Association of heart rate variability with occupational and environmental exposure to particulate air pollution.

BACKGROUND: Airborne particulate matter has been linked to excess morbidity and mortality. Recent attention has focused on the effects of particulate exposure on cardiac autonomic control. Inhaled particulates may affect the autonomic nervous system either directly, by eliciting a sympathetic stress response, or indirectly, through inflammatory cytokines produced in the lungs and released into the circulation. METHODS AND RESULTS: This longitudinal study examined the association of particulates

Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity.

FKBP ligand homodimers can be used to activate signaling events inside cells and animals that have been engineered to express fusions between appropriate signaling domains and FKBP. However, use of these dimerizers in vivo is potentially limited by ligand binding to endogenous FKBP. We have designed ligands that bind specifically to a mutated FKBP over the wild-type protein by remodeling an FKBP-ligand interface to introduce a specificity binding pocket. A compound bearing an ethyl substituent in place of a carbonyl group exhibited sub-nanomolar affinity and 1,000-fold selectivity for a mutant FKBP with a compensating truncation of a phenylalanine residue. Structural and functional analysis of the new pocket showed that recognition is surprisingly relaxed, with the modified ligand only partially filling the engineered cavity. We incorporated the specificity pocket into a fusion protein containing FKBP and the intracellular domain of the Fas receptor. Cells expressing this modified chimeric protein potently underwent apoptosis in response to AP1903, a homodimer of the modified ligand, both in culture and when implanted into mice. Remodeled dimerizers such as AP1903 are ideal reagents for controlling the activities of cells that have been modified by gene therapy procedures, without interference from endogenous FKBP.

Pharmacologic control of a humanized gene therapy system implanted into nude mice.

Systemic delivery of specific therapeutic proteins by a parenteral route of administration is a recognized practice in the management of several gene defects and acquired diseases. As an alternative to repetitive parenteral administration, gene therapy may provide a novel means for systemic delivery of therapeutic proteins while improving patient compliance and therapeutic efficacy. However, for gene therapy to be an efficacious and safe approach to the clinical management of such diseases, gene expression must be tightly regulated. These investigations demonstrate precise in vivo control of protein expression from cells that are engineered to secrete human growth hormone (hGH) in response to stimulation by rapamycin. The cells were implanted intramuscularly into nu/nu mice and stimulated by intravenous or oral administration of rapamycin. In vivo experiments demonstrate that the activity and pharmacokinetics of rapamycin determine the level of serum hGH that result from the engineered cells. In addition, responsiveness of the cells to rapamycin, number of cells implanted, hGH expression kinetics, and the pharmacokinetics of hGH itself, also influence the circulating levels of hGH after rapamycin stimulation. Controlled manipulation of several of these parameters, either independently or in combination, allows for precise regulation of circulating hGH concentration in vivo.

A humanized system for pharmacologic control of gene expression.

Gene therapy was originally conceived as a medical intervention to replace or correct defective genes in patients with inherited disorders. However, it may have much broader potential as an alternative delivery platform for protein therapeutics, such as cytokines, hormones, antibodies and novel engineered proteins. One key technical barrier to the widespread implementation of this form of therapy is the need for precise control over the level of protein production. A suitable system for pharmacologic control of therapeutic gene expression would permit precise titration of gene product dosage, intermittent or pulsatile treatment, and ready termination of therapy by withdrawal of the activating drug. We set out to design such a system with the following properties: (1) low baseline expression and high induction ratio; (2) positive control by an orally bioavailable small-molecule drug; (3) reduced potential for immune recognition through the exclusive use of human proteins; and (4) modularity to allow the independent optimization of each component using the tools of protein engineering. We report here the properties of this system and demonstrate its use to control circulating levels of human growth hormone in mice implanted with engineered human cells.

Mini-review: Mechanical factors affecting cartilage regeneration in vitro.

In the last 5 to 10 years, tissue engineering has revolutionized the way in which medical researchers and clinicians are thinking of and, in some cases, actually treating diseases involving tissue damage and destruction. One such disease, osteoarthritis, results from progressive degeneration of articular cartilage, which has a limited ability to repair itself. With tissue engineering, scientists are now able to regenerate cartilage in vitro from isolated mature chondrocytes. While the regeneration process is still not fully understood, enough has been learned that physicians are already implanting cultured chondrocytes into humans and other animals in the hopes of effecting joint repair. One aspect which has not been fully explored is the effect of mechanical stress on developing and implanted cartilage, especially over the long term. This article will review in brief what is now known about the mechanical factors affecting cartilage regeneration in vitro and what still remains to be determined for optimum tissue engineering of cartilage constructs. (c) 1996 John Wiley & Sons, Inc.

