer cell lines. Most cell lines were noninvasive into chick heart fragments, and this correlated with an epithelioid morphotype. Only cell lines COLO320DM, SW620, and variants of HCT-8 and DLD-1 were invasive and nonepithelioid. We examined in these cell lines whether invasiveness was related to changes in the structure and function of the E-cadherin/catenin complex. E-cadherin functions as an invasion suppressor and as a cell-cell adhesion molecule when linked to the cytoskeleton via alpha-catenin plus beta- or gamma-catenin. All noninvasive cell lines showed E-cadherin linked to these catenins. The E-cadherin-dependent cell-cell adhesion function in these cell lines was demonstrated by two assays in vitro. It was interesting that all invasive cell lines showed a dysfunctional E-cadherin/catenin complex. COLO320DM, SW480, and SW620 cells were defective in E-cadherin expression, whereas the invasive variants of HCT-8 and DLD-1 lacked the alpha-catenin protein. From clonal epithelioid HCT-8 cultures with functional E-cadherin/catenin complexes, we subcloned, repeatedly, round cell variants that were again invasive and expressed no alpha-catenin protein. Our data suggest that reproducible transformations toward a more invasive phenotype in HCT-8 cells are associated with down-regulation of alpha-catenin. The mechanisms of this transformation and the level of alpha-catenin down-regulation are currently investigated.

In vivo and in vitro invasion in relation to phenotypic characteristics of human colorectal carcinoma cells.

In this study we investigated the tumorigenicity, growth pattern and spontaneous metastatic ability of a series of nine human colorectal carcinoma cell lines after subcutaneous and intracaecal xenografting in nude mice. CaCo2 cells were found to be poorly tumorigenic to non-tumorigenic in either site; the other cell lines were tumorigenic in both sites. SW1116, SW480 and SW620 did not show local invasive in the NCI-H716 and LS174T cells were both invasive in the caecum, but only NCI-H716 was invasive in the subcutis. HT29 and 5583 (S and E) cells were invasive in the caecum and from that site metastatic to the lungs and/or the liver. HT29 and 5583S cells were both invasive in the subcutis, but 5583E cells were not. Of each category of in vivo behaviour in the caecum, one cell line was further investigated with regard to invasion in vitro (into embryonic chick heart fragments), E-cadherin expression in vivo and in vitro and in vitro production of u-PA and t-PA. These parameters were chosen in view of their purported role in extracellular matrix degradation and intercellular adhesion, which are all involved in the invasive and metastatic cascade. Invasion in vitro was not predictive for invasion or metastasis in vivo. In the cell line which showed invasion in embryonic chick heart tissue, heterogeneous E-cadherin expression was observed in vitro together with a relatively high production of u-PA. The non-invasive cell lines showed in vitro homogeneous expression of E-cadherin with a relatively low production of u-PA. In vivo expression of E-cadherin was either absent or heterogeneous. We conclude that: (1) colorectal carcinoma xenografts show site-specific modification of in vivo invasive and metastatic behaviour; (2) invasion in vitro does not correlate with invasion and metastasis in vivo; (3) in vitro non-invasion might be associated with homogeneous E-cadherin expression and low production of u-PA; (4) E-cadherin expression in vitro differs from E-cadherin expression in vivo. The results support the notion that the microenvironment in which cancer cells grow is one of the factors involved in the regulation of invasive and metastatic behaviour.

Micro-encapsulation of MDCK-ras-e cells prevents loss of E-cadherin invasion-suppressor function in vivo.

The invasion-suppressor molecule E-cadherin mediates Ca(2+)-dependent cell aggregation and prevents invasion. E-cadherin-positive Madin-Darby canine kidney (MDCK) cells that were non-invasive in vitro formed, upon i.p. injection, tumors that were invasive. Differentiated tubular tumor areas showed an intense immuno-signal for E-cadherin at intercellular contacts, whereas undifferentiated structures did not. Cell lines derived from such tumors turned out to be invasive in vitro and showed decreased Ca(2+)-dependent cell aggregation but no change in E-cadherin immunopositivity. This combination of phenotypes indicated a loss of the E-cadherin invasion-suppressor function. Micro-encapsulation of i.p.-injected cells prevented the loss of the E-cadherin invasion-suppressor function. We concluded that this loss in vivo was dependent upon immediate contacts between tumor cells and host cells or upon host factors that could not cross the capsule membrane.

Metastasis of Entamoeba histolytica compared to colon cancer: one more step in invasion.

