Multi-modality imaging in hypertrophic cardiomyopathy: intermodal discrepancies in key prognostic parameters

Background/Introduction Multi-modality imaging is crucial for confirming diagnosis and assessing prognosis in patients with hypertrophic cardiomyopathy (HCM). However, inter-modality discrepancies in key parameters are commonly reported. Purpose To assess real-world inter-modal reporting discrepancies between transthoracic echocardiography (TTE) and cardiac magnetic resonance (CMR) imaging in the measurement of four key parameters in HCM patients. Methods Consecutive HCM patients with TTE and CMR performed within 6 months of each other at a tertiary centre were retrospectively assessed for reported maximum wall thickness (MWT), left atrial diameter (LAd), left ventricular ejection fraction (LVEF) and presence of left ventricular apical aneurysm (LVAA). The CMR report was considered gold standard. Data are reported as mean ± standard deviation (SD) or median and interquartile range (IQR) as appropriate. Results 353 consecutive HCM patients (72% male, median age 60.9 years, IQR 49.8–71.6 years) with TTE and CMR within 6 months (median difference 1.7 months, IQR 1.1–3.4 months) were assessed between 4th January 2018 and 9th April 2019. Of 284 patients with paired MWT data, median difference was 0.0 mm (IQR −1.0 to 3.0 mm, p=0.02), likely representing a difference in distributions of MWT. TTE both over and underestimated MWT (in 36% and 46% cases respectively). Of the 94 patients with paired LAd data, mean difference was 0.4±5.7 mm (95% CI −0.8010 to 1.546, p=0.5). N=320 patients with paired LVEF data (after excluding patients with atrial fibrillation (n=20)). Median difference in LVEF was 12% (IQR 5–19% p<0.0001). TTE underestimated LVEF in 88% of cases. CMR and TTE both identified 14 (5%) patients as having LVEF <50%. There were however 8 cases of disagreement in classification of LVEF <50%, due to over (n=4) or underestimation (n=4) by TTE. LVAA was accurately identified by TTE in only 9/30 (30%) of those patients with demonstrable LVAA by CMR (p=0.0008). TTE evidence of a discreet apical chamber (paradoxical jet on spectral or colour Doppler) was present in 16/21 (76%) cases where TTE failed to overtly identify LVAA. However, apical or mid-cavity obliteration was reported in 15/21 (71%) cases where TTE failed to identify LVAA. Conclusion(s) Echocardiography and CMR measurements are often used interchangeably in clinical practice but inter-modality discrepancies can affect diagnosis and sudden cardiac death (SCD) risk assessment. This is particularly important for binary risk factors such as LVEF<50% or LVAA which are considered major SCD risk factors in the latest American Heart Association guidelines. 25 (7%) patients in our cohort had major risk factors identified by CMR that were not identified on TTE. CMR is an important, recommended tool where TTE imaging is suboptimal, but attention to more subtle elements of abnormal intracavity blood flow may be able to increase LVAA detection during TTE. FUNDunding Acknowledgement Type of funding sources: None.

Iron Transport in Intestinal Epithelial Cells Occurs by Transcytosis. A Fluorescent Metal-Sensor Study.

