Inhibition of leukotriene B4-receptor interaction suppresses eosinophil infiltration and disease pathology in a murine model of experimental allergic encephalomyelitis.

Leukotriene B4 (LTB4) is a chemotactic and cell-activating factor present at inflammatory sites in a variety of autoimmune diseases including multiple sclerosis (MS). In this study, we used a murine model of MS, experimental allergic encephalomyelitis (EAE), to assess the potential role of LTB4 on cell infiltration and paralysis. Injection of encephalogenic T cells into naive animals induced paralysis and weight loss that was completely inhibited by treatment with the selective LTB4 receptor antagonist CP-105,696 (ED50= 8.6 mg/kg orally). Although migration of lymphocytes into the central nervous system was unaffected, the efficacious effects of CP-105,696 correlated with up to a 97% decrease in eosinophil infiltration into the lower spinal cord as determined by light and electron microscopy and quantitated by levels of the specific enzyme marker eosinophil peroxidase. These results demonstrate that eosinophil recruitment in EAE is dependent on LTB4 receptor ligation and further reveal a previously unrecognized role for eosinophils in the pathogenesis of this disease.

Collagen-induced arthritis is reduced in 5-lipoxygenase-activating protein-deficient mice.

Collagen-induced arthritis in the DBA/1 mouse is an experimental model of human rheumatoid arthritis. To examine the role of leukotrienes in the pathogenesis of this disease, we have developed embryonic stem (ES) cells from this mouse strain. Here, we report that DBA/1 mice made deficient in 5-lipoxygenase-activating protein (FLAP) by gene targeting in ES cells develop and grow normally. Zymosan-stimulated leukotriene production in the peritoneal cavity of these mice is undetectable, whereas they produce substantial amounts of prostaglandins. The inflammatory response to zymosan is reduced in FLAP-deficient mice. The severity of collagen-induced arthritis in the FLAP-deficient mice was substantially reduced when compared with wild-type or heterozygous animals. This was not due to an immunosuppressive effect, because anti-collagen antibody levels were similar in wild-type and FLAP-deficient mice. These data demonstrate that leukotrienes play an essential role in both the acute and chronic inflammatory response in mice.

Transcytosis of albumin in capillary endothelium.

We have used a variety of immunocytochemical procedures to localize albumin in transit through the capillary endothelium of the murine myocardium and thereby identify endothelial cell structures involved in albumin efflux. The most informative results were obtained with a protocol that included (a) removal of endogenous albumin by perfusion of the heart with PBS supplemented with 14 mM glucose, (b) perfusion of the heart vasculature with exogenous (bovine) albumin for various short time periods, (c) fixation of the vessels by formaldehyde-glutaraldehyde mixtures, (d) processing of fixed myocardium specimens through L. R. White embedding followed by sectioning, or direct thin frozen sectioning, and (e) reacting the surface of such specimens with antialbumin antibodies followed by gold-labeled secondary antibodies. The results indicate that (a) monomeric albumin binds (with low affinity) to the luminal surface of the capillary endothelium, (b) it is restricted in transit through the endothelium to plasmalemmal vesicles, and (c) it appears in the pericapillary spaces less than 15 s after the beginning of its perfusion. No albumin concentration gradients, centered with their maxima on the exits from intercellular spaces, were detected at any time points, including the shortest ones (15 and 30 s) investigated. Additional information comparing monomeric vs. polymeric albumin transcytosis was obtained using albumin-gold complexes. The results are discussed in terms of vesicular transport of albumin across the endothelium and the relations of this type of transport to the postulated pore systems of the physiological literature.

Long-term treatment with eteplirsen in nonambulatory patients with Duchenne muscular dystrophy.

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28. All 12 patients continued onto open-label extension study 202 and received long-term treatment with eteplirsen. We compared cardiac, pulmonary, and upper limb function and dystrophin production in the nonambulatory twin patients versus the 10 ambulatory patients through 240 combined treatment weeks.Ten study patients remained ambulatory through both studies, while the identical twin patients both experienced early, rapid loss of ambulation. The twin patients had greater disease severity at baseline (6-minute walk test [6MWT], 330 and 256 m) versus the other patients (n = 10; 6MWT range, 341-418 m). They maintained cardiac and upper limb function through combined week 240, with outcomes similar to those of the patients who remained ambulatory. Dystrophin production was confirmed following eteplirsen treatment.Despite the loss of ambulation, other markers of disease progression remained relatively stable in the eteplirsen-treated twin patients and were similar to those of the ambulatory patients.

Glycemic improvement in diabetic db/db mice by overexpression of the human insulin-regulatable glucose transporter (GLUT4).

