Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose

The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin.     

Suramin inhibits cell growth and glycolytic activity and triggers differentiation of human colic adenocarcinoma cell clone HT29-D4

Suramin, a drug used in the treatment of trypanosomiasis and onchocerciasis inhibits growth factor-induced mitogenesis. In the present report, we show that suramin inhibits the growth of human colic adenocarcinoma cells HT29-D4 and rapidly induces their differentiation into enterocyte-like cells. As soon as 6 days after the addition of suramin (100 micrograms/ml) in the culture medium, the cells form a polarized monolayer of regular columnar cells with occluding junctions delimiting two distinct membrane domains (apical and basolateral) and an apical brush-border expressing alkaline phosphatase and sucrase-isomaltase. The process of differentiation is fully reversible when the drug is removed from the culture medium. We also show that suramin inhibits both glucose consumption and lactate production so that the glycolytic activity of the treated cells is lowered by 42%. This observation would shed some light on the complex mechanisms involved during the induction of HT29 cell differentiation when glucose is removed from the culture medium.     

Down regulation of hypertrophied follicular cell volume in thyroid hyperplastic gland

In the present study, changes in thyroid follicular cell volume and its regulation have been investigated during the early involution of a hyperplastic goitre. Male Wistar rats were administered an iodine deficient diet for 6 months with propylthiouracil (PTU, 0.15%) during the last two months. At the end of iodine deficiency (day 0), some rats were killed and the others received a normal iodine diet. These rats were killed after different periods of iodine refeeding. Thyroid follicular cell volume was very high in hyperplastic gland whereas thyroid protein concentration was low. Thyroid follicular cell volume quickly decreased when rats were normally iodine refed, whereas thyroid protein concentration increased. Electron microscopal observations showed that thyroid follicular cells retained their endocrine aspect in hyperplastic state and throughout the iodine refeeding period. Using concomitant stereological and biochemical techniques, it is shown that the amount of cellular iodide and an unknown iodinated compound strongly increased during the early iodine refeeding. Plasma TSH was high on day 0 and remained at this level until day 8 whereas plasma T3 and T4 were low on day 0 and remained at this low level until day 4. The present data show that the involution of thyroid follicular cell volume is induced by iodide and mediated by an iodinated compound at least in the initial phase, and is independent of plasma TSH, T3, T4, so indicating the involvement of a thyroid autoregulatory mechanism. These changes in cell volume may be of importance in ion transport, i.e. in the metabolism of thyroid follicular cell during the early involution of the hyperplastic goitre.     

A Genomic Approach to Unravel Host-Pathogen Interaction in Chelonians: The Example of Testudinid Herpesvirus 3

We report the first de novo sequence assembly and analysis of the genome of Testudinid herpesvirus 3 (TeHV3), one of the most pathogenic chelonian herpesviruses. The genome of TeHV3 is at least 150,080 nucleotides long, is arranged in a type D configuration and comprises at least 102 open reading frames extensively co-linear with those of Human herpesvirus 1. Consistently, the phylogenetic analysis positions TeHV3 among the Alphaherpesvirinae, closely associated with Chelonid herpesvirus 5, a Scutavirus. To date, there has been limited genetic characterization of TeHVs and a resolution beyond the genotype was not feasible because of the lack of informative DNA sequences. To exemplify the potential benefits of the novel genomic information provided by this first whole genome analysis, we selected the glycoprotein B (gB) gene, for detailed comparison among different TeHV3 isolates. The rationale for selecting gB is that it encodes for a well-conserved protein among herpesviruses but is coupled with a relevant antigenicity and is consequently prone to accumulate single nucleotide polymorphisms. These features were considered critical for an ideal phylogenetic marker to investigate the potential existence of distinct TeHV3 genogroups and their associated pathology. Fifteen captive tortoises presumptively diagnosed to be infected with TeHVs or carrying compatible lesions on the basis of either the presence of intranuclear inclusions (presumptively infected) and/or diphtheronecrotic stomatitis-glossitis or pneumonia (compatible lesions) were selected for the study. Viral isolation, TeHV identification, phylogenetic analysis and pathological characterization of the associated lesions, were performed. Our results revealed 1) the existence of at least two distinct TeHV3 genogroups apparently associated with different pathologies in tortoises and 2) the first evidence for a putative homologous recombination event having occurred in a chelonian herpesvirus. This novel information is not only fundamental for the genetic characterization of this virus but is also critical to lay the groundwork for an improved understanding of host-pathogen interactions in chelonians and contribute to tortoise conservation.     