Distribution and possible origins of substance P-containing nerve fibers in the rat liver.

The distribution and possible origins of substance P-containing nerve fibers in the rat liver were investigated by immunohistochemistry and nerve transection. Nerve fibers with substance P-like immunoreactivity formed a more complex network than previously known in the walls of portal vein branches. Substance P-immunoreactive fibers were seen not only in and around the walls of the hepatic artery, but also in close association with the hepatic veins and bile ducts. Transection of the greater splanchnic nerves and/or the vagus nerves indicated that substance P-immunoreactive fibers in the walls of the portal and hepatic veins enter the liver via both nerves, and that those associated with the hepatic artery and bile ducts stem from the greater splanchnic nerves. The widespread distribution of hepatic substance P and its complex innervation pattern within the liver suggest that it is involved in a variety of physiological processes in this organ.

Lymphatics and pre-lymphatics of the rabbit pericardium and epicardium with special emphasis on particulate absorption and milky spot-like structures.

The lymphatics and pre-lymphatic connective tissue of rabbit pericardium and epicardium were examined by light and electron microscopy under normal conditions and after the injection of India ink and latex particles into the pericardial cavity. A characteristic lattice structure of connective tissue was present between the small mesothelial cells and the submesothelial lymphatic capillaries in the basal region of the pericardium, but not in the epicardium. Milky spot-like structures bulging toward the pericardial cavity were found in the pericardium, similar to those in the omentum and mediastinal pleura. Within 60 minutes after injection, carbon and latex particles were directly absorbed through the intercellular clefts of the adjacent small mesothelial cells into the submesothelial layer particularly at sites of characteristic lattice structure. Carbon particles were already present in the lumens of lymphatic capillaries at this time. Macrophages in the pericardial cavity and submesothelial layers of the pericardium engulfed both carbon and latex particles. Our results suggest two possible routes of drainage of particulate matter from the pericardial cavity into the lymphatics: direct absorption and indirect absorption after phagocytosis by macrophages. Macrophages probably migrate from the milky spot-like structures described in this study. Epicardial lymphatics, in contrast, drain tissue fluid primarily from the myocardium.

Morphologic changes in detrusor muscles of patients with chronic obstruction of lower urinary tract. Electronmicroscopic and immunohistochemical findings.

The anterior bladder wall and actin- and myosin-like immunoreactivities within the detrusor muscles in patients with chronic obstruction of the lower urinary tract were examined by means of a MOP Videoplan image-processing system, electronmicroscopy, and light microscopic immunohistochemistry. The image-processing system demonstrated an excess of connective tissue elements between smooth muscle bundles in the anterior wall of the bladder similar to the results of previous studies dealing with the trabeculated posterior wall. Under electronmicroscopy, myofilaments were shown to be multidirectionally arranged in the smooth muscle cells in contrast to the regular arrangement in controls. Dense areas in the cytoplasm of the detrusor muscle also appeared to be abnormally distorted and/or elongated in the electronmicrographs. In support of these findings, actin- and myosin-like immunoreactivities in the muscle layer of the bladder were significantly less intense than in the controls. These results suggest that chronic obstruction of the lower urinary tract causes histopathologic alterations in both the intervening connective tissue and the detrusor muscle. This study raises the possibility that the aforementioned morphologic abnormalities are involved in the occurrence of uninhibited detrusor contraction and abnormal detrusor reflex in patients with lower urinary tract obstruction.

Immunoelectron microscopy of cell populations in regional and central lymph of sheep.