The effects of increased GLUT4 (insulin-regulatable muscle/fat glucose transporter) expression on glucose homeostasis in a genetic model of non-insulin-dependent diabetes mellitus were determined by expressing a human GLUT4 transgene (hGLUT4) in diabetic C57BL/KsJ-db/db mice. A genomic hGLUT4 construct was microinjected directly into pronuclear murine embryos of db/+ matings to maintain the inbred background. Four lines of hGLUT4 transgenic mice were bred to homozygosity at the db locus and all showed a marked reduction of both fasted and fed plasma glucose levels (to approximately 50 and 360 mg/dl, respectively) compared with age-matched nontransgenic db/db mice (approximately 215 and 550 mg/dl, respectively), as well as an enhanced disposal of an oral glucose challenge. In situ immunocytochemical localization of GLUT4 protein in muscle from hGLUT4 db/db mice showed elevated plasma membrane-associated GLUT4 protein in the basal state, which markedly increased after an insulin/glucose injection. In contrast, nontransgenic db/db mice had low levels of plasma membrane-associated GLUT4 protein in the basal state with a relatively small increase after an insulin/glucose challenge. Since the intracellular GLUT4 levels in db/db mice were similar to nontransgenic db/+ mice, the glucose transport defect in db/db mice is at the level of glucose transporter translocation. Together, these data demonstrate that GLUT4 upregulation overcomes the glucose transporter translocation defect and alleviates insulin resistance in genetically diabetic mice, thus resulting in markedly improved glycemic control.

Differential binding of the lectins Griffonia simplicifolia I and Lycopersicon esculentum to microvascular endothelium: organ-specific localization and partial glycoprotein characterization.

The lectins Griffonia simplicifolia I and Lycopersicon esculentum were used to assess the presence of endothelium-specific glycoproteins in the microvasculature of the rat myocardium, diaphragm and superficial cerebral cortex. Organs fixed by intravascular perfusion were processed to obtain semithin (0.5 micron) and thin (less than 0.1 micron) frozen sections that were reacted with biotinylated lectin followed by streptavidin conjugated to Texas Red, for semithin sections, or by streptavidin conjugated to 5-nm colloidal gold particles, for thin sections. Lycopersicon esculentum lectin exclusively labeled the endothelium of all small vessels in all three microvascular beds; it did not bind to components of either the parenchyma or the extracellular matrix. Griffonia simplicifolia I lectin exclusively labeled the endothelium of the entire microvasculature in the myocardium and diaphragm, but marked primarily pericytes in the cerebral microvasculature. It did not label any parenchymal or interstitial organ component. At the electron microscope level, the lectin Griffonia simplicifolia I labeling was associated with the plasmalemma proper and especially with plasmalemmal vesicles and their introits, and Lycopersicon esculentum lectin bound primarily to the luminal plasmalemma in the microvascular beds of the myocardium and diaphragm. In the cerebral cortex, labeling of the microvasculature was clearly different: Griffonia simplicifolia I bound primarily to pericytes and vascular smooth muscle cells whereas Lycopersicon esculentum labeled only the microvascular endothelium. Lysates prepared from the myocardium, diaphragm and cerebral cortex were processed through Griffonia simplicifolia I lectin affinity separation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fraction obtained. A number of putative endothelium-specific glycoproteins was detected and found to differ qualitatively and quantitatively from organ to organ. The most prominent polypeptide, approximately 97 kDa, was present in substantial amounts in the myocardium and diaphragm, but in considerably lower concentration in the cerebral cortex. The reverse applied for a approximately 55 kDa protein. The preferential distribution of the approximately 97 kDa protein parallels differences in Griffonia simplicifolia I lectin binding by fluorescence and electron microscopy on sections of the corresponding organs. The results provide further evidence for the existence of endothelial glycoproteins specific for different microvascular beds and possibly connected with local functional differentiations.

Differential and specific labeling of epithelial and vascular endothelial cells of the rat lung by Lycopersicon esculentum and Griffonia simplicifolia I lectins.

In the rat lung, we found that the Lycopersicon esculentum (LEA) lectin specifically binds to the epithelium of bronchioles and alveoli whereas Griffonia simplicifolia I (GS-I) binds to the endothelium of alveolar capillaries. The differential binding affinity of these lectins was examined on semithin (approximately 0.5 microns) and thin (less than 0.1 (microns) frozen sections of rat lung lavaged to remove alveolar macrophages. On semithin frozen sections, LEA bound to epithelial cells lining bronchioles and the alveoli (type I, but not type II epithelial cells). On thin frozen sections, biotinylated Lycopersicon esculentum (bLEA)-streptavidin-gold conjugates were confined primarily to the luminal plasmalemma of type I cells. bGS-I-streptavidin-Texas Red was detected on the endothelial cells of alveolar capillaries and postcapillary venules but not on those of larger venules, veins or arterioles. By electron microscopy, GS-I-streptavidin-gold complexes were localized primarily to the luminal plasmalemma of thick and thin regions of the capillary endothelium. Neither lectin labeled type II alveolar cells, but both lectins labeled macrophages in the interstitia and in incompletely lavaged alveoli.

