Design and function of targeted endocannabinoid nanoparticles.

Nanoparticles and nano-delivery systems are constantly being refined and developed for biomedical applications such as imaging, gene therapy, and targeted delivery of drugs. Nanoparticles deliver beneficial effects by both release of their cargo and by liberation of their constitutive structural components. The N-acylethanolamines linoleoyl ethanolamide (LEA) and oleoyl ethanolamide (OEA) both exhibit endocannabinoid-like activity. Here, we report on their ability to form nanoparticles that when conjugated with tissue-specific molecules, are capable of localizing to specific areas of the body and reducing inflammation. The facilitation of pharmacological effects by endocannabinoids at targeted sites provides a novel biocompatible drug delivery system and a therapeutic approach to the treatment, patient management and quality of life, in conditions such as arthritis, epilepsy, and cancer.

Complexation of novel thiomers and insulin to protect against in vitro enzymatic degradation - towards oral insulin delivery.

A significant barrier to oral insulin delivery is its enzymatic degradation in the gut. Nano-sized polymer-insulin polyelectrolyte complexes (PECS) have been developed to protect insulin against enzymatic degradation. Poly(allylamine) (Paa) was trimethylated to yield QPaa. Thiolation of Paa and QPaa was achieved by attaching either N-acetylcysteine (NAC) or thiobutylamidine (TBA) ligands (Paa-NAC/QPaa-NAC and Paa-TBA/QPaa-TBA thiomers). PEC formulations were prepared in Tris buffer (pH 7.4) at various polymer: insulin mass ratios (0.2:1-2:1). PECS were characterized by %transmittance of light and photon correlation spectroscopy. Insulin complexation efficiency and enzyme-protective effect of these complexes were determined by HPLC. Complexation with insulin was found to be optimal at mass ratios of 0.4-1:1 for all polymers. PECS in this mass range were positively-charged (20-40 mV), nanoparticles (50-200 nm), with high insulin complexation efficiency (>90%). Complexation with TBA polymers appeared to result in disulfide bridge formation between the polymers and insulin. In vitro enzymatic degradation assays of QPaa, Paa-NAC, and QPaa-NAC PECS showed that they all offered some protection against insulin degradation by trypsin and α-chymotrypsin, but not from pepsin. QPaa-NAC complexes with insulin are the most promising formulation for future work, given their ability to offer protection against intestinal enzymes. This work highlights the importance of optimizing polymer structure in the delivery of proteins.

In-vitro evaluation of the effect of polymer structure on uptake of novel polymer-insulin polyelectrolyte complexes by human epithelial cells.

The biocompatibility and cellular uptake of polymer, insulin polyelectrolyte complexes (PECs) prepared using polyallylamine-based polymers was evaluated in-vitro using Caco-2 cell monolayers as a predictive model for human small intestinal epithelial cells. Poly(allyl amine) (PAA) and Quaternised PAA (QPAA) were thiolated using either carbodiimide mediated conjugation to N-acetylcysteine (NAC) or reaction with 2-iminothiolane hydrochloride yielding their NAC and 4-thiobutylamidine (TBA) conjugates, respectively. The effect of polymer quaternisation and/or thiolation on the IC50 of PAA was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay carried out on Caco-2 cells (with and without a 24 h recovery period after samples were removed). Uptake of PECs by Caco-2 cells was monitored by microscopy using fluorescein isothiocyanate (FITC) labelled insulin and rhodamine-labelled polymers at polymer:insulin ratios (4:5) after 0.5, 1, 2 and 4 h incubation in growth media (±calcium) and following pre-incubation with insulin. MTT results indicated that quaternisation of PAA was associated with an improvement in IC50 values; cells treated with QPAA (0.001-4 mg mL(-1)) showed no signs of toxicity following a 24 h cell recovery period, while thiolation of QPAA resulted in a decrease in the IC50. Cellular uptake studies showed that within 2-4 h, QPAA and QPAA-TBA insulin PECs were taken up intracellularly, with PECs being localised within the perinuclear area of cells. Further investigation showed that uptake of PECs was unaffected when calcium-free media was used, while presaturating insulin receptors affected the uptake of QPAA, insulin PECs, but not QPAA-TBA PECs. The biocompatibility of PAA and uptake of insulin was improved by both thiol and quaternary substitution.

