Oral delivery of novel human IGF-1 bioencapsulated in lettuce cells promotes musculoskeletal cell proliferation, differentiation and diabetic fracture healing.

Human insulin-like growth factor-1 (IGF-1) plays important roles in development and regeneration of skeletal muscles and bones but requires daily injections or surgical implantation. Current clinical IGF-1 lacks e-peptide and is glycosylated, reducing functional efficacy. In this study, codon-optimized Pro-IGF-1 with e-peptide (fused to GM1 receptor binding protein CTB or cell penetrating peptide PTD) was expressed in lettuce chloroplasts to facilitate oral delivery. Pro-IGF-1 was expressed at high levels in the absence of the antibiotic resistance gene in lettuce chloroplasts and was maintained in subsequent generations. In lyophilized plant cells, Pro-IGF-1 maintained folding, assembly, stability and functionality up to 31 months, when stored at ambient temperature. CTB-Pro-IGF-1 stimulated proliferation of human oral keratinocytes, gingiva-derived mesenchymal stromal cells and mouse osteoblasts in a dose-dependent manner and promoted osteoblast differentiation through upregulation of ALP, OSX and RUNX2 genes. Mice orally gavaged with the lyophilized plant cells significantly increased IGF-1 levels in sera, skeletal muscles and was stable for several hours. When bioencapsulated CTB-Pro-IGF-1 was gavaged to femoral fractured diabetic mice, bone regeneration was significantly promoted with increase in bone volume, density and area. This novel delivery system should increase affordability and patient compliance, especially for treatment of musculoskeletal diseases.

Platelet activation and microfilament bundling.

Human platelets were obtained in the fully resting state by treating discoid populations with 1.5 mM tetracaine and in the activated state by treatment with 2 microM A-23187. After gel filtration or washing, respectively, platelet suspensions were lysed with 1% Triton X-100 at pH 6.8. The precipitates from resting platelets viewed by negative staining appeared predominantly granular with a few very short microfilaments. They contained polypeptides of 250, 100, 45, 38, 36.5, and 35 Kdaltons, and three small polypeptides including one with the mobility of profilin on SDS gels. Precipitates from activated platelets lacked this low molecular weight band and contained a major band at 200 Kdaltons with the mobility of myosin; these precipitates had significant K+, Ca++ ATPase activity absent from the precipitate of resting platelets. As seen in negative staining, precipitates from activated platelets contained microfilaments arranged as nets or bundles. The granular resting precipitates were transformed in vitro into microfilament bundles by washing the precipitates in buffer at higher pH (7.6) in the presence of 5 X 10(-5) M calcium chloride.

Membrane-bound and fluid-phase macromolecules enter separate prelysosomal compartments in absorptive cells of suckling rat ileum.

The absorptive cell of the suckling rat ileum is specialized for the uptake and digestion of milk macromolecules from the intestinal lumen. The apical cytoplasm contains an extensive tubulocisternal system, a variety of vesicles and multivesicular bodies (MVB), and a giant phagolysosomal vacuole where digestion is completed. To determine if sorting of membrane-bound and fluid-phase macromolecules occurs in this elaborate endocytic system, we infused adsorptive and soluble tracers into ligated intestinal loops in vivo and examined their fates. Lysosomal compartments were identified by acid phosphatase histochemistry. Native ferritin and two ferritin-lectin conjugates that do not bind to ileal membranes (Con A, UEAI) served as soluble tracers. Horseradish peroxidase binds to ileal membranes and thus was not useful as a fluid-phase tracer in this system. Cationized ferritin and a lectin that binds to terminal B-D-galactosyl sites on ileal membranes (Ricinus communis agglutinin [RCAI]-ferritin) were used as tracer ligands. All tracers entered the wide apical invaginations of the luminal cell surface and were transported intracellularly. Membrane-bound tracers were found in coated pits and vesicles, and throughout the tubulocisternal system (where cationized ferritin is released from the membrane) and later, in large clear vesicles and MVB. In contrast, fluid-phase tracers appeared within 5 min in vesicles of various sizes and were not transported through the tubulocisternae, rather, they were concentrated in a separate population of vesicles of increasing size that contained amorphous dense material. Large clear vesicles, large dense vesicles, and MVB eventually fused with the giant supranuclear vacuole. Acid phosphatase activity was present in MVB and in the giant vacuole but was not present in most large vesicles or in the tubulocisternae. These results demonstrate that membrane-bound and soluble protein are transported to a common lysosomal destination via separate intracellular routes involving several distinct prelysosomal compartments.

