[Show abstract][Hide abstract] ABSTRACT: Pharmacologic antagonism of CCR5, a chemokine receptor expressed on macrophages and activated T cells, is an effective antiviral therapy in patients with macrophage-tropic HIV infection, but its efficacy in modulating inflammation and immunity is only just beginning to be investigated. In this regard, the recruitment of CCR5-bearing cells into clinical allografts is a hallmark of acute rejection and may anticipate chronic rejection, whereas conventionally immunosuppressed renal transplant patients homozygous for a nonfunctional Delta32 CCR5 receptor rarely exhibit late graft loss. Therefore, we explored the effects of a potent, highly selective CCR5 antagonist, Merck's compound 167 (CMPD 167), in an established cynomolgus monkey cardiac allograft model. Although perioperative stress responses (fever, diminished activity) and the recruitment of CCR5-bearing leukocytes into the graft were markedly attenuated, anti-CCR5 monotherapy only marginally prolonged allograft survival. In contrast, relative to cyclosporine A monotherapy, CMPD 167 with cyclosporine A delayed alloantibody production, suppressed cardiac allograft vasculopathy, and tended to further prolong graft survival. CCR5 therefore represents an attractive therapeutic target for attenuating postsurgical stress responses and favorably modulating pathogenic alloimmunity in primates, including man.
The Journal of Immunology 09/2007; 179(4):2289-99. DOI:10.4049/jimmunol.179.4.2289 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF(1alpha) and TXB(2) aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.
[Show abstract][Hide abstract] ABSTRACT: A nonpeptidyl small molecule antagonist, compound A, to nonactivated very late antigen-4 (VLA4) was examined in lung inflammation induced by a single dose of ovalbumin challenge. Compound A presented a good pharmacokinetic property, when given intratracheally, and the blood cells from such pharmacokinetic study showed good receptor occupancy of the compound for approximately 8 hours. Compound A was then tested in an ovalbumin-induced airway inflammation model by intranasal or intravenous route of administration. There was a dose-dependent inhibition of eosinophilia in the bronchiolar lavage fluid, when compound A was given intranasally but not when it was given intravenously. For comparison, antibody to VLA4 and another compound, BIO1211, which reacts only with activated VLA4, were examined in this system. Immunohistochemical analyses of the lung tissue substantiated the findings in the bronchiolar lavage fluid. Specific staining of the major basic protein of eosinophils showed peribronchiolar infiltration of eosinophils. Some of these eosinophils were also positive for nitrotyrosine, suggesting activation of eosinophils in the lung interstitium. There was deposition of major basic protein and nitrotyrosine at the base of the perivascular endothelium, indicative of degranulation of eosinophils in the area. After intranasal treatment with compound A, eosinophils in the lungs and their activation products were substantially decreased, documenting its effectiveness in inhibiting lung inflammation.
American Journal of Respiratory and Critical Care Medicine 06/2003; 167(10):1400-9. DOI:10.1164/rccm.200207-696OC · 13.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The chemokine receptors CCR5 and CXCR4 act synergistically with CD4 in an ordered multistep mechanism to allow the binding and entry of human immunodeficiency virus type 1 (HIV-1). The efficiency of such a coordinated mechanism depends on the spatial distribution of the participating molecules on the cell surface. Immunoelectron microscopy was performed to address the subcellular localization of the chemokine receptors and CD4 at high resolution. Cells were fixed, cryoprocessed, and frozen; 80-nm cryosections were double labeled with combinations of CCR5, CXCR4, and CD4 antibodies and then stained with immunogold. Surprisingly, CCR5, CXCR4, and CD4 were found predominantly on microvilli and appeared to form homogeneous microclusters in all cell types examined, including macrophages and T cells. Further, while mixed microclusters were not observed, homogeneous microclusters of CD4 and the chemokine receptors were frequently separated by distances less than the diameter of an HIV-1 virion. Such distributions are likely to facilitate cooperative interactions with HIV-1 during virus adsorption to and penetration of human leukocytes and have significant implications for development of therapeutically useful inhibitors of the entry process. Although the mechanism underlying clustering is not understood, clusters were observed in small trans-Golgi vesicles, implying that they were organized shortly after synthesis and well before insertion into the cellular membrane. Chemokine receptors normally act as sensors, detecting concentration gradients of their ligands and thus providing directional information for cellular migration during both normal homeostasis and inflammatory responses. Localization of these sensors on the microvilli should enable more precise monitoring of their environment, improving efficiency of the chemotactic process. Moreover, since selectins, some integrins, and actin are also located on or in the microvillus, this organelle has many of the major elements required for chemotaxis.