New sources and instrumentation for neutrons in biology.

Neutron radiation offers significant advantages for the study of biological molecular structure and dynamics. A broad and significant effort towards instrumental and methodological development to facilitate biology experiments at neutron sources worldwide is reviewed.

Scattering curves of ordered mesoscopic materials.

Analytical expressions for the scattering functions of ordered mesoscopic materials are derived and compared to experimentally determined scattering curves. Ordered structures comprising spheres (fcc, bcc, hcp, sc), cylinders (hex, sq), and lamellar structures are considered. The expressions take into account particle size distributions and lattice point deviations, domain size, core/shell structures, as well as peak shapes varying analytically between Lorentzian and Gaussian functions. The expressions allow one to quantitatively describe high-resolution synchrotron small-angle X-ray (SAXS) and neutron scattering (SANS) curves from lipid and block copolymer lyotropic phases, core/shell nanoparticle superstructures, ordered nanocomposites, and ordered mesoporous materials. In addition, the diffuse out-of-plane scattering of grazing incidence GISAXS and GISANS experiments of laterally ordered thin films can be quantitatively analyzed.

Altered L-selectin expression in lymphocytes and increased adhesion to endothelium in patients with diabetic retinopathy.

AIM: To investigate L-selectin expression and shedding in patients with and without retinopathy and to determine if any observed changes are reflected by a functional change in the adhesion of leucocytes to an endothelial monolayer. METHODS: Age matched diabetic patients (26 with retinopathy, 19 without retinopathy) were compared to 24 non-diabetic controls to determine L-selectin surface protein expression, L-selectin mRNA production, and serum L-selectin levels by flow cytometry, RT-PCR, and ELISA, respectively. An adhesion assay was used to determine the binding of lymphocytes from the respective test groups to a monolayer of human endothelial cells. RESULTS: Significantly reduced (p = 0.004) L-selectin expression was demonstrated on lymphocytes (CD3+) from patients with diabetes compared to controls, the lowest levels being found in those with diabetic retinopathy (p = 0.004). L-selectin mRNA levels (p = 0.007) were significantly higher in the retinopathy group than in the no retinopathy group. Serum L-selectin levels were significantly higher (p = 0.04) in those with retinopathy compared to controls. Lymphocyte adhesion relative to control (100%) was essentially unchanged (84.0% (SD 27.7%), p = 0.15) for diabetic patients with no retinopathy and was markedly increased (192% (37.6%)) for those with retinopathy (p = 0.0001). CONCLUSION: Lymphocyte activation, reduced surface L-selectin, increased circulating L-selectin, and a corresponding increase in adhesion of patients' cells using an in vitro assay, is evident in people with diabetic retinopathy. This suggests a role for lymphocyte activation in the pathogenesis of diabetic retinopathy.

Shear-induced alignment of self-associated hemoglobin in human erythrocytes: small angle neutron scattering studies.

Small angle neutron scattering (SANS) was performed on suspensions of actively metabolising human erythrocytes in the constant shear field induced by a Couette cell. The SANS pattern recorded on a two-dimensional detector was a function of the shear rate; at zero shear, the SANS pattern had radial symmetry around the direction of the beam. The radial average of the SANS pattern consisted of a broad intensity maximum superimposed on a decay. The intensity maximum at q = 0.1 A(-1) was attributed to isotropically oriented self-associated complexes of the tetrameric oxygen transport protein hemoglobin inside the erythrocytes. A flow curve of the cell suspension was used to identify at what shear rate a suspension of uniaxially oriented ellipsoidal cells is produced. The radial symmetry of the SANS patterns persisted until the shear rate was sufficient to produce a suspension of uniaxially oriented ellipsoidal cells. Again, an intensity maximum was present in directions parallel and orthogonal to the shear axis, but this intensity maximum was superimposed upon quite different intensity decays in each direction from that of the primary neutron beam. The angular range of the SANS instrument was limited, however the results from shear-induced structural changes is consistent with a model that involves hemoglobin complexes that are aligned with respect to the plasma membranes of the elongated cells.