The immunoreactivity and the ultrastructural localization of monoclonal anti-sheep lymphocyte antibodies conjugated with colloidal gold particles were examined in free-floating cells of sheep central lymph from the thoracic duct, postnodal lymph draining either the popliteal nodes or the mesenteric nodes, and prenodal lymph draining the pregnant uterus. The monoclonal antibodies used in this study were SBU-T1 (CD5), SBU-T4 (CD4), SBU-T8 (CD8), SBU-II (anti DR antibody), and E53 which are reported to be sheep homologues of human T1, T4, T8, HLA-DR, and pan B cell antibodies, respectively. Colloidal gold particles were evenly distributed or segmentally aggregated on the surfaces of lymphocytes and macrophages incubated with monoclonal antibodies and in vesicles in the cytoplasm of anti DR antibody labeled macrophages. Not only did CD5 labeled cells show a high percentage in each regional lymph examined, but the percentage of CD4 labeled cells was consistently higher than that of CD8 labeled cells. Moreover, the immunoreactivity of CD8 labeled cells was specific among lymph from the different regions. The sum of the percentages of CD4 and CD8 labeled cells was less than the percentage of CD5 labeled cells, indicating the presence of a minor T cell subpopulation which was CD5+, CD4-, and CD8-. A characteristic finding was a high percentage of CD8 labeled cells and many abnormal eosinophils in uterine prenodal lymph in pregnant sheep. Taken together the results showed that variously labeled immunoreactive cells are distributed somewhat differently in lymph derived from different organ sites.

Fine structure of substance P-containing nerve fibers in the rat lateral septum; simultaneous localization in pre- and postsynaptic elements.

The fine structure of nerve fibers with substance P (SP)-like immunoreactivity in the rat lateral septum (LS) was investigated by preembedding immunoelectron microscopy. SP axon terminals frequently made synapses with non-immunoreactive neuronal soma and dendrites in the LS. Occasionally, two closely apposed nerve endings with SP immunoreactivity were presynaptic to the soma. A small number of immunopositive axon terminals formed synapses not only with neuronal perikarya but also with small dendrites in the vicinity of the perikarya. There were also some SP dendrites in contact with immunoreactive as well as non-immunoreactive nerve endings. These findings may provide a morphological basis for the complexity of SP actions on septal neurons.

Distribution of peptidergic nerve fibers in rat bronchus-associated lymphoid tissue: light microscopic observations.

The localization of neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) in the nerve fibers of rat bronchus-associated lymphoid tissue (BALT) was investigated by light microscopic immunohistochemistry. Nerve fiber bundles revealing NPY-like immunoreactivity were shown to enter the BALT together with pulmonary artery branches. They frequently reached the central zone of the BALT to give rise to fine, tortuous fibers. On the other hand, nerve fibers immunoreactive for SP and CGRP seemed to distribute in the subepithelial zone of the BALT after dissociating from fiber networks in the walls of bronchi, although small numbers of SP and CGRP fibers were also seen in the BALT central zone. CGRP fibers formed a more intense network than SP fibers in the BALT. Scattered VIP fibers were found only in the subepithelial zone of the BALT. These findings not only suggest that the four kinds of peptidergic fibers act on BALT in multiple ways, but also that these neuropeptides may be involved in the control of mucosal immunity, lymphocyte migration and proliferation within the BALT.

Lymphatics, intraepithelial lymphocytes and endometrial lymphoid tissues in the rabbit uterus: an electron microscopic and immunohistological study.

Rabbit uterine intraepithelial lymphocytes, endometrial lymphoid aggregates and lymphatic capillaries were examined electron-microscopically and immunohistochemically at well-defined intervals after the injection of human chorionic gonadotropin (hCG). Lymphatic capillaries originated near the bases of the glands in the uterine cervix and in the border zone between the lamina propria mucosae and myometrium in the uterine body. The lymphatic capillaries were maximally dilated, and their endothelial cells were thinnest 8 hours after hCG injection. Patent junctions between the adjacent endothelial cells of lymphatics in the uterine body were observed in good accordance with the appearance of stromal edema and lymphatic dilatation. The numbers of intraepithelial lymphocytes and macrophages changed cyclically after the induction of ovulation. They were highest 11 hours after hCG injection when lymphocytes were seen occasionally in the lumens of lymphatics located in the lamina propria mucosae of the uterine body. Most of the intraepithelial and interstitial lymphocytes were cells labeled with T cell serum but some were labeled with IgA serum and were occasionally seen beneath the epithelium. Lymphoid aggregates were uniformly present in the stratum basalis and consisted of lymphocytes and macrophages. They had no germinal centers, surrounding lymphatics or high endothelial venules (HEV). The results suggest that lymphatic capillaries are the main route for the removal of edema fluid and for migratory lymphoid cells in the rabbit uterus.

Immunogold-silver staining method for light and electron microscopic detection of lymphocyte cell surface antigens with monoclonal antibodies.