Journal of Virology 05/2001; 75(8):3779-90. DOI:10.1128/JVI.75.8.3779-3790.2001 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prostaglandins are synthesized by cyclooxygenases (COX)-1 and -2. The expression and cellular localization of COX-1 and COX-2 in normal human colon and inflammatory bowel disease (IBD) surgical resections were studied.
COX-1 and COX-2 protein expression and cellular localization were assessed by Western blotting and immunohistochemistry.
COX-1 protein was expressed at equal levels in normal, Crohn's disease, and ulcerative colitis colonic epithelial cells. COX-2 protein was not detected in normal epithelial cells but was detected in Crohn's disease and ulcerative colitis epithelial cells. Immunohistochemistry of normal, Crohn's colitis, and ulcerative colitis tissue showed equivalent COX-1 expression in epithelial cells in the lower half of the colonic crypts. COX-2 expression was absent from normal colon, whereas in Crohn's colitis and ulcerative colitis, COX-2 was observed in apical epithelial cells and in lamina propria mononuclear cells. In Crohn's ileitis, COX-2 was present in the villus epithelial cells. In ulcerative colitis, colonic epithelial cells expressing COX-2 also expressed inducible nitric oxide synthase.
COX-1 was localized in the crypt epithelium of the normal ileum and colon, and its expression was unchanged in IBD. COX-2 was undetectable in normal ileum or colon, but it was induced in apical epithelial cells of inflamed foci in IBD.
[Show abstract][Hide abstract] ABSTRACT: Objective
It has long been proposed that stromelysin is one of the major degradative matrix metalloproteinases responsible for the loss of cartilage in rheumatoid arthritis (RA) and osteoarthritis (OA). This hypothesis was tested by examining the arthritic paws of stromelysin 1 (SLN1)-deficient mice for loss of cartilage and for generation of neoepitopes that would be indicative of aggrecan cleavage.Methods
The SLN1 gene was inactivated in murine embryonic stem cells, and knockout mice deficient in SLN1 activity were bred onto the B10.RIII background. The incidence and severity of collagen-induced arthritis (CIA) were compared in wild-type and knockout mice. Paws from mice with CIA were examined for loss of cartilage and for proteoglycan staining, as well as for the generation of the neoepitope FVDIPEN341.ResultsSLN1-deficient mice developed CIA, as did the wild-type N2 mice. Histologic analyses demonstrated no significant differences among the B10.RIII, wild-type, and knockout mice in loss of articular cartilage and proteoglycan staining. No decrease in the FVDIPEN341 epitope was observed in the SLN1-deficient mice.Conclusion
Disruption of the SLN1 gene neither prevents nor reduces the cartilage destruction associated with CIA. Moreover, SLN1 depletion does not prevent the cleavage of the aggrecan Asn341-Phe342 bond.
[Show abstract][Hide abstract] ABSTRACT: To analyze the roles of two classes of proteinases, 'aggrecanase', and matrix metalloproteinases (MMPs), in chondrodestruction during murine collagen-induced arthritis (CIA).
Generation of the 'aggrecanase' neo-epitope (NITEGE373), and the MMP neo-epitope (VDIPEN341) within aggrecan was studied by immunoperoxidase microscopy using specific anti-peptide antibodies in normal and stromelysin-1 (SLN-1) deficient knockout mice with CIA.
High levels of NITEGE373 and VDIPEN341 neo-epitopes were observed in foci within CIA paw articular cartilage exhibiting depletion of glycosaminoglycans, in advance of significant cartilage erosion. The highest concentrations of NITEGE373 and VDIPEN341 labeling were observed and often co-distributed in the chondrocyte pericellular matrix, suggesting that stimulated chondrocytes can synthesize and/or activate both enzymes. Other regions of the cartilage frequently exhibited either NITEGE373 or VDIPEN341 labeling, but not both neo-epitopes simultaneously, suggesting that 'aggrecanase' and MMP cleavages of aggrecan may be generated independently. No detectable differences were observed in expression or distribution of either neo-epitope in SLN-1 knockout versus wild-type mice. In addition, in vitro digestion of joint sections with SLN-1 did not alter the expression of cartilage NITEGE373, while markedly increasing VDIPEN341 labeling. Peripheral nerves and brains of naive mice also exhibited intense anti-NITEGE373 labeling.