High glucose concentration decreases insulin-like growth factor type 1-mediated mitogen-activated protein kinase activation in bovine retinal endothelial cells.

Clinical trials have incontrovertibly demonstrated that the onset and progression of diabetic retinopathy (DR) is influenced by the control of glucose levels in patients. In the present study, we examined the effect of glucose concentration on the responsiveness of bovine retinal endothelial cells (BREC) to insulin-like growth factor type 1 (IGF-1). Retinal endothelial cells were isolated from bovine retina and cultured in 5 or 20 mmol/L glucose with or without 100 ng/mL IGF-1. The level of cell growth and p42/44 and p38 mitogen-activated protein kinase (MAPK) activation was determined using the alamarBlue (Serotech) assay and Western blotting, respectively. IGF-1 significantly enhanced cell growth in BREC exposed to 5 mmol/L glucose but not in cells exposed to high glucose concentrations (20 mmol/L). IGF-1 induced a transient activation of p42/44 MAPK, with peak activation at 15 minutes in cells exposed to 5 mmol/L glucose; however, no increase in p42/44 MAPK was evident at the higher glucose concentration of 20 mmol/L. There was no significant change in the level of p38 MAPK during the time period examined when IGF-1 was also present. However, high glucose concentrations alone increased the level of p38 MAPK after 60 minutes and the level of p42/44 MAPK after only 15 minutes exposure in 20 mmol/L glucose. Thus, BREC exposed to high glucose concentrations are not sensitive to IGF-1 and this is due, at least in part, to a reduced activation of the p42/44 MAPK pathway. Furthermore, the presence of IGF-1 appears to exert a protective effect on the cells in high glucose concentration by preventing progression through the cell cycle.

Does the medial temporal lobe bind phonological memories?

The medial temporal lobes play a central role in the consolidation of new memories. Medial temporal lesions impair episodic learning in amnesia, and disrupt vocabulary acquisition. To investigate the role of consolidation processes in phonological memory and to understand where and how, in amnesia, these processes begin to fail, we reexamined phonological memory in the amnesic patient HM. While HM's word span performance was normal, his supraspan recall was shown to be markedly impaired, with his recall characterized by a distinctive pattern of phonological errors, where he recombined phonemes from the original list to form new response words. These were similar to errors observed earlier for patients with specifically semantic deficits. Amnesic Korsakoff's patients showed a similar, though much less marked, pattern. We interpret the data in terms of a model of lexical representation where temporal lobe damage disrupts the processes that normally bind semantic and phonological representations.

Regulation of plasminogen activation by TGF-beta in cultured human retinal endothelial cells.

BACKGROUND/AIMS: Regulation of plasmin mediated extracellular matrix degradation by vascular endothelial cells is important in the development of angiogenesis. The aim was to determine whether transforming growth factor beta (TGF-beta) affected the regulation of components of the plasminogen system by human retinal endothelial cells, in order to define more clearly the role of TGF-beta in retinal angiogenesis in the context of diabetes mellitus. METHODS: Human retinal endothelial cells (HREC) were isolated from donor eyes and used between passages 4-8. The cells were cultured in medium supplemented with 2, 5, 15, or 25 mM glucose, plus or minus TGF-beta (1 ng/ml). The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitor type 1 (PAI-1) in cell conditioned medium were determined by ELISA and the level of PAI-1 mRNA was determined using northern hybridisation. Cell associated plasminogen activity was determined using a clot lysis assay and a chromogenic assay. RESULTS: Under basal conditions (5 mM glucose), HREC produced PAI-1, t-PA, and trace amounts of u-PA. Cell surface plasminogen activation observed by lysis of fibrin or by cleavage of chromogenic substrate, was mediated by t-PA. Glucose at varying concentrations (2-25 mM) had no significant effect on t-PA mediated clot lysis. In contrast, treatment with TGF-beta resulted in increased synthesis of PAI-1 protein and mRNA. The increased expression of the PAI-1 mRNAs by TGF-beta did not occur uniformly, the 2.3 kb mRNA transcript was preferentially increased in comparison with the 3.2 kb mRNA (p<0.05). CONCLUSIONS: These data demonstrate that TGF-beta increases PAI-1 and decreases cell associated lysis. This is sufficient to decrease the normal lytic potential of HREC.

