Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolayers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.

/pdf/culture-of-human-endothelial-cells-derived-from-umbilical-3vt4ihjkgj.pdf

Culture of Human Endothelial Cells Derived from Umbilical Veins. IDENTIFICATION BY MORPHOLOGIC AND IMMUNOLOGIC CRITERIA

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

The acute respiratory distress syndrome is a common, devastating clinical syndrome of acute lung injury that affects both medical and surgical patients. Since the last review of this syndrome appeared in the Journal, 1 more uniform definitions have been devised and important advances have occurred in the understanding of the epidemiology, natural history, and pathogenesis of the disease, leading to the design and testing of new treatment strategies. This article provides an overview of the definitions, clinical features, and epidemiology of the acute respiratory distress syndrome and discusses advances in the areas of pathogenesis, resolution, and treatment. Historical Perspective and Definitions . . .

/pdf/the-acute-respiratory-distress-syndrome-4dksnctehq.pdf

The acute respiratory distress syndrome

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Allometric scaling relations, including the 3/4 power law for metabolic rates, are characteristic of all organisms and are here derived from a general model that describes how essential materials are transported through space-filling fractal networks of branching tubes. The model assumes that the energy dissipated is minimized and that the terminal tubes do not vary with body size. It provides a complete analysis of scaling relations for mammalian circulatory systems that are in agreement with data. More generally, the model predicts structural and functional properties of vertebrate cardiovascular and respiratory systems, plant vascular systems, insect tracheal tubes, and other distribution networks.

https://science.sciencemag.org/content/sci/276/5309/122.full.pdf

A general model for the origin of allometric scaling laws in biology

Commentaries on viewpoint: use of mean airspace chord length to assess emphysema. To be or not to be-accurate

Physiologists are bound to test their scientific theories in experiments on living matter and, ultimately, on living organisms—animals or humans. This confronts the physiologist with ethical dilemm...

The Physiologist's Ethical Dilemmas

Influence of Environmental Po2 on the Growth of the Pulmonary Gas Exchange Apparatus

https://popups.ulg.ac.be/0351-580x/index.php?id=2805&file=1&pid=2801

STEPone - An interactive program for manual stereology

Fractals in biology and medicine. Volume II , edited by Gabriele A. Losa, Danilo Merlini, Theo F. Nonnenmacher and Ewald R. Weibel. Pp. 369. SFr98. 1997. ISBN 3 7643 5715 0(Birkhäuser).

Ewald R. Weibel

Papers

Commentaries on viewpoint: use of mean airspace chord length to assess emphysema. To be or not to be-accurate

The Physiologist's Ethical Dilemmas

Influence of Environmental Po2 on the Growth of the Pulmonary Gas Exchange Apparatus

STEPone - An interactive program for manual stereology

Fractals in biology and medicine. Volume II , edited by Gabriele A. Losa, Danilo Merlini, Theo F. Nonnenmacher and Ewald R. Weibel. Pp. 369. SFr98. 1997. ISBN 3 7643 5715 0(Birkhäuser).