Mammal responses to global changes in human activity vary by trophic group and landscape.

Wildlife must adapt to human presence to survive in the Anthropocene, so it is critical to understand species responses to humans in different contexts. We used camera trapping as a lens to view mammal responses to changes in human activity during the COVID-19 pandemic. Across 163 species sampled in 102 projects around the world, changes in the amount and timing of animal activity varied widely. Under higher human activity, mammals were less active in undeveloped areas but unexpectedly more active in developed areas while exhibiting greater nocturnality. Carnivores were most sensitive, showing the strongest decreases in activity and greatest increases in nocturnality. Wildlife managers must consider how habituation and uneven sensitivity across species may cause fundamental differences in human-wildlife interactions along gradients of human influence.

Pituitary iron and volume imaging in healthy controls.

BACKGROUND AND PURPOSE: Patients with transfusional iron overload develop iron deposits in the pituitary gland, which are associated with volume loss and HH. The purpose of this study was to characterize R2 and volumetric data in a healthy population for diagnostic use in patients with transfusional iron overload. MATERIALS AND METHODS: One hundred healthy controls without iron overload between the ages of 2 and 48 were recruited to have MR imaging of the brain to assess their pituitary R2 and volume. Pituitary R2 was assessed with a 8-echo spin-echo sequence, and pituitary volumes, by a 3D spoiled gradient-echo sequence with 1-mm(3) resolution. A 2-component continuous piecewise linear approximation was used for creating volumetric and R2 nomograms. Equations were generated from regression relationships for convenient z-score calculation. RESULTS: Pituitary R2 rose weakly with age (r(2) = 0.19, P < .0001). Anterior and total pituitary volumes increased steadily up to 18 years of age, after which volume slightly decreased. Females had larger pituitary glands, most likely representing their larger lactotroph population. CONCLUSIONS: From these data, a clinician can calculate the z scores for R2 and pituitary volume in patients with iron overload. Normal ranges are well-differentiated from values previously associated with endocrine disease in transfusional siderosis; this finding suggests that preclinical iron overload can be recognized and appropriately treated.

Quantitative analysis and modelling of hepatic iron stores using stereology and spatial statistics.

Hepatic iron overload is a common clinical problem resulting from hyperabsorption syndromes and from chronic transfusion therapy. Not only does iron loading vary between reticuloendothelial stores and hepatocytes, but iron is heterogeneously distributed within hepatocytes as well. Since the accessibility of iron particles to chelation may depend, in part, on their distribution, we sought to characterize the shape and scale of iron deposition in humans with transfusional iron overload. Toward this end, we performed a histological analysis of iron stores in liver biopsy specimens of 20 patients (1.3-57.8 mg iron/g dry tissue weight) with aid of electron and light microscopy. We estimated distributions related to variability in siderosomal size, proximity of iron centres and inter-cellular iron loading. These distributions could be well modelled by Gamma distribution functions over most of the pathologic range of iron concentrations. Thus, for a given liver iron burden, a virtual iron-overloaded liver could be created that served as a model for the true histologic appearance. Such a model may be helpful for understanding the mechanics of iron loading or in predicting response to iron removal therapy.

Sickle cell disease: selected aspects of pathophysiology.

Sickle cell disease (SCD), a genetically-determined pathology due to an amino acid substitution (i.e., valine for glutamic acid) on the beta-chain of hemoglobin, is characterized by abnormal blood rheology and periods of painful vascular occlusive crises. Sickle cell trait (SCT) is a typically benign variant in which only one beta chain is affected by the mutation. Although both SCD and SCT have been the subject of numerous studies, information related to neurological function and transfusion therapy is still incomplete: an overview of these areas is presented. An initial section provides pertinent background information on the pathology and clinical significance of these diseases. The roles of three factors in the clinical manifestations of the diseases are then discussed: hypoxia, autonomic nervous system regulation and blood rheology. The possibility of a causal relationship between these three factors and sudden death is also examined. It is concluded that further studies in these specific areas are warranted. It is anticipated that the outcome of such research is likely to provide valuable insights into the pathophysiology of SCD and SCT and will lead to improved clinical management and enhanced quality of life.