The role of speech production in auditory-verbal short-term memory: evidence from progressive fluent aphasia.

We report investigations of auditory-verbal short-term memory (AVSTM) in a patient with progressive fluent anomic aphasia. Despite having apparently normal AVSTM as measured by digital span, FM was significantly impaired in immediate serial recall of short sequences of familiar words, and even in reproducing a single word after a filled delay of just a few seconds. In both tasks, unlike normal subjects, she produced numerous phonological errors, often consisting of phonological segments from the intended target word concatenated with segments from other words in the stimulus sequence. Her success in these tasks was modulated (i) consistently by word frequency (high > low), (ii) inconsistently by word imageability (high > low), and (iii) most dramatically by 'nameability': that is, FM was much more likely to reproduce a word correctly in AVSTM if it was a word that she could also produce successfully in picture-naming tasks. On the basis of these and additional experiments designed to exclude other interpretations, we conclude that AVSTM may be crucially supported by activation of the lexical phonological representations responsible for production of content words in speech.

Regulation of transforming growth factor-beta, basic fibroblast growth factor, and vascular endothelial cell growth factor mRNA in peripheral blood leukocytes in patients with diabetic retinopathy.

In the present study, we examined the effect of glucose concentration on the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-beta) mRNA using reverse transcriptase-polymerase chain reaction (RT-betaCR) in normal healthy leukocytes in vitro and in leukocytes from patients with type 1 diabetes mellitus. In vitro, the level of TGF-beta mRNA was altered in response to the glucose concentration (maximum at 10 mmol/L), while bFGF mRNA remained relatively constant and VEGF mRNA varied with no clear correlation with the glucose concentration. Leukocytes from type 1 patients showed no difference in bFGF or TGF-beta mRNA levels compared with age-matched healthy controls. However, VEGF mRNA was significantly lower in type 1 patients compared with controls (P < .05). When the patients were subtyped according to the severity of retinopathy, the level of TGF-beta mRNA was elevated selectively in patients with evidence of active new retinal vessels (P < .01) and VEGF121 mRNA was reduced in patients with mild to moderate retinopathy. Thus, leukocyte growth factor mRNAs respond to acute changes in the glucose concentration in vitro, and are differentially expressed in type 1 diabetic patients during the course of the disease.

A model system for the study of human retinal angiogenesis: activation of monocytes and endothelial cells and the association with the expression of the monocarboxylate transporter type 1 (MCT-1).

AIMS/HYPOTHESIS: The growth of retinal vessels is associated with a number of disease conditions, including diabetic retinopathy and proliferative vitreo-retinopathy. In this study we describe a model of human retinal angiogenesis and show how this may be used to explain the mechanisms that are associated with the growth of new retinal vessels. METHODS: A 4 mm diameter disc of retinal tissue was placed within a fibrin matrix and the appearance was monitored daily by light microscopy. Immunohistochemical techniques were used for the detection of, glial fibrillary acidic protein, CD68, the Ki-67 antigen, vascular endothelial growth factor, monocarboxylate transporter type 1 and von Willebrand's factor. RESULTS: Vessels were evident extending from the periphery of the explant and the activation of endothelial cells was shown by immuno-peroxidase staining of paraffin embedded sections of the explants for the expression of the Ki-67 antigen, a marker of cell proliferation. The expression of glial fibrillary acidic protein and von Willebrand's factor increased with duration in culture and the presence of activated macrophages or microglia or both was shown by positive immunoreactivity for CD68 and Ki-67 and were identified by day 3. The presence of endogenous vascular endothelial growth factor and the activation of monocarboxylate transporter type 1 by vascular endothelial growth factor, showed the involvement of specific growth factors. CONCLUSION/INTERPRETATION: The explant model provides evidence for the involvement of macrophages and glial fibrillary acidic protein activation in human retinal angiogenesis and for the expression of monocarboxylate transporter type 1, which is likely to be important in the use of lactate in the hypoxic retina.

