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Chemical investigations into solid phase cultivations of an Australian sheep station pasture plant-derived Streptomyces sp. CMB-PB042, yielded the rare enamine naphthopyranoquinones BE-54238A (1) and BE-54238B (2), together with four new analogues, glenthenamines B–D (4–6) and F (8), and two handling artifacts, glenthenamines A (3) and E (7). Single crystal X-ray analyses of 1–2 resolved configurational ambiguities in the scientific literature, while detailed spectroscopic analysis and biosynthetic considerations assigned structures inclusive of absolute configuration to 3–8. We propose a plausible sequence of biosynthetic transformations linking structural and configurational features of 1–8, and apply a novel Schiff base "fishing" approach to detect a key deoxyaminosugar precursor. These enamine naphthopyranoquinone disclose a new P-gp inhibitory pharmacophore capable of reversing doxorubicin resistance in P-gp overexpressing colon carcinoma cells.

Successful wound healing requires adequate transcutaneous oxygen tension (tcpO(2) ). TcpO(2) may not commonly be incorporated in clinical assessments because of variable measurement response at different sensory temperatures. This study aims to assess the relationship between changes in tcpO(2) , measured under basal (39°C) and stimulated (44°C) conditions and healing rate of chronic wounds over 4 weeks, to determine whether tcpO(2) measurement can predict delayed wound healing. TcpO(2) (Radiometer TCM400) measurements at sensor temperatures 39 and 44°C were recorded (twice, 4 weeks apart) adjacent to the ulcer site, and at a mirror image site on the contralateral leg. Ulcer outline was traced on clear acetate and perimeter and area measured (Visitrak™, Smith and Nephew). TcpO(2) measured at 44 and 39°C adjacent to all 13 wounds were lower compared to the contralateral site, significant at 44°C (P = 0·008). Significant correlation (r(2) = 0·8) occurred between wound healing rate and increased tcpO(2) at 44°C over 4 weeks. Importantly, the ratio of 39/44°C tcpO(2) , measured at the initial appointment, appeared to predict normal or delayed healing rate. TcpO(2) may provide clinicians with information regarding anticipated healing ability of wounds at the initial appointment, and hence identify wounds requiring early implementation of adjuvant therapies to accelerate healing.

This study aimed to determine the role of early and late glycation products in modulating inflammation in early diabetes.Sprague-Dawley rats (130-170 g) were injected with streptozotocin (75 mg/kg, ip) and treated with daily aminoguanidine (AG, 25 mg/kg, ip) or vehicle for 2 or 4 weeks.The base of a vacuum-induced blister raised on the hind paw was perfused with substance P (SP, 1 microM) and sodium nitroprusside (SNP, 100 microM). Changes in blood flow and plasma extravasation (PE) were measured. Amadori (1 mg/ml), advanced glycation end products (AGEs, 10 mg/ml), and anti-RAGE IgG (antibody against AGE receptors, 100 microg/ml) were individually perfused prior to SP.In diabetic rats, responses to SNP and SP were reduced by 60% and 70%, respectively (P.05). Amadori increased responses to SNP by 50% and 90% and to SP by 70% and 80% in control and diabetic rats, respectively (both P.05). SP responses were significantly increased after anti-RAGE IgG (70%) or AG treatments (175%) with PE responses normalized.Amadori and anti-AGE agents enhance peripheral vascular responses in diabetes and may ameliorate microvascular damage.