Abnormal autonomic cardiac response to transient hypoxia in sickle cell anemia.

The objective of this study was to non-invasively assess cardiac autonomic control in subjects with sickle cell anemia (SCA) by tracking the changes in heart rate variability (HRV) that occur following brief exposure to a hypoxic stimulus. Five African-American SCA patients and seven healthy control subjects were recruited to participate in this study. Each subject was exposed to a controlled hypoxic stimulus consisting of five breaths of nitrogen. Time-varying spectral analysis of HRV was applied to estimate the cardiac autonomic response to the transient episode of hypoxia. The confounding effects of changes in respiration on the HRV spectral indices were reduced by using a computational model. A significant decrease in the parameters related to parasympathetic control was detected in the post-hypoxic responses of the SCA subjects relative to normal controls. The spectral index related to sympathetic activity, on the other hand, showed a tendency to increase the following hypoxic stimulation, but the change was not significant. This study suggests that there is some degree of cardiovascular autonomic dysfunction in SCA that is revealed by the response to transient hypoxia.

Multisite microelectrode arrays for measurements of multiple neurochemicals.

Multisite microelectrode arrays designed for electrochemical measures of neurochemicals in CNS tissues are presented. The arrays have platinum recording sites insulated with polyimide on a ceramic substrate. Most designs include 4 recording sites, however arrays with 5 to 8 recording sites have been fabricated. Enzyme coatings have been developed to measure glutamate, choline, lactate, and glucose. Electroactive compounds such as dopamine, norepinephrine, and O/sub 2/ can also be measured. The multiple recording sites can be exploited for interferent or noise removal and measures of multiple compounds using a single microelectrode array.

Periodic formation of nascent lamellae is driven by changes in the stable F-actin pool of polymorphonuclear neutrophils after stimulation with chemotactic peptide and cross-linking of CD18 or CD61.

Cell motility and changes in cell shape are largely powered by actin polymerization and depolymerization. Eight to ten second periodic changes in human polymorphonuclear neutrophil (PMN) shape were detected by video-image analysis of PMN crawling on a surface and by right angle light scattering (RALS) in suspended PMN. However, sustained RALS oscillations in suspended PMN requires pre-treatment with an inhibitor of phosphatidylinositol 3-kinase or an activator of protein kinase C. Here, we show that cross-linking of the beta(2) (CD18) or beta(3) (CD61), but not beta(1) (CD 29) integrins in the presence of a low dose of formyl-Methionyl-Leucyl-Phenylalanine (fMLP) enables similar 8-s periodic RALS oscillations in suspended PMN in response to stimulation with two consecutive doses of chemoattractants. This effect did not appear to be due to increased surface expression of CD18 or CD61. RALS oscillations occurred in phase with 8-s oscillations in the stable F-actin pool and peaks in F-actin correlated with predominance of cells exhibiting a nascent lamella. Thus, simulation of surface attachment by CD18 and CD61 cross-linking after exposure to fMLP in suspended cells supports shape oscillations that are the result of actin-driven cyclic extension/retraction of nascent lamellae at the same frequency as the shape changes previously observed in crawling PMN.

Function of the cytoskeleton in human neutrophils and methods for evaluation.

The cytoskeleton plays a critical role in the determination of cell shape and serves as a scaffold for critical cellular enzymes and adhesion molecules. It provides structural integrity for the cell and regulates the function of many biochemical events that are critical to cellular function. The microfilamentous cytoskeleton participates in force generation necessary for shape change and motion. In neutrophils and other motile cells, polymerization of actin likely drives extension of the lamellae and participates in force generation through interaction with myosin, by polymerization alone and by osmotic mechanisms. Here, we will focus on the microfilamentous cytoskeleton in the neutrophil and briefly review its function as well as some direct and indirect methods that have been used to asses its role in neutrophil function. The discussion will address general approaches and leaves the details of the methods to the references.

An in vitro model of human red blood cell production from hematopoietic progenitor cells.

