Designing tailored biomaterial surfaces to direct keratinocyte morphology, attachment, and differentiation.

Precisely engineering the surface chemistry of biomaterials to modulate the adsorption and functionality of biochemical signaling molecules that direct cellular functions is critical in the development of tissue engineered scaffolds. Specifically, this study describes the use of functionalized self-assembled monolayers (SAMs) as a model system to assess the effects of biomaterial surface properties on controlling fibronectin (FN) conformation and concentration as well as keratinocyte function. By systematically analyzing FN adsorption at low and saturated surface densities, we distinguished between SAM-dependent effects of FN concentration and conformation on presenting cellular binding domains that direct cellular functions. Quantitative image analyses of immunostained samples showed that modulating the availability of the FN synergy site directly correlated with changes in keratinocyte attachment, spreading, and differentiation, through integrin-mediated signaling mechanisms. The results of this study will be used to elucidate design features that can be incorporated into dermal equivalents and percutaneous implants to enhance the rate of re-epithelialization and tissue regeneration. Furthermore, these findings indicate that SAM-based model systems are a valuable tool for designing and investigating the development of scaffolds that regulate the conformation of extracellular matrix cues and cellular functions that accelerate the rate of tissue regeneration.

Joint cytokine quantification in two rodent arthritis models: kinetics of expression, correlation of mRNA and protein levels and response to prednisolone treatment.

Biomarker quantification in disease tissues from animal models of rheumatoid arthritis (RA) can help to provide insights into the mechanisms of action of novel therapeutic agents. In this study we validated the kinetics of IL-1beta, TNF-alpha and IL-6 mRNA and protein expression levels in joints from DBA/1OlaHsd murine collagen-induced arthritis (CIA) and Lewis rat Streptococcal cell wall (SCW)-induced arthritis by real-time polymerase chain reaction (PCR) TaqMan and Enzyme-linked immunosorbent assay (ELISA). Prednisolone was used as a reference to investigate any correlation between clinical response and cytokine levels at selected time-points. To our knowledge this is the first report showing a close pattern of expression between mRNA and protein for IL-1beta and IL-6, but not for TNF-alpha, in these two models of RA. The kinetics of expression for these biomarkers suggested that the optimal sampling time-points to study the effect of compounds on both inflammation and cytokine levels were day 4 postonset in CIA and day 3 after i.v challenge in SCW-induced arthritis. Prednisolone reduced joint swelling through a mechanism associated with a reduction in IL-1beta and IL-6 protein and mRNA expression levels. At the investigated time points, protein levels for TNF-alpha in arthritic joints were lower than the lower limit of detection of the ELISA, whereas mRNA levels for this cytokine were reliably detected. These observations suggest that RT-PCR TaqMan is a sensitive technique that can be successfully applied to the quantification of mRNA levels in rodent joints from experimental arthritis models providing insights into mechanisms of action of novel anti-inflammatory drugs.

Effects of a PEGylated soluble TNF receptor type 1 (PEG sTNF-RI) on cytokine expression in adjuvant arthritis.

OBJECTIVE: To investigate the effects of TNF blocking therapy on synovial immune activity in rat adjuvant arthritis (AA) by measuring mRNA expression of key macrophage and T cell cytokines during PEG sTNF-RI treatment (10mg/kg) on days 8, 10 and 12. METHODS: Paw volume was assessed every 3-4 days. Ankles were removed for quantitative radiology and histology and synovial membrane removed to determine cytokine mRNA expression using semi-quantitative RT-PCR. T cells in joints were quantified by immunohistochemistry. RESULTS: Paw volume was significantly decreased in rats treated with PEG STNF-RI from days 12 to 17. Histology scores and synovial T cell numbers were reduced on days 13 and 17 and radiology scores significantly reduced on day 13. Expression of synovial TNF, IFN-gamma, IL-17, IL-2 and IL-4 mRNA was unchanged in treated rats and TGF-beta expression was significantly increased at day 13. CONCLUSIONS: PEG sTNF-RI attenuates AA and disease recurs after treatment ceases, similar to human rheumatoid arthritis. Continued TNF production and/or ongoing T cell activity, may explain the recrudescence of disease once treatment is stopped.