We used the immunogold-silver staining method (IGSS) for detection of lymphocyte cell surface antigens with monoclonal antibodies in light and electron microscopy and compared this procedure with the immunogold staining method. Two different sizes of colloidal gold particles (5 nm and 15 nm) were used in this study. Immunolabeling on cell surfaces was visualized as fine granules only by IGSS in light microscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold particles on cell surfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time of the physical developer but by the size of the gold particles. However, the development of shells of silver-enhanced gold particles correlated with the enhancement time of the physical developer rather than the size of the colloidal gold particles. Five-nm gold particles enhanced with the physical developer for 3 min were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure of identical positive cells detected in 1-micron sections counterstained with toluidine blue by electron microscopy, in addition to the percentage of positive cells by light microscopy.

Co-localization of arginine vasopressin- and enkephalin-like immunoreactivities in nerve cells of the rat hypothalamus.

The co-localization of arginine vasopressin- and enkephalin-like immunoreactivities in nerve cells of the rat paraventricular hypothalamic nucleus and adjacent areas was investigated by the simultaneous application of immuno-beta-galactosidase staining and the peroxidase-antiperoxidase method to sections. Arginine vasopressin-like immunoreactive cells were stained blue with immuno-beta-galactosidase staining and enkephalin-like immunoreactive cells brown with the peroxidase-antiperoxidase method. Double-labeled cells with overlap of blue and brown immunoreaction products were identified in the anterior, medial, and lateral parvocellular parts of the paraventricular hypothalamic nucleus as well as in the previously indicated posterior magnocellular part. Other regions that contained double-labeled cells were the lateral hypothalamic area, anterior hypothalamic nucleus, area between the lateral hypothalamic area and anterior hypothalamic nucleus, suprachiasmatic nucleus, and bed nucleus of the stria terminalis, medial division, posterolateral part. These findings suggest that nerve cells with both arginine vasopressin- and enkephalin-like immunoreactivities may be more actively involved in neuroendocrine regulation and neural transmission than previously considered. They may provide a morphological basis for an increase in enkephalin-like immunoreactivity within the anterior pituitary in cases of hemorrhagic shock which is presumably accompanied by arginine vasopressin hypersecretion.

Somatostatin co-localizes with tyrosine hydroxylase in the nerve cells of discrete hypothalamic regions in rats.

The co-expression of somatostatin (SOM)- and tyrosine hydroxylase (TH)-like immunoreactivities in nerve cells of the rat hypothalamus was investigated by the simultaneous application to the same sections of immuno-beta-galactosidase staining and the peroxidase-antiperoxidase (PAP) method. SOM-like immunoreactive cells stained blue with immuno-beta-galactosidase staining and TH-like immunoreactive cells stained brown with the PAP method. Double-labeled cells with overlapping blue and brown immunoreaction products were frequently identified in the preoptic periventricular nucleus (pope). These double-labeled cells were seen in clusters within the ventral half of the rostral pope. The periventricular hypothalamic nucleus at the level of the anterior hypothalamic nucleus contained only scattered nerve cells with both SOM- and TH-like immunoreactivities, despite the presence of many nerve cells immunoreactive for either SOM or TH in this nucleus. Double-labeled cells were also observed in some regions of the medial-basal hypothalamus, including the boundary between the ventromedial hypothalamic nucleus and the arcuate hypothalamic nucleus, and areas dorsal and lateral to the ventromedial hypothalamic nucleus. These findings may provide insight into the mechanisms underlying previously described catecholamine-mediated modulation of SOM release from the hypothalamus.

Immunoelectron-microscopic localization of peptidergic nerve fibers around lymphatic capillaries in the rat liver.

The localization of neuropeptide Y (NPY)-, substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing nerve fibers around lymphatic capillaries (initial lymphatics) in the interlobular connective tissue of the rat liver was investigated by preembedding immunoelectron-microscopy. Nerve terminals with NPY were frequently seen in close apposition to the abluminal surface of lymphatic endothelium. A small number of NPY fibers without a glial (Schwann cell) covering at the tip ran toward lymphatic capillaries in the interlobular connective tissue. Nerve fibers immunoreactive for SP were present within unmyelinated fiber bundles that ran close to lymphatic capillaries in the interlobular connective tissue. Besides these immunoreactive nerve fibers, many of which appeared to pass through the subendothelial regions of lymphatic capillaries, scattered SP nerve endings were seen in areas contiguous to lymphatic endothelium. CGRP terminals were rarely found around lymphatic capillaries, although nerve fiber bundles containing CGRP components traversed close to some lymphatic capillaries. These findings suggest that NPY and SP, if released from nerve terminals into the subendothelial areas of adjacent lymphatic capillaries, are more likely to affect the metabolic activity of lymphatic endothelium and the flow (or formation) of lymph than CGRP. SP and CGRP, as possible mediators of sensory transmission, might be involved in the conveyance of information on the hydrostatic pressures of hepatic lymphatics and surrounding tissue fluid to the central nervous system.