The multistep invasion process of metastasis is a well-conserved mechanism of normal symbiosis and pathogenic parasitosis. To illustrate this statement, we have compared cellular and molecular aspects of metastasis from colon cancer and enteric amebiasis caused by Entamoeba histolytica. Both types of pathogenic cell, cancer cells and amebic trophozoites, follow the same metastatic route for the liver and other organs. Both establish invasion microecosystems implicating similar kinds of host cells and molecules belonging to closely related families. We have recently found indications that molecules released by trophozoites immediately after adhesion to epithelial monolayers bind to elements of the epithelial intercellular junctions. This phenomenon may lead to the functional disturbance of these junctions. Down-modulation of cell-cell adhesion molecules promotes invasion of colon cancer and other cancer cells. It is our opinion that comparative studies of apparently unrelated systems may reveal cellular and molecular mechanisms of invasion and metastasis that are of general biological importance.

Insulin-like growth factor I activates the invasion suppressor function of E-cadherin in MCF-7 human mammary carcinoma cells in vitro.

The calcium-dependent cell-cell adhesion molecule E-cadherin has been shown to counteract invasion of epithelial neoplastic cells. Using three monoclonal antibodies, we have demonstrated the presence of E-cadherin at the surface of human MCF-7/6 mammary carcinoma cells by indirect immunofluorescence coupled to flow cytometry and by immunocytochemistry. Nevertheless, MCF-7/6 cells failed to aggregate in a medium containing 1.25 mM CaCl2, and they were invasive after confrontation with embryonic chick heart fragments in organ culture. Treatment of MCF-7/6 cells with 0.5 microgram ml-1 insulin-like growth factor I (IGF-I) led to homotypic aggregation within 5 to 10 min and inhibited invasion in vitro during at least 8 days. The effect of IGF-I on cellular aggregation was insensitive to cycloheximide. However, monoclonal antibodies that interfered with the function of either the IGF-I receptor (alpha IR3) or E-cadherin (HECD-1, MB2) blocked the effect of IGF-I on aggregation. The effects of IGF-I on aggregation and on invasion could be mimicked by 1 microgram ml-1 insulin, but not by 0.5 microgram ml-1 IGF-II. The insulin effects were presumably not mediated by the IGF-I receptor, since they could not be blocked by an antibody against this receptor (alpha IR3). Our results indicate that IGF-I activates the invasion suppressor role of E-cadherin in MCF-7/6 cells.

Differential regulation of gelatinase B and tissue-type plasminogen activator expression in human Bowes melanoma cells.

A comparison of the production of tissue-type plasminogen activator (t-PA) and gelatinases A and B was made at the mRNA and protein levels in human Bowes melanoma cells treated with phorbol myristate acetate (PMA). Immunocytochemical analysis confirmed previous quantitative data on PMA-mediated induction of t-PA. It also showed that t-PA immunoreactivity can be restrained to the local environment of the producing cell, most probably by interaction with extracellular matrix components. Zymographical analysis showed that gelatinase B protein was induced by PMA, whereas gelatinase A remained at the constitutive level. Protein kinase C (PKC) appeared to be involved in this regulation since, after PMA treatment (1) the PKC activity was found to be translocated from the cytosol to the particulate fraction of the cells and (2) addition of staurosporine and H-7 blocked the gelatinase B increase. Northern-blot hybridization showed a transient rise in t-PA and gelatinase B mRNA levels whereas gelatinase A mRNA levels remained unchanged. When c-fos and c-jun mRNAs were investigated, only that of c-fos was affected by PMA. Activation by PMA can be kinetically ordered as follows: translocation of PKC to the membrane fraction, transcription of the c-fos gene and eclipsing of gelatinase B mRNA, increase in steady-state mRNA levels of t-PA and gelatinase B and, finally, secretion of t-PA and gelatinase B glycoproteins. Our data also suggest that various proteases that are known to cooperate in the remodeling of the extracellular matrix can be differently regulated in one tumor-cell type.

Arrest of MCF-7 cell migration by laminin in vitro: possible mechanisms.

Laminin, a major basement membrane component, arrested the migration of MCF-7/AZ human breast adenocarcinoma cells that were not invasive in vitro. Migration of invasive MCF-7/6 cells was not affected by laminin. Both cell types expressed the 67 kD laminin receptor, at both mRNA and protein level, but did not express the alpha 6 subunit of the VLA-6 integrin-type laminin receptor. The presence of YIGSR peptides (100 micrograms/ml), reported to block the interaction between laminin and its 67 kD receptor, did not change the migratory response of MCF-7/AZ or MCF-7/6 cells when meeting laminin lanes. In addition, the migration of these cell types was not affected by the presence of 17-beta-estradiol (10(-6) M) or all-trans retinoic acid (10(-6) M), which were both reported to increase the number of 67 kD receptors. We could therefore not assign an involvement of the 67 kD receptors in migration of MCF-7 cells on laminin, nor did we find evidence that conditioned medium of MCF-7/6 cells contains factors that are able to initiate migration of MCF-7/AZ cells on laminin.

Spontaneous acquisition of tumorigenicity and invasiveness by mouse lens explant cells during culture in vitro.