The in vivo effects of tumour necrosis factor blockade on the early cell mediated immune events and syndrome expression in rat adjuvant arthritis.

Anti-TNF therapy is effective in rheumatoid arthritis (RA); however, its mechanisms of action are incompletely understood. T cell-driven mechanisms are thought to play an important role in RA and the effects of TNF blockade on these mechanisms are unclear. Adjuvant arthritis (AA) is a T cell dependent model of inflammatory arthritis. The aims of this study were to investigate the effects of TNF blockade on in vivo T cell cytokine expression and to clarify the role of TNF in the inguinal lymph nodes (ILN) in early arthritis. AA was induced in male DA rats. Rats received either 3 mg/kg or 10 mg/kg PEG sTNF-RI at days 0, 2 and 4 postinduction or 10 mg/kg anti-TNF antibody on day of arthritis induction. Control rats received either saline or normal sheep serum. Paw volume was assessed every 3-4 days. Rats were sacrificed on days 0, 6, 13 and 21 postinduction. Ankles were removed for quantitative radiology and histology. Synovium and ILN were removed for cell culture and to determine mRNA expression of cytokines using semiquantitative RT-PCR. TNF and IFN-gamma protein production was measured using a bioassay and an ELISA. TNF blockade did not suppress mRNA expression of T cell cytokines in the ILN of rats in the early phase of AA, suggesting ongoing T cell activity. TNF protein production by ILN cells in culture was reduced in PEG sTNF-RI treated rats, although mRNA expression was increased in the ILN prior to culture. Early administration of PEG sTNF-RI did not attenuate AA, in contrast to an anti-TNF antibody, which suppressed disease. A shorter half-life for the PEG sTNF-RI compared with the anti-TNF antibody or the development of anti-PEG sTNF-RI antibodies may account for these results.

The kappa-opioid agonist, asimadoline, alters cytokine gene expression in adjuvant arthritis.

OBJECTIVE: We have previously found that the kappa-opioid agonist, asimadoline, attenuates adjuvant arthritis in a dose-dependent, antagonist-reversible manner. To elucidate possible mechanisms, we investigated the effects of asimadoline (5 mg/kg/day i.p.) or vehicle on in vivo cytokine expression and T-cell recruitment in adjuvant arthritis. METHODS: Arthritis severity was assessed every 3-4 days for 21 days. Rats were killed on days 0, 13 and 21 post-induction and synovial membrane and inguinal lymph nodes were removed for mRNA extraction. Changes in cytokine mRNA expression were measured using reverse transcription-polymerase chain reaction (RT-PCR) and densitometry. T cells in joints were quantified by immunohistochemistry. RESULTS: Asimadoline significantly decreased arthritis severity at day 13, with a concomitant decrease in synovial membrane expression of cytokines interleukin-17 and transforming growth factor-beta (TGF-beta) mRNA at day 13, and no change in T cell numbers in the joints of arthritic rats. By contrast, in the inguinal lymph nodes, expression of tumour necrosis factor was increased at day 13 and TGF-beta mRNA was increased throughout. CONCLUSION: An altered balance, therefore, in the pro- and anti-inflammatory effects of TGF-beta by asimadoline might explain its striking anti-arthritic actions.

Cytokine expression and synovial pathology in the initiation and spontaneous resolution phases of adjuvant arthritis: interleukin-17 expression is upregulated in early disease.