These data indicate that NITEGE373 and VDIPEN341 aggrecan neo-epitopes are sensitive and specific markers of early joint pathology, and are consistent with the hypothesis that SLN-1 does not have 'aggrecanase' activity, and that 'aggrecanase' is distinct from the MMPs which cleave aggrecan at the MMP site.
Osteoarthritis and Cartilage 11/1997; 5(6):407-18. DOI:10.1016/S1063-4584(97)80045-3 · 4.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inducible nitric oxide synthase (iNOS) is generated in several cell types by treatment with lipopolysaccharides or cytokines. Earlier studies suggested that ulcerative colitis is associated with increased NO produced by iNOS; however, the cellular source of the NO synthesis was not identified. A possible mechanism of NO-induced cellular damage is through its interaction with superoxide to produce peroxynitrite, which reacts with tyrosine to form nitrotyrosine in cellular proteins.
Using immunoperoxidase microscopy with a new monospecific human iNOS antibody (NO-53), the cellular distribution of iNOS and nitrotyrosine was examined using human colonic mucosa from normal bowel, ulcerative colitis, Crohn's disease, and diverticulitis.
Intense focal iNOS labeling was localized to the inflamed colonic epithelium in ulcerative colitis, Crohn's disease, and diverticulitis but was not detectable in the uninflamed epithelium. Nitrotyrosine labeling was also observed in the inflamed colonic epithelium and was associated with nearby iNOS staining; nitrotyrosine was undetectable in normal mucosal epithelium. iNOS and nitrotyrosine were also detected in lamina propria mononuclear cells and neutrophils.
These findings suggest that iNOS is induced in the inflamed human colonic epithelium and is associated with the formation of peroxynitrite and the nitration of cellular proteins.
[Show abstract][Hide abstract] ABSTRACT: The destruction of articular cartilage in immune inflammatory arthritic disease involves the proteolytic degradation of its extracellular matrix. The role of activated matrix metalloproteinases (MMPs) in the chondrodestructive process was studied by identifying a selective cleavage product of aggrecan in murine arthritis models initiated by immunization with either type II collagen or proteoglycan. We conducted semiquantitative immunocytochemical studies of VDIPEN341 using a monospecific polyclonal antibody requiring the free COOH group of the COOH-terminal Asn for epitope detection. This antibody recognizes the aggrecan G1 domain fragment generated by MMP [i.e., stromelysin (SLN) or gelatinase A] cleavage of aggrecan between Asn341-Phe342 but does not recognize intact aggrecan. VDIPEN was undetectable in normal mouse cartilage but was observed in the articular cartilage (AC) of mice with collagen-induced arthritis 10 d after immunization, without histological damage and clinical symptoms. This aggrecan neoepitope was colocalized with high levels of glycosaminoglycans (GAGs) in pericellular matrices of AC chondrocytes but was not seen at the articular surface at this early time. Digestion of normal (VDIPEN negative) mouse paw cryosections with SLN also produced heavy pericellular VDIPEN labeling. Computer-based image analysis showed that the amount of VDIPEN expression increased dramatically by 20 d (70% of the SLN maximum) and was correlated with GAG depletion. Both infiltration of inflammatory cells into the synovial cavity and early AC erosion were also very prominent at this time. Analysis of adjacent sections showed that both induction of VDIPEN and GAG depletion were strikingly codistributed within sites of articular cartilage damage. Similar results occurred in proteoglycan-induced arthritis, a more progressive and chronic model of inflammatory arthritis. These studies demonstrate for the first time the MMP-dependent catabolism of aggrecan at sites of chondrodestruction during inflammatory arthritis.