The lens of the eye is one of the rare organs in which tumors do not occur spontaneously. It therefore appeared to us that lens cells would not present the background of spontaneous transformation toward malignancy found with many other cell cultures. We have cultured C3H/HeA mouse lens explant (MLE) cells for 70 wk and analyzed changes in malignancy-related phenotypes in function of the number of passages. In vitro, we studied morphology, colony forming efficiency on tissue culture plastic substrate (CFEtc) and in soft agar, population doubling time, saturation density, and invasiveness into precultured chick heart fragments. In vivo, tumorigenicity, invasion, and metastasis were analyzed after injection of cell suspensions subcutaneously and intraperitoneally, after implantation of cells aggregated to collagen sponges under the renal capsule and after implantation of cell aggregates subcutaneously into the tail and into the pinna. The CFEtc, population doubling time, and saturation density increased as the number of passages of culture in vitro increased, but colony formation in soft agar was never observed. MLE cells till passage 16 were not invasive in vitro, but hereafter consistently were found to be invasive. After about 17 passages, corresponding to 25 wk of culture, MLE cells acquired the capacity to form tumors in syngeneic mice. These tumors were invasive but metastases were not observed. We concluded that MLE cells acquired in an apparently spontaneous way a number of malignancy-related phenotypes, without, however, reaching the stage of metastasis.

Metastatic T-cell hybridoma cells display target preference for invasion ('homing') in tissue culture models.

BW-O-Li1 murine T-lymphoma cells display target preference for invasion in two different in vitro models. In the precultured chick heart fragment (PHF) assay, BW-O-Li1 preferentially invaded into aggregates of MCF-7 mammary carcinoma cells rather than into PHFs. In a monolayer invasion assay (MIA), BW-O-Li1 cells preferentially invaded under 10 T1/2 mouse embryo cell monolayers rather than under MCF-7 monolayers. Thus, although BW-O-Li1 cells were perfectly able to invade into any of the targets presented, they migrated and accumulated preferentially in one of the targets when a choice was offered. We suggest that this in vitro 'homing' phenomenon can be exploited to investigate certain aspects of organ-specific metastasis.

Down-regulation of E-cadherin expression in Madin Darby canine kidney (MDCK) cells inside tumors of nude mice.

The 120-kDa cell-cell adhesion molecule E-cadherin is localized at the epithelial junctional complex and participates in the organization and maintenance of epithelia. The Madin Darby canine kidney (MDCK) cell line expresses E-cadherin in a stable way and forms polarized epitheloid structures in vitro. Harvey-murine-sarcoma-virus-transformed derivatives (MDCK-ras) produce malignant (i.e., invasive and metastatic) tumors in nude mice. We obtained evidence that E-cadherin is down-regulated in nude mouse tumors and that this down-regulation is reversible. MDCK-ras-e cell lines were cloned in vitro from MDCK-ras cell cultures. They showed an epithelioid morphotype and expressed E-cadherin at homogeneously high level. This characteristic has been conserved for at least 60 passages in vitro. MDCK-ras-e cells were not invasive in vitro. When injected into nude mice, however, they produced invasive and metastatic tumors. Primary tumors as well as large metastases were heterogeneous, showing E-cadherin-positive well differentiated epithelial structures and E-cadherin-negative undifferentiated areas. Metastasis-derived cell cultures contained both E-cadherin-positive and E-cadherin-negative MDCK-ras-e cells during early passages in vitro. During further culture, however, they regained the homogeneous E-cadherin-positive characteristic of the original MDCK-ras-e cell line. The behavior of MDCK-ras-e cells in vitro, as compared with its in vivo behavior, points to the existence of host factors which are able to down-regulate E-cadherin expression. We hypothesize that this down-regulation plays a basic role in invasion.

Influence of basement membrane molecules on directional migration of human breast cell lines in vitro.

Spheroidal cell aggregates were prepared from four tumorigenic human breast cell lines (HBL-100 and three MCF-7 variants). Cells from these aggregates were allowed to migrate towards lanes of basement membrane components coated on a glass substratum. Matrigel (reconstituted basement membrane) lanes permanently arrested the migration of one MCF-7 cell line, while migration of the others was permitted. Amongst several purified basement membrane constituents only laminin, not collagen type IV or fibronectin, was found to cause the same arrest of migration. Within the laminin molecule only the pepsin P1, not the elastase E8 fragment, efficiently arrested migration of that cell line. Although migration was inhibited by these components, time-lapse video recordings revealed that arrested cells still proliferated and actively ruffled on top of the coatings. These data suggest that, amongst several basement membrane components, laminin can function as a stop signal for cell migration. Within laminin, this activity seems to be mainly associated with the P1 fragment. We conclude that laminin is the major determinant of the barrier-function of the basement membrane, to which some cell types have become insensitive.