The aim of this study was to understand the immune processes controlling the initiation and spontaneous resolution of adjuvant arthritis (AA). We investigated synovial T-cell recruitment and mRNA expression of IL-17 and other important disease related cytokines, IFN-gamma, IL-2, IL-4, TNF and TGF-beta in inguinal lymph node (ILN) and synovial membrane (SM). Arthritis severity was assessed by a numerical rating score and rats were sacrificed every 3--4 days postadjuvant induction. Further assessment involved quantitative radiology and histology of the ankle joints on each day, and the ILN and SM were removed for RNA extraction. Cytokine mRNA expression was measured using RT-PCR and densitometry. Paraffin sections of rat ankle joints were stained for T-cells (CD3) by immunohistochemistry. In the ILN, there was an increase in IL-17, TNF and IFN-gamma expression in the early stages of disease, with a secondary sustained increase in IFN-gamma expression. In the SM, there was expression of T-cell cytokines in early arthritis (day 13), and prolonged TNF and TGF-beta expression, which reflected disease progression. IL-4 mRNA expression increased in the later stages of AA. Synovial T-cell numbers transiently increased at day 6, and remained high from days 13--28. Increased pro-inflammatory cytokine expression, including IL-17, in the ILN reflects the initiating events in the early stage of disease. IL-17 may therefore play an important role in the pathogenesis of AA. The increase in IL-4 (an anti-inflammatory cytokine) in the SM in the later stages of AA suggests that IL-4 is involved in the spontaneous resolution of AA. The initial increase in IFN-gamma in the ILN may reflect a pro-inflammatory response, while the prolonged secondary increase may indicate activation of regulatory T-cells.

Effects of the peripherally selective kappa-opioid asimadoline, on substance P and CGRP mRNA expression in chronic arthritis of the rat.

We have previously shown that the kappa-opioid agonist, asimadoline, produces time-dependent changes in neuropeptide concentrations in the joints of rats with chronic arthritis. We hypothesized that asimadoline acts on peripheral terminals to modulate substance P (SP) release. To address this hypothesis, here we have examined neuropeptide expression in their source cells in dorsal root ganglia (DRG) that innervate the joint, as well as in non-neuronal tissue, after treatment with asimadoline. We found an increased production of SP and CGRP in untreated chronic arthritic animals which supports our previous finding of increased SP content in the joint. More importantly, the kappa-opioid asimadoline reduced the expression of both SP and calcitonin gene-related peptide-alpha (alpha-CGRP) in DRG cells but had no effect on the very low expression of neuropeptides in non-neuronal tissue. The fact that SP synthesis is attenuated by asimadoline accords with our hypothesis that the increased tissue levels of SP result from kappa-mediated pre-synaptic inhibition of release leading to augmented tissue stores. These in vivo data confirm literature findings that opioids inhibit SP release from peripheral endings of primary afferent fibres.

Hydrophobic amino acids define the carboxylation recognition site in the precursor of the gamma-carboxyglutamic-acid-containing conotoxin epsilon-TxIX from the marine cone snail Conus textile.

To identify the amino acid sequence of the precursor of the Gla-containing peptide, epsilon-TxIX, from the venom of the marine snail Conus textile, the cDNA encoding this peptide was cloned from a C. textile venom duct library. The cDNA of the precursor form of epsilon-TxIX encodes a 67 amino acid precursor peptide, including an N-terminal prepro-region, the mature peptide, and four residues posttranslationally cleaved from the C-terminus. To determine the role of the propeptide in gamma-carboxylation, peptides were designed and synthesized based on the propeptide sequence of the Gla-containing conotoxin epsilon-TxIX and used in assays with the vitamin K-dependent gamma-glutamyl carboxylase from C. textile venom ducts. The mature acarboxy peptide epsilon-TxIX was a high K(M) substrate for the gamma-carboxylase. Synthetic peptides based on the precursor epsilon-TxIX were low K(M) substrates (5 microM) if the peptides included at least 12 residues of propeptide sequence, from -12 to -1. Leucine-19, leucine-16, asparagine-13, leucine-12, leucine-8 and leucine-4 contribute to the interaction of the pro-conotoxin with carboxylase since their replacement by aspartic acid increased the K(M) of the substrate peptide. Although the Conus propeptide and the propeptides of the mammalian vitamin K-dependent proteins show no obvious sequence homology, synthetic peptides based upon the structure of pro-epsilon-TxIX were intermediate K(M) substrates for the bovine carboxylase. The propeptide of epsilon-TxIX contains significant alpha-helix, as estimated by measurement of the circular dichroism spectra, but the region of the propeptide that plays the dominant role in directing carboxylation does not contain evidence of helical structure. These results indicate that the gamma-carboxylation recognition site is defined by hydrophobic residues in the propeptide of this conotoxin precursor.