Hemoglobinopathies, such as beta-thalassemias and sickle cell anemia (SCA), are among the most common inherited gene defects. Novel models of human erythropoiesis that result in terminally differentiated red blood cells (RBCs) would be able to address the pathophysiological abnormalities in erythrocytes in congenital RBC disorders and to test the potential of reversing these problems by gene therapy. We have developed an in vitro model of production of human RBCs from normal CD34(+) hematopoietic progenitor cells, using recombinant growth factors to promote terminal RBC differentiation. Enucleated RBCs were then isolated to a pure population by flow cytometry in sufficient numbers for physiological studies. Morphologically, the RBCs derived in vitro ranged from early polylobulated forms, resembling normal reticulocytes to smooth biconcave discocytes. The hemoglobin pattern in the in vitro-derived RBCs mimicked the in vivo adult or postnatal pattern of beta-globin production, with negligible gamma-globin synthesis. To test the gene therapy potential using this model, CD34(+) cells were genetically marked with a retroviral vector carrying a cell-surface reporter. Gene transfer into CD34(+) cells followed by erythroid differentiation resulted in expression of the marker gene on the surface of the enucleated RBC progeny. This model of human erythropoiesis will allow studies on pathophysiology of congenital RBC disorders and test effective therapeutic strategies.

Sickle erythrocytes adhere to polymorphonuclear neutrophils and activate the neutrophil respiratory burst.

The vasoocclusive process in patients with sickle cell disease (SCD) is complex and involves interactions among sickle erythrocytes (SS-RBC), vascular endothelium, and plasma and cellular components. The role of neutrophils (PMN) in vasoocclusion has not been examined. Patients with SCD appear to have chronically activated PMN. Because the first step in PMN activation is particle recognition, we explored whether normal PMN recognize SS-RBC and whether this recognition results in PMN monolayers, significantly more SS-RBC adhered to the PMN than did normal erythrocytes (AA-RBC; P < .001). Preincubation of erythrocytes with autologous plasma significantly increased the adherence of SS-RBC to PMN but had no effect on AA-RBC (P < .001). When adhesion of density fractionated SS-RBC was performed, dense SS-RBC showed greater adherence to the PMN monolayers than did light SS-RBC (P < .001). To determine mechanisms of this adhesion, IgG and Arg-Gly-Asp-Ser (RGDS) receptor sites on PMN were saturated. IgG inhibited adherence of dense SS-RBC, whereas RGDS inhibited adherence in both fractions, although to a greater extent in the light fraction. We measured SS-RBC activation of PMN by incubating SS-RBC with 2', 7'-Dichloro-fluroescin Diacetate (DCF)-labeled PMN. Incubation of PMN with SS-RBC resulted in a significant increase in fluorescence compared to AA-RBC. We show here that PMN recognize SS-RBC through multiple mechanisms and that this recognition results in activation of PMN. These findings contribute to the understanding of vasoocclusive crisis in patients with SCD and may have therapeutic implications.

Shape oscillations of human neutrophil leukocytes: characterization and relationship to cell motility.

When neutrophil leukocytes are stimulated by chemotactic factors or by substratum contact, they change their shape. Shape changes are a prerequisite for cellular migration and typically involve the extrusion of thin, veil-like lamellipods and the development of morphological polarity. Stimulation also leads to changes in the neutrophil content of filamentous actin (F-actin), which is the major cytoskeletal component. Suspensions of human neutrophils stimulated with chemoattractants exhibit sinusoidal light-scattering oscillations with a period of approximately 8 s at 37 degrees C. These oscillations arise from periodic fluctuations in the cell body size caused by lamellipod extension and retraction cycles. The light-scattering oscillations are paralleled by corresponding oscillations in F-actin content. This raises the interesting possibility that cyclic actin polymerization constitutes the driving force for shape oscillations of suspended neutrophils. Similar periodic shape changes are present in neutrophils crawling on a surface, suggesting that shape oscillations are important for neutrophil motion. This review summarizes our present knowledge about shape oscillations in suspended and crawling neutrophils and discusses a possible role for these oscillations in neutrophil motility.

Behavioral aspects of neutrophil motility.