Detection of collagenase-induced damage of collagen by 9A4, a monoclonal C-terminal neoepitope antibody.

To determine whether the collagen network is compromised by collagenase during acute inflammation, a monoclonal antibody (9A4) was developed with specificity for the C-terminal neoepitope sequence generated by collagenase-cleavage of type II collagen (Gly-Pro-Pro-Gly-Pro-Gln-Gly-COOH). 9A4 was shown to detect the collagen collagenase-cleavage neoepitope with a K = 1.7 x 10(-7) M (type II) and K = 2 x 10(-6) M (type I). It does not recognize uncleaved native or denatured collagen. Articular cartilage from control animals is unstained by 9A4. During acute inflammation elicited in hamsters by intra-articular LPS, positive staining for the 9A4 neoepitope indicated the collagen was damaged. Wheel running exercise was used to apply stress to control cartilage and cartilage from animals with damaged collagen. After 6 months of running, the cartilage from normal animals was unaffected. By contrast, in the group with damaged collagen, the cartilage was fibrillated in all animals and in half of those, the cartilage failed and bony eburnation resulted.

A survey of the binding of polycationic ferritin in several fenestrated capillary beds: indication of heterogeneity in the luminal glycocalyx of fenestral diaphragms.

Polycationic ferritin (PCF) was injected into umbilical veins of 17- and 21-day-gestation rats and saphenous veins of young, male, adult Sprague-Dawley rats and allowed to circulate for 1 min or less followed by excision of tissues and immersion fixation. Fetal liver, intestine, and kidney, as well as adult liver, intestine, pancreas, kidney, adrenal cortex, bone marrow, and pituitary were examined. Nearly all fenestral diaphragms, but no subendothelial structures, were labeled with a tuft of particles in fetal intestine, adult intestine, pancreas, pituitary, and renal peritubular capillaries. A fraction of the diaphragms covering fenestrae in the thyroid, adrenal cortex, and fetal kidney glomerulus, but none in the bone marrow and fetal liver, had associated PCF. In these tissues some diaphragms appeared to be permeable to PCF, because tufts were observed immediately beneath unlabeled fenestral diaphragms either in the basal lamina (fetal kidney, adult thyroid, and adrenal cortex) or on other subendothelial structures (fetal liver, adult bone marrow). Also periodic concentrations of PCF were observed in the lamina rara interna of the adult glomerular basement membrane. PCF binding to the fenestral diaphragms and the basal lamina in the adult intestine and the renal glomerulus, respectively, have been reported by Simionescu et al. ((1981a) J. Cell. Biol. 90, 605-613) and Kanwar and Farquhar ((1979) J. Cell. Biol. 81, 137-153) as indicating accumulations of anionic material. In this study we have demonstrated not only high-specificity binding of PCF to fenestral diaphragms in capillaries of organs other than intestine and pancreas, but also considerable variation of PCF stainability of fenestral diaphragms in other organs.

Fetal and neonatal rat intestinal capillaries: a TEM study of changes in the mural structure.

The development of intestinal mucosal capillaries was studied in Wistar rats from the fourteenth day of gestation through the second postnatal day with the transmission electron microscope. The microvessels develop first as continuous capillaries without a basal lamina and become fenestrated during later stages of gestation. In the thickened areas of the cytoplasm of endothelial cells at early stages, there is a paucity of pinocytotic vesicles; but some vesicles are observed which are larger than those in the adult. Later in gestation these larger vesicles can still be seen, but the frequency of their occurrence decreases with the maturation of the vessels. The intercellular junctions vary in length and shape, and the space between the outer leaflets of the apposed cell membranes is usually 20 nm. Surrounding the abluminal surface of the endothelium, the incipient basal lamina appears attenuated and incomplete until at least the second day of neonatal life.

Fetal and neonatal rat intestinal capillaries: permeability to carbon, ferritin, hemoglobin, and myoglobin.