Uptake and transepithelial transport of nerve growth factor in suckling rat ileum.

Nerve growth factor (NGF) is necessary for the development of sympathetic and some sensory neurons. Milk may be a source of NGF for suckling young, but sites of intestinal absorption of the protein have not been identified. To determine whether NGF is transported across the absorptive epithelium of suckling rat ileum, we assessed binding, uptake, and transport of 125I-NGF by light microscopy and EM autoradiography. Blood and tissue extracts were analyzed by biochemical and immunological methods to determine whether NGF was taken up structurally intact. NGF binding sites were identified on microvilli and apical invaginations of ileal absorptive cells in vitro. Injected into ileal loops in vivo, NGF radioactivity retained by fixation was evident after 20 min in apical regions of absorptive cells, in endocytic tubules (which mediate the uptake of membrane-bound ligands), in vesicles (which mediate nonspecific endocytosis), and in the supranuclear lysosomal vacuole. At 1 and 2 h, radiolabel in these compartments increased and silver grains were evident at the basal cell surface, and in cells, matrix, and vessels of the lamina propria. In blood and liver, radiolabeled molecules that were immunologically and electrophoretically indistinguishable from NGF and that co-eluted with NGF on gel filtration columns were detected, confirming that some NGF was transported across the epithelium structurally intact. Thus, absorptive cells of suckling rat ileum can take up NGF by both receptor-mediated and nonspecific endocytosis, and direct NGF either to the lysosome for degradation, or into a transepithelial transport pathway.

Transepithelial transport of epidermal growth factor by absorptive cells of suckling rat ileum.

Epidermal growth factor (EGF), an acid-stable peptide present in rodent and human milk, is absorbed and promotes intestinal growth when fed to suckling rats. To determine whether absorptive cells of suckling rat ileum conduct selective transepithelial transport of EGF, we followed uptake of 125I-EGF from ileal loops by autoradiography and biochemical methods. Specific binding sites for 125I-EGF were localized by electron microscope autoradiography on apical membranes of ileal epithelial sheets in vitro. During uptake in vivo, radiolabeled molecules were concentrated in apical endosomal compartments and were also associated with lysosomal vacuoles, basolateral cell surfaces, and lamina propria. Excess cold EGF reduced basolateral label by 44% and TCA precipitable serum label by 38%. After 30 and 60 min of continuous uptake, radiolabeled molecules in epithelium, denuded mucosa, blood, and liver were analyzed under reducing conditions by reversed-phase high-pressure liquid chromatography (HPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Although considerable degradation of 125I-EGF occurred after uptake from the lumen, a portion of radiolabel in epithelium and mucosa represented 125I-EGF which eluted somewhat more rapidly from C18 HPLC columns and showed a slight decrease in apparent molecular weight by SDS-PAGE. All radiolabel in blood and liver represented breakdown products. Thus, EGF is selectively transported across the ileal epithelium in suckling rats but is modified during transport. Milk EGF may accumulate in the lamina propria where it could influence growth and maturation of the suckling intestine.

Mechanisms of recovery from experimental autoimmune encephalomyelitis: T cell deletion and immune deviation in myelin basic protein T cell receptor transgenic mice.

Experimental autoimmune encephalomyelitis (EAE) is a Th1-type cell-mediated autoimmune disease directed against central nervous system (CNS) myelin antigens such as myelin basic protein (MBP). EaE is usually characterized by spontaneous remission of clinical disease and immune pathology despite the persistence of self myelin antigens in the central nervous system. Following induction of an acute episode of EAE, spontaneous remission also occurs in MBP T cell receptor (TCR) transgenic mice even through most T cells express a TCT specific for MBP. To investigate the mechanisms of recovery associated with EAE, we examined the behavior of MBP-specific T cells in the MBP TCR transgenic mouse model during disease progression and recovery. We found that recovery from EAE was associated with three major immunologic changes: (1) deletion of encephalitogenic T cells in the brain; (2) deviation of MBP-specific transgenic (Tg+) T cells both in the periphery and in the central nervous system from INF- gamma secretin Th1 type cells to cells that secrete IL-4, IL-10, and TGF- beta ; and (3) deletion of Tg+ T cells in the thymus through apoptosis. Thus spontaneous recovery from a classic Th1 type organ specific autoimmune disease is associated with two mechanisms of immune tolerance, deletion of autoreactive cells and immune deviation of autoreactive cells to a non-pathogenic phenotype.