The molecular mechanisms ensuring directionality of iron transport across the intestinal epithelium are still poorly understood. Iron is transported across the brush-border membrane (BBM) by the divalent metal transporter 1 (DMT1) and then must be targeted to specific organelles and other transporters for transport across the basolateral membrane (BLM). We have previously shown that in Caco2 cells grown in bicameral chambers with the addition of iron to the apical surface, DMT1 on the BBM undergoes endocytosis and fuses with endocytic vesicles derived from the basal surface. However, which cytosolic proteins mediate the endocytosis of DMT1 and the interaction of apical derived vesicles with basal derived vesicles are still unknown. The present study focuses on trying to confirm our hypothesis that iron absorption in the intestinal epithelium is through endocytic processes and involves a pathway in which apical derived vesicles fuse with basolateral-derived vesicles. Calcein is a fluorescent metal sensor whose fluorescence is quenched with chelation of iron. Calcein, which is hydrophilic and membrane-impermeable, is taken up by cells by endocytosis (Glickstein H et al. Blood First Edition Paper, prepublished online July 14, 2005). To monitor the flux of iron via transcytosis we took advantage of the properties of calcein and used Caco2 cells grown as a polarized cell layer in bicameral chambers as a model system to study iron flux. In the Caco2 model the apical chamber baths the brush border surface of the cells and the basal chamber baths the basolateral surface. When calcein was offered in the basal chamber along with apo-transferrerin (apo-Tf), calcein was found to undergo endocytosis and co-localize with apo-Tf in the sub-apical cytoplasm with a coefficient of co-localization of 36.8 ± 3.6 %. Under these conditions offering ferrous iron in the apical chamber caused the calcein to be quenched and the coefficient of co-localization decreased to 22.5 ± 5.7 %. The addition of a permeable iron chelator restored calcein fluorescence and the co-localization increased to 58.6 ± 20.5 %. Iron chelated to calcein, which markedly quenches calcein fluorescence, was then offered in the apical chamber as a source of iron. Apo-Tf was offered simultaneously in the basal chamber. After internalization calcein fluorescence was subsequently restored and calcein was observed to co-localize in vesicles with apo-Tf with a co-localization coefficient was 26.4 ± 5.8 %. These studies strongly suggest that iron is transported across the intestinal epithelium by transcytosis with apical derived vesicles fusing with basolateral-derived vesicles. Early endosome antigen 1 (EEA1) and the small GTPase, Rab5, are required for endosomal transport and fusion in mammalian cells and might provide directionality to vesicular transport from the membrane to the early endosomes. We examined if both EEA1 and Rab5 are involved in the transcytosis of DMT1 and iron and found that after iron feeding that there was increased co-localization of DMT1 with both EEA1 and Rab5 in the sub-apical compartment. Taken together these studies support our hypothesis that iron absorption in the intestinal epithelium is through endocytic processes and involves a transcytosis pathway.

Development and characterization of secretin-stimulated secretion of cultured rat cholangiocytes

We sought to develop a cholangiocyte cell culture system that has preservation of receptors, transporters, and channels involved in secretin-induced secretion. Isolated bile duct fragments, obtained by enzyme perfusion of normal rat liver, were seeded on collagen and maintained in culture up to 18 wk. Cholangiocyte purity was assessed by staining for γ-glutamyl transpeptidase (γ-GT) and cytokeratin-19 (CK-19). We determined gene expression for secretin receptor (SR), cystic fibrosis transmembrane conductance regulator, Cl−/HCO[Formula: see text] exchanger, secretin-stimulated cAMP synthesis, Cl−/HCO3exchanger activity, secretin-stimulated Cl− efflux, and apical membrane-directed secretion in polarized cells grown on tissue culture inserts. Cultured cholangiocytes were all γ-GT and CK-19 positive. The cells expressed SR and Cl−/HCO[Formula: see text] exchanger, and secretin-stimulated cAMP synthesis, Cl−/HCO[Formula: see text] exchanger activity, and Cl− efflux were similar to freshly isolated cholangiocytes. Forskolin (10−4 M) induced fluid accumulation in the apical chamber of tissue culture inserts. In conclusion, we have developed a novel cholangiocyte line that has persistent HCO[Formula: see text], Cl−, and fluid transport functions. This cell system should be useful to investigators who study cholangiocyte secretion.

Modulation of Cytosolic Ca2+ Concentration in Airway Epithelial Cells by Pseudomonas aeruginosa

ABSTRACT Modulation of cytosolic (intracellular) Ca2+ concentration (Cai) may be an important host response when airway epithelial cells are exposed to Pseudomonas aeruginosa. We measured Cai in Calu-3 cells exposed from the apical or basolateral surface to cytotoxic and noncytotoxic strains of P. aeruginosa. Apical addition of either noncytotoxic strains or cytotoxic strains failed to affect Cai over a 3-h time period, nor were changes observed after basolateral addition of noncytotoxic strains. In contrast, basolateral addition of cytotoxic strains caused a slow increase in Cai from 100 nM to 200 to 400 nM. This increase began after 20 to 50 min and persisted for an additional 30 to 75 min, at which time the cells became nonviable. P. aeruginosa-induced increases in Cai were blocked by the addition of the Ca channel blocker La3+ to the basolateral but not to the apical chamber. Likewise, replacing the basolateral but not the apical medium with Ca-free solution prevented P. aeruginosa-mediated changes in Cai. With isogenic mutants of PA103, we demonstrated that the type III secretion apparatus, the type III-secreted effector ExoU, and type IV pili were necessary for increased Cai. We propose that translocation of ExoU through the basolateral surface of polarized airway epithelial cells via the type III secretion apparatus leads to release of Ca stored in the endoplasmic reticulum and activation of Ca channels in the basolateral membranes of epithelial cells.

The transcytosis of divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium

Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis.