[Show abstract][Hide abstract] ABSTRACT: The murine anti-CD18 mAb 1B4 has been humanized using CDR grafting. Three VH (Gal, Jon, and New) and two VL (Rei and Len) human frameworks, whose selection was based exclusively on their sequence identity with m1B4, were used to construct five human gamma 4/kappa recombinant antibodies: Gal/Rei, Gal/Len, Jon/Rei, and New/Rei, and a "hemichimeric" antibody pairing the VH of m1B4 with grafted Rei. Each of these h1B4 constructs completely inhibited the binding of m1B4 to activated human leukocytes with avidities (IC50) ranging from 1.5 to 8.0 nM, compared to 0.5 nM for m1B4. Replacement of three VH residues in the best VH framework, Gal, with the corresponding m1B4 "packing" (nonsolvent exposed) residues gave an h1B4 (mutant Gal/Rei) with the same avidity as m1B4. Avidity correlated with overall percent identity between the human and murine VH frameworks and, in particular, with conservation of "packing" residues. Rei and Len VL frameworks proved to be interchangeable. Further characterization showed that the Gal/Rei prototype was equipotent to m1B4 in blocking adhesion of polymorphonuclear leukocytes and monocytes to human vascular endothelium in vitro, and polymorphonuclear leukocyte extravasation into C5a-injected rabbit or monkey skin sites. Dual-label immunofluorescence microscopy of bone marrow cells with Gal/Rei h1B4 and m1B4 demonstrated that the fine specificity of the combining sites had not been altered by humanization. Reduced immunogenicity was demonstrated in rhesus monkeys that tolerated weekly treatment with h1B4 for 6 wk, whereas m1B4 induced profound anaphylaxis at 3 wk. Anti-1B4 titers in h1B4-treated rhesus were 50 to 66% lower and developed 1 wk later than in m1B4-treated monkeys. Crucially, the anti-h1B4 antibodies were anti-idiotypic while the anti-m1B4 antibodies were directed against constant and framework regions. We conclude that sequence identity searches are sufficient to identify suitable human frameworks for CDR-grafting of m1B4, yielding functionally equivalent humanized antibodies that are tolerated better in primates.
The Journal of Immunology 05/1993; 150(7):2844-57. · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and a potent hypocholesterolemic agent, induces a hyperplastic thickening of the rat forestomach mucosa after oral administration of its active form, a hydroxyacid. We studied the effects of lovastatin on the intracellular accumulation of HMG-CoA reductase immunostaining and the accompanying morphological changes in rat forestomach keratinocytes by immunofluorescence microscopy and transmission electron microscopy (TEM). Administration of lovastatin hydroxyacid induced increases in HMG-CoA reductase levels within forestomach keratinocytes that were dose and time dependent and reversible. The adjacent glandular stomach epithelium did not exhibit induction of reductase. A pharmacologically inactive epimer of lovastatin hydroxyacid did not increase keratinocyte reductase accumulation, and lovastatin lactone induced minimal forestomach reductase. TEM of forestomachs from rats given lovastatin hydroxyacid demonstrated profound alterations in epidermal lamellar bodies (organelles that transport lipids and steroids to the intercellular spaces of the stratum corneum). Treated cells lacked internal lipid lamellae and failed to secrete sheets of lipid material into the intercellular spaces of the stratum corneum. We hypothesize that sustained inhibition of HMG-CoA reductase in rat forestomach keratinocytes induces accumulation of HMG-CoA reductase and hyperplasia by inhibiting sterol synthesis, assembly of lamellar bodies, and formation of intercellular lipid sheets.
Arteriosclerosis and thrombosis: a journal of vascular biology / American Heart Association 09/1991; 11(5):1156-65. DOI:10.1161/01.ATV.11.5.1156
[Show abstract][Hide abstract] ABSTRACT: Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs post-transfection into non-lymphoid mammalian cells. The application of these new approaches enables the expression of a recombinant humanized antibody just 6 weeks after initiating the cDNA cloning of the murine mAb.