Glucose-mediated regulation of transforming growth factor-beta (TGF-beta) and TGF-beta receptors in human retinal endothelial cells.

PURPOSE: Diabetic retinopathy is a micro-angiopathy affecting predominantly small vessels of the retina. Clinical trials have demonstrated a strong association between tight glucose control and a reduction in the incidence and the severity of diabetic retinopathy. Transforming growth factor beta (TGF-beta) is involved in the control of endothelial cell proliferation, adhesion, and deposition of extracellular matrix, thus TGF-beta may play a role in the control of endothelial cell proliferation seen in the disease. We wished to investigate the regulation of transforming growth factor beta and its receptors (type I and II) in human retinal endothelial cells exposed to a range of glucose concentrations. METHODS: Human retinal endothelial cells were isolated from donor eyes, cultured in vitro and exposed to a range of glucose concentrations (0-25 mmol/l). TGF-beta protein and mRNA levels were determined by ELISA and Northern analysis, respectively. The binding affinities and TGF-beta receptor numbers were defined using a binding assay. RESULTS: Northern hybridisation and ELISA showed that after 8 hours, the level of TGF-beta mRNA and protein was significantly higher at 15mmol/l compared to 5, 20 or 25mmol/ l. Binding assays showed that for high glucose (25 mmol/l), human retinal endothelial cells express a population of TGF-beta receptors with higher affinity for its ligand than at 5 or 15 mmol/l. CONCLUSIONS: These results demonstrate that glucose regulates TGF-beta mRNA and protein production and also TGF-beta receptor expression in human retinal endothelial cells. Thus, the glucose-mediated changes that occur in diabetic patients may expose human retinal endothelial cells to potential angiogenic factors which may influence disease progression.

Cytokine regulation of RANTES production by human retinal pigment epithelial cells.

The mechanism whereby inflammatory cells gain access to the retina in posterior intraocular inflammatory disease remains unclear. The chemokine RANTES has the potential to influence the migration of memory T cells and monocytes across the blood-retinal barrier during inflammatory eye disease. We have therefore examined the production of RANTES by cultured human retinal pigment epithelial cells (RPE), which form a part of the blood-retinal barrier, in response to cytokines likely to be present in the microenvironment. IL-1 beta and TNF alpha stimulated RANTES production by these cells. IFN-gamma acted synergistically with TNF alpha to increase RANTES production. In contrast, IL-4 downregulated RANTES production stimulated by TNF alpha. RT-PCR studies showed that RANTES mRNA from RPE followed the same pattern of expression in response to cytokines as did RANTES production indicating that RANTES production was controlled at, or prior to, transcription. RANTES is produced in vitro by RPE in response to the proinflammatory cytokines IL-1 beta, TNF alpha, and IFN-gamma and is therefore likely to play a role in the development of the inflammatory eye disease endogenous posterior uveitis.

Evidence for atrial natriuretic factor induced natriuretic peptide receptor subtype switching in rat proximal tubular cells during culture.