Platelet shape change and cytoskeletal assembly: effects of pH and monovalent cation ionophores.

The monovalent cation ionophores monensin and nigericin cause platelet shape change at a rate of approximately 1/20 of that caused by ADP. The effect of monensin was studied further. Shape change caused by monensin is pH dependent, increasing in rate as extracellular pH increases. Monensin induced shape change is not blocked by 30 microM cinanserin which completely inhibits serotonin induced shape change. Also, the amount of serotonin secreted by monensin treated platelets is below the threshold required to induce shape change. 100 microM ATP which inhibits ADP induced shape change does not affect monensin induced shape change. Amiloride, a sodium transport blocker, inhibits both the rate of ADP induced shape change and platelet spreading on poly-lysine coated glass. Amorphous platelet cytoskeletons isolated from resting platelets at pH 6.8 with Mg++ but not Ca++ can be transformed into filament bundles by subsequent incubation at pH 7.6. We conclude that platelet shape change is at least in part triggered by changes in cellular Na+ and pH.

Effect of oral beta interferon on subsequent immune responsiveness.

Oral administration of myelin antigens reduces the incidence and severity of EAE in rat and mouse models and decreases the frequency of MBP-reactive cells and the frequency of attacks in some patients with multiple sclerosis. Low-dose oral tolerance has been shown to be mediated by Th2-type regulatory cells that secrete TGFbeta and IL-4/IL-10. Adjuvants and cytokines may modulate oral tolerance. The addition of betaIFN to the experimental therapy regimen, either orally or by intraperitoneal injection, has been shown to enhance the suppressive effects of oral myelin antigens when either are fed the suboptimal dosing regimen to suppress EAE. The current studies were conducted to elucidate the mechanism of the observed in vivo synergy of betaIFN and antigen feeding. Analysis of the in vitro proliferative response and cytokine production by lymphocytes from fed animals in response to specific antigen in culture shows that the synergistic effect may be related to both independent suppression of the immune response by oral betaIFN and enhanced production of TGFbeta and IL-4/IL-10. There was an unexpected increase in the production of gammaIFN by lymphocytes in vitro after three doses of oral betaIFN in vivo. These observations have important implications for the use of cytokines to modulate oral tolerance.

Glycoconjugate distribution and mobility on apical membranes of absorptive cells of suckling rat ileum in vivo.

The luminal membrane of ileal absorptive cells in suckling rats includes two domains: microvillar membranes and deep invaginations between microvilli. We examined the fates of foreign macromolecules that bind to anionic or saccharide sites on these domains after infusion into ligated loops in vivo. Cationized ferritin (CF) and ferritin-RCAI (beta-galactosyl) binding sites were distributed over the entire apical membrane. Ligands bound to apical invaginations were rapidly endocytosed, but ligands on microvilli were not. After CF binding, anionic sites on microvilli were mobile in the plane of the membrane and formed CF clusters at the tip and base of each microvillus. RCAI binding sites did not cluster. Wheat germ agglutinin (WGA, sialic acid) labeling was restricted to microvillus tips of mature cells but was dispersed over the microvillar surfaces of lower villus cells. Ferritin conjugates of Concanavalia ensiformis (Con A), Ulex europaeus agglutinin (UEA), and Dolichos biflorus agglutinin (DBA) did not bind to cell surfaces in vivo. Aldehyde fixation dramatically altered lectin binding patterns, resulting in unmasking and labeling of Con A, WGA, and DBA binding sites that were unavailable in vivo.

Co-localization of class II antigen and exogenous antigen in the rat enterocyte.