The in vivo effects of milrinone on the airways of cystic fibrosis mice and human subjects.

Previous studies have indicated that milrinone, a specific type III phosphodiesterase inhibitor, may be able to induce chloride secretion in cystic fibrosis (CF) tissues. We have now assessed the effect of this agent in vivo on the nasal epithelium of CF mutant mice and also in the nose and lungs of human subjects with CF. Wild-type mice showed a small hyperpolarization of the nasal potential difference (PD) in response to milrinone (100 microM, 1.6 +/- 0.6 mV, n = 8, P < 0.05). In contrast, CF mice carrying either the most common human mutation of the gene for the CF transmembrane regulator (CFTR), DeltaF508 (protein mislocalized), or the G551D mutation (protein normally localized) failed to demonstrate this response. Milrinone perfused alone had no significant effect on the baseline nasal PD of human subjects without CF (14.7 +/- 4.0 mV preperfusion; 15.3 +/- 4.6 mV postperfusion), but significantly (P < 0.05) augmented the hyperpolarization induced by a subsequently perfused low-chloride solution (with milrinone, 36.8 +/- 3.0 mV, n = 6; without milrinone, 18.1 +/- 2.2 mV, n = 19). In contrast, in human subjects with CF (n = 6), milrinone alone significantly (P < 0. 05) altered the nasal baseline PD (52.2 +/- 3.3 mV preperfusion; 57. 4 +/- 4.2 mV, postperfusion) but not the subsequent responses to the low-chloride solution (with milrinone, 1.1 +/- 2.2 mV, n = 4; without milrinone, 0.6 +/- 0.5 mV, n = 28) or to isoproterenol (100 microM). In a separate study in subjects (n = 6) with the DeltaF508 mutation, nasal coadministration of milrinone with isoproterenol produced no effect in the presence of amiloride and a low-chloride solution (-0.8 +/- 0.5 mV). This was also the case in the nasal epithelium of CF subjects (n = 4) carrying at least one G551D allele (-0.3 +/- 0.8 mV). Similarly, milrinone did not hyperpolarize the PD of either the tracheal (n = 6) or segmental (n = 6) airways of CF subjects (DeltaF508) when applied topically in vivo in the presence of amiloride, isoproterenol, or adenosine triphosphate (all 100 microM) in a low-chloride solution. These data do not support the use of milrinone to induce chloride secretion in CF airways in vivo.

Illumination coherence effects in laser-speckle imaging: modeling and experimental demonstration.

Coherent imaging techniques rely on the coherence properties of backscattered radiation to form an image of an illuminated object. These techniques are sensitive to the degree of coherence of the illuminating source. We present an approach for simulating the effects that partial temporal coherent illumination has on these techniques. Computer simulation and laboratory experimental results are presented that illustrate illumination coherence effects on imagery obtained with the technique known as imaging correlography.

Photo-reversible binding of thrombin to avidin by means of a photolabile inhibitor.

Human alpha-thrombin and Factor Xa were acylated at their active site serine hydroxyls with biotin-substituted cinnamate derivatives 1b-1c. These acyl enzymes (2) showed no enzyme activity in the absence of light. On irradiation with light of wavelength 366 nm for 6 min, however, up to 80% of pre-inhibition activity was regained. This photo-deacylation of the modified enzyme results in the formation of active enzyme and a coumarin by-product (3). In addition, the acyl enzyme that results from incubation of 1c with thrombin was capable of binding to avidin, both immobilized and free in solution. Furthermore, the complex formed between the thrombin acyl enzyme (2c) and avidin was capable of binding to immobilized biotin.