Glycolysis inhibition by palmitate in renal cells cultured in a two-chamber system

A major shortcoming of renal proximal tubular cells (RPTC) in culture is the gradual modification of their energy metabolism from the oxidative type to the glycolytic type. To test the possible reduction of glycolysis by naturally occurring long-chain fatty acids, RPTC were cultured in a two-chamber system, with albumin-bound palmitate (0.4 mM) added to the basolateral chamber after confluency. Twenty-four hours of contact with palmitate decreased glycolysis by 38% provided that carnitine was present; lactate production was decreased by 38%, and the decrease in glycolysis resulted from a similar decrease of basolateral and apical net uptake of glucose. In contrast to the previously described effect of the nonphysiological oxidative substrate heptanoate, palmitate promoted a long-term decrease in lactate production and sustained excellent cellular growth. After 4 days of contact, decreased glycolysis was maintained even in the absence of carnitine and resulted from a decrease of basolateral uptake only, suggestive of long-term regulation different from the earlier effects. Thus, although cultured RPTC lost their oxidative phenotype, they exhibited a type of regulation (Randle effect) that is found in the oxidative-type but not in the glycolytic-type tissues, therefore unmasking a regulative capacity barely detectable in fresh RPTC. Low Po 2 (50 mmHg in the apical chamber) could be a major cause of elevated glycolysis and could hinder the effects of palmitate.

Transepithelial acidification by cultures of rabbit proximal tubules grown on filters

Transepithelial acidification in the proximal tubule occurs by the simultaneous actions of the Na(+)-H+ exchanger in the brush border and the basolateral Na(+)-HCO3- cotransporter. The presence of these systems has been demonstrated for cultured cells; however, their contributions to the transepithelial movement of acid equivalents has not been confirmed in monolayers. To examine transepithelial acidification by intact cells, tubules were grown on membrane filters. Confluent cultures developed a transepithelial pH gradient within 6 h by decreasing the pH of medium in the apical chamber (6.66 +/- 0.03) while raising the basolateral pH to 7.40 +/- 0.02. Cells maintained on plastic did not acidify the medium during this time. Amiloride (10-100 microM) inhibited development of the gradient only when placed in the top chamber. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 10-100 microM), which inhibits basolateral Na(+)-HCO3- cotransport, decreased the gradient only when added to the bottom. These results demonstrate that cultured proximal tubule cells can develop a transepithelial pH gradient and that the polarized distribution of the transport systems is maintained in vitro.

Catenicellid Cheilostome Bryozoa II.* Introduction to Ovicell Complexes

Catenicellid ovicell complexes are of two types, 'terminal' and 'subterminal'. The former, found at the end of a branch, generally consists of a female zoid with ovicell, together with vestigial ovicell zoid and apical chamber. Subterminal ovicell complexes bud distally from part of the complex and are generally composed of female zoid with ovicell, lateral zoid, and ovicell zoid with apical chamber. For all 13 species described, the position of the ovicell complexes in the budding pattern is shown diagrammatically. Frontal walls and associated structures of some species not described in a previous study are included.

The Manitoba Horse Fly Trap

The development of the Manitoba Horse Fly Trap was motivated initially by the need to control tabanid flies in spite of a lack of effective and safe insecticides. This objective could be realized only through a study of orientation behaviour in the field. Starting with the old observation that tabanids are attracted to darkcoloured objects (‘targets’), the influence of colour of target, colour of background, size, shape and movement of target were investigated. The most attractive target proved to be a glossy black (or red) sphere about 24 inches diameter, about three feet above the ground. In the course of its development the trap has provided interesting insights into visual perception of tabanids which will require physiological confirmation. The attractant principle is overwhelmingly visual. An initial surmise that thermal radiation contributes to the attraction significantly has been superseded, although a thermal stimulus may be involved in the behaviour of the flies after they reach the trap. Since only the hemato- phagous sex (females) is attracted to the trap, the behaviour involved appears to be teleologically related to host seeking activity. On arrival at the trap the insects fly in the space around the target and eventually move upward into the no-return chamber at the apex. When the trap is used for fly control the apical chamber is not needed if the interior of the trap is treated with a powerful, residual insecticide. In addition to its potential uses in conjunction with insecticides for control of tabanids, the Manitoba Horse Fly Trap can be used to study the correlation of flight activity with meteorological conditions as well as the seasonal and geographical distribution of tabanid species. The trap can be used effectively in the study of Stomoxys behaviour and control. If the sphere is replaced by a wick containing organic sulfides, blow flies can be attracted to it in considerable numbers.