Culture and natriuretic peptide dependent changes in the expression of the natriuretic peptides atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) and the natriuretic peptide receptors A, B, and C in primary cultures of rat proximal tubular cells were demonstrated using polymerase chain reaction analysis and cyclic guanosine monophosphate response to ANF and CNP. Freshly isolated cells expressed mRNA coding for the natriuretic peptide receptor C only, with no expression of the natriuretic peptides or the natriuretic peptide receptors A or B. At confluence natriuretic peptide receptor C expression was lost, while mRNA transcripts for both ANF and BNP and the A and B receptors became apparent. The appearance of mRNA transcripts for the natriuretic peptide receptors A and B during cell growth correspond with a significant increase in the cyclic guanosine monophosphate response to both ANF and CNP, confirming the presence of functionally active guanylate cyclase linked A and B natriuretic peptide receptors. The observed changes in peptide receptor expression during culture were preceded by changes in natriuretic peptide mRNA expression, suggesting the possibility that natriuretic peptide receptor subtype switching may be under the control of endogenous peptide release. Incubation of freshly isolated proximal tubular cells with ANF, BNP, or CNP for 3 h induced similar changes in receptor expression. Incubation with ANF induced expression of the natriuretic peptide receptor B and CNP while inhibiting natriuretic peptide receptor C. Incubation with BNP induced expression of the natriuretic peptide receptor B and CNP. Incubation with CNP induced expression of the natriuretic peptide receptors A and B and CNP. These results suggest that primary cultures of rat proximal tubular cells may experience natriuretic peptide and natriuretic peptide receptor subtype switching as they approach confluence under the control of endogenously expressed natriuretic peptides.

Glucose-dependent regulation of DNA synthesis in bovine retinal endothelial cells.

PURPOSE: Clinical trials have demonstrated that the onset and progression of diabetic retinopathy is influenced by the glucose control of the patient. The disease is characterised by the coexistence of impaired cell growth and excessive cell proliferation, and we wished to determine the effect that glucose has upon these parameters. METHODS: Bovine retinal endothelial cells were exposed to a range of glucose concentrations from 0-25 mmol/l. The level of DNA synthesis and cell number was then determined using pulse labelling with tritiated thymidine and a Coomassie blue dye-based assay, respectively. RESULTS: The level of DNA synthesis declined significantly as the concentration of glucose increased. DNA synthesis was further decreased by the presence of an inhibitor of PI3 kinase (Wortmannin). The decline in DNA synthesis was abrogated by the presence of a protein kinase C (PKC) inhibitor or by incubating the cells with antibodies specific for the GLUT-1 and GLUT-3 specific isoforms of glucose transporter proteins. TGF-beta antibody significantly increased the level of DNA synthesis in cells exposed to high concentrations of glucose. The changes that are observed in the level of DNA synthesis was not coincident with any significant changes in cell number as measured by the Coomassie blue assay. CONCLUSIONS: This demonstrated that the decline in DNA synthesis is dependent upon the entry of glucose into the cells and that this is mediated via a PKC dependent pathway.

Insulin and insulin-like growth factor-I responsiveness and signalling mechanisms in C2C12 satellite cells: effect of differentiation and fusion.

In proliferating C2C12 myoblasts, serum and physiological concentrations of insulin and IGF-I stimulated protein synthesis and RNA accretion. After fusion, the multinucleated myotubes remained responsive to serum but not to insulin or IGF-I, even though both insulin and type-I IGF receptor mRNAs increased in abundance. Protein synthetic responses to insulin and IGF-I in myoblasts were not inhibited by dexamethasone, ibuprofen or Ro-31-8220, thus phospholipase A2, cyclo-oxygenase and protein kinase C did not appear to be involved in the signalling mechanisms. Neither apparently were polyphosphoinositide-specific phospholipase C or phospholipase D since neither hormone increased inositol phosphate, phosphatidic acid, choline or phosphatidylbutanol production. Only the phosphatidylinositol-3-kinase inhibitor, wortmannin, and the 70 kDa S6-kinase inhibitor, rapamycin, wholly or partially blocked the effects of insulin and IGF-I on protein synthesis. 2-deoxyglucose uptake remained responsive to insulin and IGF-I after fusion and was also inhibited by wortmannin. The results suggest that the loss of responsiveness after fusion is not due to loss of receptors, but to the uncoupling of a post-receptor pathway, occurring after the divergence of the glucose transport and protein synthesis signalling systems, and that, if wortmannin acts at a single site, this is prior to that point of divergence.