The role of class II major histocompatibility antigens in classical antigen-presenting cells has been described (Unanue (1984) Annu. Rev. Immunol. 2, 395-428; Watts and McConnell (1987) Rev. Immunol. 5, 461-475). Whether enterocytes, which also express class II antigens, can act as antigen-presenting cells in vivo is not known. One pre-requisite for a role for enterocytes in antigen presentation is an interaction between exogenous antigen and class II antigens. Our results demonstrate that class II antigen and exogenous antigen absorbed from the gastrointestinal tract are co-localized within endocytic compartments and along the basolateral membranes of enterocytes.

Inhibitory effect of calcitonin gene-related peptide and calcitonin on opossum esophageal smooth muscle.

Calcitonin gene-related peptide (CGRP) is widely distributed in the gastrointestinal nerves, including those of the esophagus. The present investigation was undertaken to examine the effect and the mechanism of action of CGRP on the lower esophageal sphincter and esophageal contractions. This peptide caused dose-dependent relaxation of the lower esophageal sphincter. The D50 for inhibitory effect of intraarterial CGRP on the sphincter was 5.0 X 10(-13) mol/kg. Calcitonin gene-related peptide is 3000 times more potent than calcitonin. The effect of CGRP on the lower esophageal sphincter was partially antagonized by tetrodotoxin or black widow spider venom. The inhibitory effect of CGRP on the sphincter appears to be exerted at two levels: (a) at the sphincteric smooth muscle, and (b) at the noncholinergic, nonadrenergic inhibitory neurons. Calcitonin gene-related peptide also exerts a potent inhibitory effect on the peristaltic contraction of the esophageal body in response to swallowing and vagal efferent stimulation. Using immunohistochemical studies we also showed the presence of CGRP-immunoreactive neurons within the myenteric ganglia of the esophagus. These studies suggest that CGRP may play an important role as an inhibitory neurotransmitter in the esophagus.

Diffuse necrotizing retinochoroiditis in a child with AIDS and toxoplasmic encephalitis.

We examined a child with a human immunodeficiency virus (HIV) infection who at 15 months of age developed acute encephalitis, followed 1 week later by a diffuse, uniocular retinochoroiditis. The clinical picture in the right eye was characterized by the occurrence of some intraretinal hemorrhages; punctate, yellow-white, outer retinal lesions temporal to the macula; and a quadrantal, white area of necrotic retina located superotemporally. - The vitreous was remarkably clear, and the left eye was normal. Fluorescein angiography revealed small spots of late hyperfluorescence, vasculitis in the posterior pole, and a persistently hypofluorescent quadrantal superotemporal area. Toxoplasma IgM antibodies that were absent 1 week after birth became detectable in the serum and the cerebrospinal fluid. Serological testing for cytomegalovirus was negative. Neurological signs improved on a specific therapy (pyrimethamine and sulfamethopirazine), but the patient died 2 months later of disseminated cytomegalovirus infection.

Prolactin is transported across the epithelium of the jejunum and ileum of the suckling rat.

Milk prolactin is transferred from the gastrointestinal tract to the circulation of the suckling rat. To identify the site of prolactin penetration and to determine the mechanism by which the hormone traverses the mucosal barrier, we followed the uptake of prolactin from ligated loops of jejunum or ileum in vivo by three methods: autoradiography, transport of prolactin-gold conjugates, and immunocytochemistry. Autoradiographic studies demonstrated specific binding sites for 125I-prolactin on apical membranes of the jejunum and ileum. Excess cold prolactin reduced radiolabel in apical and basal compartments. Gel autoradiography of portal sera showed the presence of intact prolactin and a prolactin fragment following jejunal transport but only a prolactin fragment following ileal transport. Uptake of prolactin-gold conjugates demonstrated that, in the jejunum, label was present at the luminal surface, within endosomal compartments and lysosomes, in basal coated and smooth vesicles, within basal coated pits, and beyond the basolateral surface. In the ileum, label was found at the luminal surface; within the tubulocisternae, endosomal vesicles, lysosomes, and basal smooth vesicles; and beyond the basolateral surface. Immunoreactive prolactin was present throughout the transepithelial pathways. This study demonstrates that prolactin is selectively and nonselectively absorbed in the jejunum and ileum and that the hormone is directed either to the lysosome for degradation or across the epithelium by means of a transcellular pathway.

B cell-deficient (mu MT) mice have alterations in the cytokine microenvironment of the gut-associated lymphoid tissue (GALT) and a defect in the low dose mechanism of oral tolerance.