Nucleic Acids Research 06/1991; 19(9):2471-6. DOI:10.1093/nar/19.9.2471 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have localized several major extracellular matrix protein receptors in the specific granules of human polymorphonuclear (PMN) and monocytic leukocytes using double label immunoelectron microscopy (IEM) with ultrathin frozen sections and colloidal-gold conjugates. Rabbit antibodies to 67-kD human laminin receptor (LNR) were located on the inner surface of the specific granule membrane and within its internal matrix. LNR antigens co-distributed with lactoferrin, a marker of specific granules, but did not co-localize with elastase in azurophilic granules of PMNs. Further, CD11b/CD18 (leukocyte receptor for C3bi, fibrinogen, endothelial cells, and endotoxin), mammalian fibronectin receptor (FNR), and vitronectin receptor (VNR) antigens were also co-localized with LNR in PMN specific granules. A similar type of granule was found in monocytes which stained for LNR, FNR, VNR, CD18, and lysozyme. Activation of PMNs with either PMA, f-met-leu-phe (fMLP), tumor necrosis factor (TNF), or monocytic leukocytes with lipopolysaccharide (LPS), induced fusion of specific granules with the cell membrane and expression of both LNR and CD18 antigens on the outer cell surface. Further, stimulation led to augmented PMN adhesion on LN substrata, and six- to eightfold increases in specific binding of soluble LN that was inhibited by LNR antibody. These results indicate that four types of extracellular matrix receptors are located in leukocyte specific granules, and suggest that up-regulation of these receptors during inflammation may mediate leukocyte adhesion and extravasation. We have thus termed leukocyte specific granules adhesomes.
The Journal of Cell Biology 01/1990; 109(6 Pt 1):3169-82. · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin-coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate-bound vitronectin.
The Journal of Cell Biology 07/1988; 106(6):2171-82. · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Double-label immunofluorescence microscopy and immunoelectron microscopy were performed on stationary cultures of Nil 8 fibroblasts to determine if fibronectin and basement membrane heparan sulfate proteoglycans play coordinated roles in cell-to-substrate adhesion. Relationships between subcellular matrix fibers containing fibronectin plus proteoglycan, and focal contacts associated with microfilament bundles, were studied simultaneously using interference reflection microscopy, differential interference contrast microscopy, and immunofluorescence microscopy. Cells maintained in 0.3% FBS were doubly stained with monospecific anti-fibronectin IgG and antibodies against a basement membrane proteoglycan purified from the EHS (Engelbreth-Holm-Swarm) tumor. Coincident patterns of fibronectin and proteoglycan-containing fibers were found to codistribute with focal contacts and microfilament bundles in both early (6-h) and late (24-h) cultures. The early cells showed doubly-stained fibers colinear with substrate adhesion sites in 43% of the sample, while 100% of the later cells exhibited these coaligned matrix-cytoskeletal attachment complexes. Immunoelectron microscopy showed that both of these antigens were situated in the same type of extracellular matrix fiber that appeared to be loosely associated with the cell surface membrane. We hypothesize that the appearance of proteoglycan in subcellular matrix fibers of these fibroblasts might stabilize fibronectin-containing cell-to-substrate contacts.
Experimental Cell Research 01/1988; 173(2):558-71. DOI:10.1016/0014-4827(87)90295-3 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cultured fibroblasts form focal contacts (FCs) associated with actin microfilament bundles (MFBs) during attachment and spreading on serum- or fibronectin (FN)-coated substrates. To determine if the minimum cellular adhesion receptor recognition signal Arg-Gly-Asp-Ser (RGDS) is sufficient to promote FC and MFB formation, rat (NRK), hamster (Nil 8), and mouse (Balb/c 3T3) fibroblasts in serum-free media were plated on substrates derivatized with small synthetic peptides containing RGDS. These cultures were studied with interference reflection microscopy to detect FCs, Normarski optics to identify MFBs, and immunofluorescence microscopy to observe endogenous FN fiber formation. By 1 h, 72-78% of the NRK and Nil 8 cells plated on RGDS-containing peptide had focal contacts without accompanying FN fibers, while these fibroblasts lacked FCs on control peptide. This early FC formation was followed by the appearance of coincident MFBs and colinear FN fibers forming fibronexuses at 4 h. NRK and Nil 8 cultures on substrates coated with native FN or 75,000-D FN-cell binding fragment showed similar kinetics of FC and MFB formation. In contrast, the Balb/c 3T3 mouse fibroblasts plated on Gly-Arg-Gly-Asp-Ser peptide-derivatized substrates, or on coverslips coated with 75,000-D FN cell-binding fragment, were defective in FC formation. These results demonstrate that the apparent binding of substrate-linked RGDS sequences to cell surface adhesion receptors is sufficient to promote early focal contact formation followed by the appearance of fibronexuses in some, but not all, fibroblast lines.