Biomechanical roles of medial pooling of glycosaminoglycans in thoracic aortic dissection

Spontaneous dissection of the human thoracic aorta is responsible for significant morbidity and mortality, yet this devastating biomechanical failure process remains poorly understood. In this paper, we present finite element simulations that support a new hypothesis for the initiation of aortic dissections that is motivated by extensive histopathological observations. Specifically, our parametric simulations show that the pooling of glycosaminoglycans/proteoglycans that is singularly characteristic of the compromised thoracic aorta in aneurysms and dissections can lead to significant stress concentrations and intra-lamellar Donnan swelling pressures. We submit that these localized increases in intramural stress may be sufficient both to disrupt the normal cell-matrix interactions that are fundamental to aortic homeostasis and to delaminate the layered microstructure of the aortic wall and thereby initiate dissection. Hence, pathologic pooling of glycosaminoglycans/proteoglycans within the medial layer of the thoracic aortic should be considered as a possible target for clinical intervention.     

Follicle-like structures formed by intestinal cell lines derived from the HT29-D4 adenocarcinoma cell line: morphological and functional characterization.

When cultured in high glucose containing medium, the human colon carcinoma cell line HT29-D4 and a clone derived by transfection with the MDR1 cDNA (MDR31) form multilayers of unorganized cells which are not polarized and are linked by desmosomes. Within these multilayers appear spontaneously large multicellular follicle-like-structures (FLS) where polarized cells linked by tight junctional complexes surround a lumen. Electron microscopy showed that some FLS display well developed brush borders with densely packed microvilli. Others have irregularly oriented microvilli of various lengths or are even completely devoid of apical differentiation. The lumen contains a variable amount of amorphous osmiophilic material. The apical surface of FLS forming cells express dipeptidylpeptidase IV, carcinoembryonic antigen, the mucin MUC1 and for the transfected cells the gp-170 protein. The organic anion fluorescein is transported from the cell to the lumen of FLS. Rhodamine 123, a substrate of the gp-170 ABC transporter is also concentrated in the lumen formed by MDR31 cells. Verapamil and cyclosporine A inhibited this last transport. Cyclic AMP stimulates the formation of these structures since treatment of post-confluent multilayers dramatically increased the number of FLS in HT29-D4 and MDR31 cell cultures within 24 h. The spontaneous formation of these morphologically and functionally polarized structures appeared at random and might respond to the coincidence of fluctuating parameters of the regulatory pathways (cAMP, Ca2+).     

Contribution of left ventricular residual stress by myocytes and collagen: existence of inter-constituent mechanical interaction

We quantify the contribution of myocytes, collagen fibers and their interactions to the residual stress field found in the left ventricle (LV) using both experimental and theoretical methods. Ring tissue samples extracted from normal rat, male and female, LV were treated with collagenase and decellularization to isolate myocytes and collagen fibers, respectively. Opening angle tests were then performed on these samples as well as intact tissue samples containing both constituents that served as control. Our results show that the collagen fibers are the main contributor to the residual stress fields found in the LV. Specifically, opening angle measured in collagen-only samples (106.45\(^\circ \) ± 23.02\(^\circ \)) and myocytes-only samples (21.00\(^\circ \) ± 4.37\(^\circ \)) was significantly higher and lower than that of the control (57.88\(^\circ \) ± 12.29\(^\circ \)), respectively. A constrained mixture (CM) modeling framework was then used to infer these experimental results. We show that the framework cannot reproduce the opening angle found in the intact tissue with measurements made on the collagen-only and myocytes-only samples. Given that the CM framework assumes that each constituent contributes to the overall mechanics simply by their mere presence, this result suggests the existence of some myocyte–collagen mechanical interaction that cannot be ignored in the LV. We then propose an extended CM formulation that takes into account of the inter-constituent mechanical interaction in which constituents are deformed additionally when they are physically combined into a mixture. We show that the intact tissue opening angle can be recovered in this framework.     

Quantifying whole bladder biomechanics using the novel pentaplanar reflected image macroscopy system

Optimal bladder compliance is essential to urinary bladder storage and voiding functions. Calculated as the change in filling volume per change in pressure, bladder compliance is used clinically to characterize changes in bladder wall biomechanical properties that associate with lower urinary tract dysfunction. But because this method calculates compliance without regard to wall structure or wall volume, it gives little insight into the mechanical properties of the bladder wall during filling. Thus, we developed Pentaplanar Reflected Image Macroscopy (PRIM): a novel ex vivo imaging method to accurately calculate bladder wall stress and stretch in real time during bladder filling. The PRIM system simultaneously records intravesical pressure, infused volume, and an image of the bladder in five distinct visual planes. Wall thickness and volume were then measured and used to calculate stress and stretch during filling. As predicted, wall stress was nonlinear; only when intravesical pressure exceeded ~ 15 mmHg did bladder wall stress rapidly increase with respect to stretch. This method of calculating compliance as stress vs stretch also showed that the mechanical properties of the bladder wall remain similar in bladders of varying capacity. This study demonstrates how wall tension, stress and stretch can be measured, quantified, and used to accurately define bladder wall biomechanics in terms of actual material properties and not pressure/volume changes. This method is especially useful for determining how changes in bladder biomechanics are altered in pathologies where profound bladder wall remodeling occurs, such as diabetes and spinal cord injury.