The function of the mural components involved in vascular exchange (pinocytotic vesicles, fenestrations, intercellular junctions, and the basal lamina) was assessed by intravascular injection of tracers (carbon, carbon plus histamine, ferritin, hemoglobin, and myoglobin) on separate gestational days in fetal and neonatal rat intestinal capillaries. The continuous capillaries present through day 17 of gestation were impermeable, even at junctions, to all the tracers within the maximum circulation time studied (3 minutes). Most of the luminal pinocytotic vesicles were labeled within 3 minutes with myoglobin (3.3-nm diameter) or hemoglobin (5.5-nm diameter), while only occasional vesicles contained ferritin (11-nm diameter) and none contained carbon (25-50nm diameter). Even though luminal pinocytotic vesicles contained the smaller tracers, no vesicular transport and discharge were ever seen. The fenestrated vessels present from day 18 of gestation allowed a rapid extravasation of ferritin and hemoglobin via diaphragmed fenestrations. At no time period did extravasation of carbon take place, nor did the carbon plus histamine solution induce vascular leakage. Once a tracer gained access to the extravascular space, the developing basal lamina did not seem to retard its movement.

Lectin and immunolabeling of microvascular endothelia.

A number of recently developed localization techniques are beginning to be applied in the study of endothelial cells and their structural components. In this article we will review a number of these cytochemical approaches as well as their advantages and disadvantages and their applications. The methods will be presented for processing tissues for either L.R. White embedding or semi-thin and thin frozen sections followed by subsequent lectin and immunolabeling for fluorescence and electron microscopic examination. These techniques are easily applied in the localization of perfused exogenous proteins and of endogenous endothelial-associated proteins. The results that can be obtained from such studies are presented and discussed.

The endothelial pocket. A new structure in fenestrated endothelia.

A new endothelial cell structure, named the endothelial pocket, has been found by combined transmission and scanning electron microscopic studies of renal peritubular capillaries. Transmission EM observations made on these and other fenestrated capillaries demonstrated that each pocket consists of an attenuated fold of fenestrated endothelium that projects approximately 200 nm into the lumen above the rest of the endothelial surface. Beneath this luminal fold, there is a space and then another layer of fenestrated endothelium which abuts the basal lamina. The linear density of endothelial pockets was measured in the capillaries of the kidney cortex, intestinal mucosa and exocrine pancreas in mice and determined to be 0.067, 0.017 and 0.007 pockets X micron-1 respectively. Cationic ferritin decoration of the anionic sites on the luminal surface of the endothelium in these capillary beds revealed that both unlabelled and labelled diaphragms are clustered. In such specimens, the majority of the luminal diaphragms on endothelial pockets did not have cationic ferritin binding sites detectable by either scanning or transmission EM. On this account as well as on account of their general morphology, endothelial pockets appear to be multifold versions of the simple transendothelial channel.

Changes in Mobility of the Golden Hamster with Induction of an IL-1-Induced Arthritis.

Many studies in animals have examined biochemical, immune and histological changes during arthritis; however, the study of the effects of arthritis on mobility has been largely neglected. Interleukin-1, administered by the intraarticular route into hamster knee joints, resulted in inhibition of spontaneous wheel running activity; however, the effect was transient, lasting only through the evening following IL-1 administration. A further injection of IL-1 2 days later showed still greater inhibition of running. The effect again did not extend beyond the first evening after injection. IL-1alpha and IL-1beta showed equivalent effects on mobility, and no evidence was seen for cooperative interaction between them. A 50% inhibition of running occurred at a dose of approximately 10 ng/knee of IL-1alpha. The effect appeared not to be systemic since intraperitoneal injection required microgram amounts of IL-1 for an equivalent inhibition. At the time mobility had been restored to normal, histological examination showed the continued presence of inflammatory cells, soft tissue swelling and cartilage proteoglycan loss. These results suggest a lack of correlation between inhibition of mobility and histopathological changes in cartilage and soft tissue.

Some factors affecting inhibition and restoration of mobility after induction of an acute arthritis in the hamster.

Mobility is impaired during arthritis. In order to study the causes of the mobility impairment, we have examined hamsters with a LPS arthritis in which running is inhibited over a 4-5 day period. Parameters have been examined to determine which correlate with running impairment. Knee diameter, as well as cell infiltration into the surrounding synovial tissue and the accompanying edema, failed to resolve by the time normal mobility was reestablished. Other factors must be primary determinants of mobility.

Extracellular matrix specificity for the differentiation of capillary endothelial cells.