The Journal of Cell Biology 04/1987; 104(3):573-84. · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Because the small bowel is a site of significant cholesterol synthesis, we determined the ileal distribution of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme of the cholesterol biosynthetic pathway. Immunofluorescence microscopy on unfixed snap-frozen sections of ileum and jejunum from untreated rats or dogs showed HMG-CoA reductase in the absorptive villus epithelial cells and this appeared to be strikingly localized in their apical cytoplasm. This pattern of HMG-CoA reductase staining approximated a gradient along the villus-crypt axis with the distal villi labeling most intensely. Treatment of rats with mevinolin and/or cholestyramine for 12 days induced a 5- to 11-fold increase in ileal HMG-CoA reductase activity, and yielded a higher intensity of immunostaining without altering the pattern of enzyme distribution observed in control intestines. Also, rats with maximal induction of ileal HMG-CoA reductase exhibited a twofold increase in the number of epithelial villus cells containing prominent stacks of smooth-surfaced membranes in their apical cytoplasm as seen with electron microscopy. These observations suggest that the distal villus absorptive epithelial cells of the ileum contain high concentrations of HMG-CoA reductase, and therefore might be capable of contributing significant quantities of cholesterol to the circulation.
[Show abstract][Hide abstract] ABSTRACT: The distribution of laminin was studied during pulmonary fibrosis induced in rodents by bleomycin sulfate. Large accumulations of laminin associated with basement membranes were seen in thickened lung interstitial spaces by immunofluorescence microscopy, starting at 7 days (32-75% increases) and persisting through 28 days (66-79% increase). By electron microscopy, these laminin concentrations were skeinlike masses of reduplicated basement membranes localized at the surface of alveolar capillary endothelial cells. Numerous macrophages were also associated with this basement membrane material. These findings suggest that bleomycin-induced damage to lung cells causes massive local accumulations of basement membranes, which might be involved in the expansion of the interstitial stroma by stimulating attachment and activation of certain inflammatory cells.
American Journal Of Pathology 12/1986; 125(2):258-68. · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied cell surface interactions between the fibronectin (FN)-containing extracellular matrix and the actin cytoskeleton of normal porcine synoviocytes in vitro, using electron microscopic methods. These type B synovial cells were distinguishable from dermal fibroblasts co-isolated from the same organism, because of their very long cellular processes and their ability to synthesize prostaglandin E2 after stimulation with interleukin-1. With plastic sections, we found end-to-end (tandem) and track-like (lateral) transmembrane associations of extracellular fibers and cortical 5-nm microfilaments localized along the attenuated synoviocyte processes in postconfluent cultures. Very similar FN-actin complexes, termed fibronexus (FNX), have been observed on cultured fibroblasts and on granulation tissue myofibroblasts in vivo. Using double-label immunoelectron microscopy with monospecific antibodies applied to ultrathin frozen sections of synoviocytes cut in situ, we proved that these FNX were indeed composed of associated FN and actin filaments. The striking finding of numerous FNX in cultured type B synoviocytes strongly suggests that the FNX is a major cell surface adhesion site in normal synovium, which may play an important role in pannus formation, connective tissue remodeling, and synoviocyte proliferation in patients with rheumatoid arthritis.
[Show abstract][Hide abstract] ABSTRACT: An intimate transmembrane complex of fibronectin-containing extracellular fibers and actin microfilaments termed the fibronexus has been observed at the adhesive surface of fibroblasts in vitro  and along the plasmalemma of myofibroblasts in vivo . Although the observation of coincident actin and fibronectin immunofluorescence patterns in the latter work strongly suggested that the fibronexus is localized at the myofibroblast surface, we only obtained morphological evidence for its existence with electron microscopy. Therefore, in the present study, we have utilized a double-label immunoelectron microscopic technique to localize fibronectin and actin simultaneously in the putative fibronexuses of myofibroblasts within guinea pig granulation tissue, formed 7 to 9 days after skin wounding. This method employed rabbit antifibronectin and mouse anti-actin antibodies, followed by species-specific secondary antibodies conjugated to colloidal gold particles of different sizes. These probes were applied to the surfaces of ultrathin frozen sections mounted on grids. We found that fibronectin and actin were specifically localized on the respective external and internal components of myofibroblast fibronexuses. Our results suggest that specific transmembranous fibronectin-cytoskeletal complexes play an important role in the cohesion of granulation tissue.
European Journal of Cell Biology 08/1985; 38(1):94-101. · 3.83 Impact Factor