Peripheral immune tolerance following i.v. administration of Ag has been shown to occur in the absence of B cells. Because different mechanisms have been identified for i.v. vs low dose oral tolerance and B cells are a predominant component of the gut-associated lymphoid tissue (GALT) they may play a role in tolerance induction following oral Ag. To examine the role of B cells in oral tolerance we fed low doses of OVA or myelin oligodendrocyte glycoprotein to B cell-deficient ( microMT) and wild-type C57BL/6 mice. Results showed that the GALT of naive wild-type and microMT mice was characterized by major differences in the cytokine microenvironment. Feeding low doses of 0.5 mg OVA or 250 microg myelin oligodendrocyte glycoprotein resulted in up-regulation of IL-4, IL-10, and TGF-beta in the GALT of wild-type but not microMT mice. Upon stimulation of popliteal node cells, in vitro induction of regulatory cytokines TGF-beta and IL-10 was observed in wild-type but not microMT mice. Greater protection against experimental autoimmune encephalomyelitis was found in wild-type mice. Oral tolerance in microMT and wild-type mice was found to proceed by different mechanisms. Anergy was observed from 0.5 mg to 250 ng in microMT mice but not in wild-type mice. Increased Ag was detected in the lymph of microMT mice. No cytokine-mediated suppression was found following lower doses from 100 ng to 500 pg in either group. These results demonstrate the importance of the B cell for the induction of cytokine-mediated suppression associated with low doses of Ag.

alpha-Actinin and vinculin in normal and thrombasthenic platelets.

Recently, the contractile protein alpha-actinin was identified in normal human platelets by its antigenic cross-reaction with a monospecific antibody to purified muscle alpha-actinin. In this study, we extend that preliminary identification of platelet alpha-actinin. Amino acid analysis, one-dimensional peptide maps, and silver stain analysis on polyacrylamide gels demonstrate that human platelet alpha-actinin shows a greater degree of similarity to smooth muscle alpha-actinin than to striated muscle alpha-actinin. There is no evidence to suggest that alpha-actinin is a glycoprotein. In addition, we find that thrombasthenic platelets, which are deficient in glycoproteins IIb and IIIa (GPIIb and GPIIIa) contain normal amounts of alpha-actinin, confirming the recent finding that alpha-actinin and GPIIIa are different proteins in human platelets. We demonstrate that both normal and thrombasthenic platelets also contain vinculin, a 130,000-dalton polypeptide found in many cell types at sites of end-on attachment of microfilaments to the plasma membrane. Thus, the thrombasthenic defect in GPIIb and GPIIIa does not diminish the content of either alpha-actinin or vinculin.

Uptake and transport of epidermal growth factor by the small intestinal epithelium of the fetal rat.

Epidermal growth factor may play an important part in postnatal gastrointestinal development. However, little is known of its role prenatally. The aim of this study was to detect epidermal growth factor in amniotic fluid and to study its uptake and transfer across the epithelium of fetal rat small intestine. Anesthetized 20-day gestation rats underwent caesarean section. Three fetuses were exteriorized, their abdomens were opened, and ligated loops of proximal and distal small intestine were infused with 100 micrograms epidermal growth factor. Infused segments were removed 30 min later and processed for electron microscopy, and tissue was embedded in LR gold resin. Sections were treated with rabbit anti-rat epidermal growth factor antibody, followed by 5 nm gold-labeled goat anti-rabbit immunoglobulin before staining. Epidermal growth factor was measured in amniotic fluid by radioimmunoassay. In the proximal and distal small intestine epidermal growth factor was found membrane-associated along the luminal surface of microvilli, within apical invaginations and endosomal compartments, free from the membrane in multivesicular bodies, within large clear vesicles, basal vesicles and in association with the basolateral membrane and beyond. Epidermal growth factor was found in amniotic fluid at a concentration of 0.38 +/- 0.07 (SD) ng/ml. This study shows that epidermal growth factor is present in amniotic fluid and is transported across the epithelium of fetal rats by an endocytotic process in both the upper and lower small intestine. It is likely that amniotic fluid epidermal growth factor plays a part in intestinal mucosal development, and may be active systemically after transepithelial passage in utero.