Capillary endothelial cells in a fenestrated vasculature contain openings in attenuated areas of the cytoplasm which are often covered with one or two diaphragms. However, due to endothelial cell dedifferentiation upon culturing, such indicative membrane structure are often not expressed. The expression of a more differentiated phenotype can be regulated by the extracellular matrix to which the cells attach. In addition, during angiogenesis endothelial cell migration enables their interaction with other cell types and matrices which may directly affect endothelial cell growth and function. We report the ability to modify the expression of diaphragmed fenestrations and transcapillary channels in capillary endothelial cells by specific extracellular matrices. When the number of membrane openings is measured, growth of the cells on Madin--Darby canine kidney cell matrix results in the greatest number while other matrices or isolated matrix components are only partially active. Production of such a biologically active matrix not only allows an avenue to identify fenestrae-associated proteins but also provides an easy culture method to more closely mimic the in vivo phenotype of endothelial cells.

Surface densities of diaphragmed fenestrae and transendothelial channels in different murine capillary beds.

Fenestrated capillaries are provided with two types of regular discontinuities: fenestrae with negatively charged diaphragms, and transendothelial channels fitted with two diaphragms, of which the luminal one is uncharged. These structures are expected to affect macromolecular exchanges on the basis of size and charge. We have detected variations in the surface density of fenestrations and transendothelial channels (normalized to 1 micron of capillary endothelial profiles in sections) in selected areas of different murine organs, i.e., kidney cortex, duodenal mucosa, and exocrine pancreas. The survey was limited to endothelial segments less than or equal to 400 nm thick, and covered a total length of endothelial profiles of 1180 microns, 730 microns, and 1189 microns in the exocrine pancreas, intestinal mucosa, and kidney cortex, respectively. At least 1000 transendothelial openings were recorded by scoring for easily recognizable fenestrations, transendothelial channels, and unknowns, the latter representing either of the above in grazing sections. The linear density of all transendothelial openings taken together was found to vary among the different capillary beds (kidney cortex greater than intestinal mucosa greater than exocrine pancreas). This same sequence was observed for the linear densities of fenestrations, transendothelial channels, and unknowns considered individually. The values obtained were as follows: kidney cortex, 1.35 fenestrations per micron, 0.25 transendothelial channels per micron, 0.52 unknowns per micron; intestinal mucosa, 0.92 fenestrations per micron, 0.10 transendothelial channels per micron, 0.38 unknowns per micron; exocrine pancreas, 0.58 fenestrations per micron, 0.04 transendothelial channels per micron, 0.27 unknowns per micron. The differences in distribution of transendothelial openings among fenestrated capillary beds probably reflect capillary permeability modulations connected with the functions of these various organs.

The formation of fenestrations and channels by capillary endothelium in vitro.

Microvascular endothelial cells isolated from fenestrated capillaries have been shown to form tubes in vitro, thereby demonstrating that they retain the ability to express some degree of their in vivo differentiated phenotype. However, some of their physiologically important structural features, such as transendothelial openings (i. e., diaphragmed fenestrations and transendothelial channels) are lost or are greatly reduced in number. In this study, cloned bovine adrenal cortex endothelial cells were cultured on plastic or on a basal lamina produced by Madin-Darby canine kidney (MDCK) cells for up to 17 days postconfluence. All cultures were then routinely fixed and processed for electron microscopic morphometry. For cells grown on plastic for 17 days postconfluence, the linear density of transendothelial openings in endothelial profiles less than 400 nm thick was found to be 0.007 openings per micron. On MDCK matrix, however, the linear density of transendothelial openings in endothelial profiles less than 400 nm thick was found to be 0.157 per micron. Occasionally some cells formed "tube-like" structures that also contained diaphragmed fenestrations and transendothelial channels on both sides of the tubes. These findings suggest that the substrate on which endothelial cells are grown can affect their differentiation.

Histology and tissue chemistry of tidemark separation in hamsters.

Adult articular cartilage is divided by the tidemark into a deep calcified layer and a more superficial uncalcified layer. Histologic examination of articular cartilage from the knee joint of golden Syrian hamsters 123 days of age or older revealed defects at the tidemark in the tibia. Defects ranged from small separations of the calcified and uncalcified layers along the tidemark to progressively larger defects apparently formed by dissolution. These larger defects appeared as cavities in the noncalcified cartilage, had smooth rather than rough edges, frequently contained coalesced debris, and often resulted in a bulge in the articular surface. Occasionally, these large defects broke through the articular surface. Defects were not observed in tibial cartilage of younger (<90 days old) hamsters or in femoral cartilage from hamsters of any age. Exercise neither protected against nor increased the severity of the defects. Collagen cross-linking by pyridinoline was examined as a function of age and increased from 1,090 to 3,062 micromoles of pyridinoline/mole of hydroxyproline over the period of 1-9 months of age but was not correlated with defect formation. With increasing age, these focal tidemark defects could lead to osteoarthrosis-like cartilage lesions.