The ability to move is one of the most basic and critical functions of the neutrophil. The neutrophil changes its shape from round to highly polar and glides along the surface, pausing every 45 to 60 seconds to reestablish its direction. Iterations of this sequence of small-scale motion, or shape change, sum to form the large-scale trajectories that have fascinated many investigators over the years. Recent studies of neutrophil motion using novel stimuli and high-temporal resolution measurements of motion have unveiled extremely regular behaviors with periods of approximately 8 seconds. These and previous studies suggest that neutrophil shape change consists of high-frequency ruffling and lower-frequency development of morphologic polarity. These components of shape change are superimposed, because of separate cytoskeletal mechanisms, and are regulated differently. The fundamental motor of the neutrophil seems to be nonrandom and driven by two clocks, one with a highly regular period of 8 seconds and another with a period of 45 to 60 seconds.

ADP-ribosylation of Rho enhances actin polymerization-coupled shape oscillations in human neutrophils.

Stimulated neutrophils exhibit coordinated sinusoidal oscillations in filamentous actin content and cellular shape. We investigated the effect of inhibition of the small G protein Rho on neutrophil actin polymerization, shape changes and oscillations using a genetically engineered toxin that enters cells and selectively ADP-ribosylates endogenous Rho. This treatment increased the amplitudes and frequencies of shape oscillations and duration of the oscillating transient. However, it had no effect on the initial actin polymerization and shape changes induced by N-formyl-Met-Leu-Phe. Regulation of these oscillations may be important for the control of neutrophil motility.

Shape oscillations: a fundamental response of human neutrophils stimulated by chemotactic peptides?

Neutrophils undergo periodic cytoskeletal rearrangements that lead to cycles of shape change, ultimately resulting in cell translocation. Repeated stimulation of resting neutrophils with subsaturating chemoattractant doses induced transient sinusoidal oscillations in neutrophil filamentous actin content at the second and subsequent stimulations. Oscillation frequencies increased with increasing concentration of the first stimulus. In contrast, neutrophils pretreated with the phosphatidylinositol 3-kinase inhibitor (17-hydroxy)wortmannin displayed shape oscillations with the first stimulation, and the frequencies were independent of agonist type and dose. We demonstrate that oscillations in filamentous actin, which may be critical for neutrophil motility, can be induced in untreated cells by natural peptide chemoattractants.

The fundamental motor of the human neutrophil is not random: evidence for local non-Markov movement in neutrophils.

The search for a fundamental mechano-chemical process that results in net cell motion has led investigators to fit neutrophil tracking data to well described physical models in hopes of understanding the functional form of the driving force. The Ornstein-Uhlenbeck (OU) equation for mean square displacement describes a locally persistent and globally random process and is often used as a starting point for analysis of neutrophil displacements. Based upon the apparently close fit of neutrophil tracking data to this equation and the nature of its derivation, biologists have inferred that the motor of the neutrophil is best represented as a random process. However, 24 of 37 neutrophil paths that we investigated preferentially display programmatic rather than Markov short term correlations between displacements or turn angles. These correlations reflect a bimodal rather than a uniform distribution of subpath correlations in the two variables, and are strongly sampling rate-dependent. Significant periodic components of neutrophil shape change are also detected at the same time scale using either Fourier or elliptical Fourier transform-based descriptors of the neutrophil perimeter. Oscillations in neutrophil velocity have the same period. Taken together, these data suggest a nonstochastic, and perhaps periodic, component to the process driving neutrophil movement.

Mechanism of cigarette smoke condensate induced adhesion of human monocytes to cultured endothelial cells.