Immunohistochemical differentiation between lymphatic vessels and blood vessels--use of anti-basement membrane antibodies and anti-factor VIII-related antigen.

Several immunohistochemical methods using Factor VIII-Related antigen (FVIIIR:Ag), laminin, Type IV collagen and fibronectin antisera were applied for the purpose of differentiating rat lymphatics from blood vessels by light and electron microscopy. Weibel-Palade bodies (WPB) were demonstrated in both types of vessels by conventional electron microscopy. The immunoreactivity to laminin and Type IV collagen in blood vessels showed a strong, continuous, linear subendothelial staining pattern in contrast to lymphatic vessels in which immunoreactivity was absent or weak in paraffin-embedded sections stained with the indirect immunoperoxidase technique. A positive reaction for fibronectin was observed in all extra-vascular tissue spaces as well as in lymphatics and blood vessels. FVIIIR:Ag and WPB were present in both lymphatic and blood endothelial cells. FVIIIR:Ag antiserum labeled with gold particles was observed only in the vacuoles which were assumed to be identical with WPB as demonstrated by our conventional electron microscopy. We conclude that the immunohistochemical method using laminin and Type IV collagen antisera is a reliable and practical way to differentiate lymphatic vessels from blood vessels by light microscopy.

Lymphatics and lymphoid tissue of the fallopian tube: immunoelectronmicroscopic study.

Lymphoid tissue of the human fallopian tube consists of follicles, lymphoepithelium, and lymphatic and blood capillaries and is located consistently in the interstitial part of the human fallopian tube. Using an immunoelectronmicroscopic technique, we have elucidated the ultrastructure of the lymphoid tissue of the human fallopian tube and the fine distribution and ultrastructure of the lymphatics associated with the rabbit fallopian tube. Lymphatic capillaries arise in the lamina propria mucosa and the periphery of follicles, where they are sparsely distributed, run through the muscular layer, and form a dense network in the subserosa. Characteristic features of the ultrastructure are aggregations of smooth muscle cells, alternating areas of densely and sparsely distributed collagen fibers, and unmyelinated nerve fibers beneath the lymphatic endothelium. Immunoelectronmicroscopic analysis has demonstrated an obvious difference in the distribution of T- and B-lymphocytes in the lymphoid tissue of the human fallopian tube. Many T-lymphocytes are present in the follicles and epithelium, but B-lymphocytes are either absent or rarely found. T-lymphocytes sometimes infiltrate into the basal lamina of the epithelium lying in close contact with the follicles. We conclude that the lymphoid tissue is constantly located in the interstitial part of the human fallopian tube and that intraepithelial lymphocytes, mainly T-lymphocytes, migrate via the basal lamina of the epithelium from follicles. Lymphatic capillaries in the fallopian tube may be the main migratory route of intraepithelial lymphocytes. The intraepithelial lymphocytes and epithelial cells of the fallopian tube have attracted considerable interest as a result of immunological studies of the recognition of spermatozoal antigens and the fertilized ovum.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphology of arachnoid granulations and villi in the region of the human sella turcica--light microscopy and three-dimensional image analysis.

The distribution and structure of the arachnoid granulations and villi in the region of the sella turcica in human adult brains were observed under light microscopy. In order to study the interrelationships between the arachnoid projections, hypophysis, cavernous sinus, and sella turcica, we performed a three-dimensional reconstruction and analysis of the region with the use of a digital image-processing method. Arachnoid projections penetrating the dura were villous when they were few in number, but more frequently granular when they were numerous. No relationship was observed between the type of projections and the age or the underlying disease of the subjects. Brains which showed no or few arachnoid granulations or villi had a small and deep hypophyseal fossa with a poorly-developed intercavernous sinus or venous plexus. Three-dimensional image analysis revealed that arachnoid granulations and villi form a maze in the dura or connective tissue between the venous plexuses.