Cigarette smoking is ranked among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanisms, however, that link cigarette smoking to increased incidence of atherosclerosis are not understood. The adherence of circulating monocytes to the endothelium, migration into the subendothelium, and subsequent formation of foam cells are principal initial events in the development of atherosclerosis. We therefore determined whether cigarette smoke caused increased adherence of monocytes to endothelial cells and the cellular mechanism of this increased adherence. Cigarette smoke condensate (CSC), the particulate fraction of cigarette smoke derived from 2R1 standard research cigarettes, at a concentration of 25-30 micrograms/ml (average yield of CSC is 26.1 mg/cigarette), augmented (70-90%) basal adherence of human peripheral blood monocytes to a cultured monolayer of endothelial cells derived from bovine aorta (BAEC) and human umbilical vein (HUVEC). There was a concomitant increase in the expression of CD11b ligand on the surface of monocytes as determined by flow cytometry, utilizing FITC conjugated Mab MO-1 (CD11b). However, nicotine (1-15 micrograms/ml) and cadmium sulfate (10 micrograms/ml), constituents of CSC, individually or in combination had no effect either on CD11b expression or adherence of monocytes to endothelial cells. Treatment of HUVEC with CSC for 60 min also resulted in an increased expression of ICAM-1 and ELAM-1 as determined by mean fluorescence intensity of ICAM-1 and ELAM-1 labeled cells in flow cytometric analysis. The CSC induced expression of CD11b in monocytes was optimal at 25-30 min and was inhibited by protein kinase C inhibitors, staurosporine and H-7, and also by baicalein, a lipoxygenase inhibitor. Similarly, CSC induced ICAM-1 and ELAM-1 expression in HUVEC was inhibited by protein kinase C inhibitors. CSC stimulated the adherence of human monocytes but not the monocytic cell lines HL-60, U937, and THP-1 to endothelial cells. The CSC stimulated adherence of human monocytes was inhibited (80%) by MAb to CD11b and 50% by Mab to ICAM-1 and ELAM-1. These results suggest that cigarette smoke particulate constituents activate protein kinase C, leading to increased surface expression of adhesive ligand CD11b on peripheral blood monocytes and counter receptor(s) ICAM-1 and ELAM-1 in endothelial cells. The expression of ligand and counter receptor leads to potentiated adherence of monocytes to endothelial cells, an initial event in the pathogenesis of cigarette smoke induced inflammatory response in the vessel wall.

Development of a shape vector that identifies critical forms assumed by human polymorphonuclear neutrophils during chemotaxis.

Human polymorphonuclear neutrophils undergo characteristic shape changes that are critical to their ability to move and ingest their targets. We present here the construction of a simple shape vector, calculated from the coordinates of the cell perimeter, that can identify critical forms that a neutrophil assumes during the course of ameboid movement. The vector can be used to find neutrophils of a specific shape from the image analyzer data produced during a typical neutrophil tracking experiment.

Comparison of buffy coat preparation to direct method for the evaluation and interpretation of bone marrow aspirates.

Bone marrow differential and French, American, British (FAB) classification of buffy coat preparation (BCP) was compared to direct method (DM) in 69 pediatric patients with various hematologic and oncologic disorders. The marrow evaluation differed significantly in 12 of 69 patients (17.4%). The differential counts were discordant in 9 out of 69 patients (13%), and the FAB classifications were discordant in 3 out of 25 patients (12%) with acute lymphoblastic leukemia (ALL). Underestimation of the percent blasts occurred with buffy coat preparation in patients with acute non-lymphoblastic leukemia (ANLL). While buffy coat preparations can facilitate morphologic evaluation in marrow malignancies, significant errors can occur in determination of the differential count. Direct smear should be used in conjunction with buffy coat smears in the evaluation of bone marrow aspirates.

Analysis of multi-parameter video measurements of human neutrophil movement and its relation to cell shape and cytosolic calcium.

We have developed a software system for the analysis of multiparameter video image data derived from cell tracking experiments. The software is part of an integrated system for the study of human neutrophil motility. Input is taken from a file produced by a separate tracking program. The present software calculates several analytic parameters related to movement as well as novel relations between the distribution of fluorescent probes within the cell and vectors which represent cell movement. The software is designed to facilitate data analysis on several platforms by taking input and producing output as ASCII files.

An integrated system for quantitation of chemotaxis using a 48-well millipore filter assay.

We have established a computerized system for quantification of human neutrophil motility using a 48-well chemotaxis assay. The software is primarily written in the Unix C-shell and is designed to integrate with standard statistical and graphics packages and permit analysis either under Unix or MS-DOS. We demonstrate how simple image analysis techniques may be used to count neutrophils that have traversed a polycarbonate filter. Methods of optical optimization, cell counting and integration with the Statistical Analysis System (SAS) are presented.