Low frequency transcutaneous sensory nerve stimulation (LF-SNS) [International Patent PCT/AU2004/001079:“nerve function and tissue healing” (Khalil, Z.)] improves sensory nerve function and accelerates wound healing of older animals. Intervention: Double blind, placebo controlled randomised trial of LF-SNS for 5 minutes, twice daily for up to 12 weeks, on healing of chronic venous leg ulcers in older people. Four layer compression bandaging was the standard therapy. Outcome measures (methods): Wound healing and the rate of epithelialisation (calculated from serial wound area estimation), microvascular blood flow (measured using LASER Doppler flowmetry), transcutaneous oxygen tension (measured using a transcutaneous oxygen monitor, TCM Radiometer). Sensory nerve activity (assessed via measuring electrical cutaneous perception threshold using the Neurometer®CPT and the flare response to 5% capsaicin (a selective activator of C fibres). Participants: 14 older people with chronic venous ulcers randomly allocated to active (mean age 74.8±2.3 years) and 15 to Sham nerve stimulation (mean age 76.5±2.6 years). Results: Microvascular blood flow improved in all participants. A high proportion of wounds healed (∼ 60%). There were trends for better C-fibre function and faster healing rates in the Active group (1.1±0.3 cm2/wk) compared to the Sham group (0.6±0.2 cm2/wk) but failed to reach statistical significance due to the small sample size. Conclusions: The improvement in microvascular blood flow in both groups was an unexpected finding that has not previously been described. Most likely this was due to the four layer compression bandaging provided to all participants. Improved microvascular blood flow may be a significant contributor to wound healing. The observed trends to increased healing rates and improvements in C-fibre function in the actively stimulated group compared to the sham group warrant further studies of LF-SNS as an adjunct therapy for chronic venous leg ulcers. An exploration of the possibility that LFSNS induced-improvement in C-fibre function could protect against future ulceration is also warranted.

To assess the effect of sensory nerve stimulation in older people with peripheral neuropathy associated with diabetes (DPN).A randomized, placebo controlled, double blind trial was used to assess the effect of 12 weeks of low frequency sensory nerve stimulation (LF-SNS) in the lower limb [International Patent Application No. PCT/AU2004/001079: 'nerve function and tissue healing' (Z. Khalil)]. Response to capsaicin, basal microvascular blood flow, electric cutaneous threshold and oxygen tension were assessed pre- and post-treatment and between limbs.People 55 years of age or older diagnosed with DPN: 35 active and 31 placebo participants.Between groups comparisons: no significant differences occurred between stimulation groups. Within subject comparisons: in the active LF-SNS group, comparing stimulated to contralateral legs, there were significant increases in size of capsaicin flare [t(1,33)=3.65, p0.05] and capillary blood flow [t(1,34)=-0.33, p0.05]. There was a trend to improvement in time to initial flare response [t(1,34)=-1.86, p=0.07]. No changes were evident in the placebo group. RESPONDER ANALYSES: In a group of 'responders', the time to initial flare response (p0.05, r=0.64), size of capsaicin flare (p0.05 r=1.0) and microvascular blood flow (p0.05, r=0.60) improved significantly after LF-SNS.The observed data suggest that LF-SNS improves nerve function in a subset of people with DPN. Targeting toward probably 'responders' may deliver the greatest benefit from short-term therapy. Testing optimal application in others seems warranted.

Objectives: Venous leg ulcers represent a major clinical problem, with poor rates of healing. Ideal treatment is compression bandaging. The effect of compression on neurovascular tissues involved in wound repair is unclear. This study aims to assess the effect of four-layer compression therapy (40 mmHg) on neurovascular function and wound healing in people with chronic venous leg ulcers – 15 people (55 years or older) with venous leg ulcers for more than six weeks. Methods: Basal microvascular perfusion measurement (MPM), oxygen tension (tc pO2) measured at sensor temperatures of 39°C and 44°C and sensory nerve function using electrical cutaneous perception thresholds (ECPT) at 5, 250 and 2000 Hz (corresponding to C, A δ and A β fibres) were assessed adjacent to the ulcer site, and at a mirror location on the non-ulcerated limb. Testing was undertaken before and after therapy for 5–12 weeks of four-layer compression bandaging. Results: There was significant improvement in tc pO2 at 44°C and ECPT at 2000 Hz ( P2 at 39°C and ECPT at 5 and 250 Hz after compression therapy did not reach statistical significance. Conclusion: Four-layer compression bandaging in people with venous leg ulcers improved some components of neurovascularture in people with chronic venous leg ulcers. Whether this improvement has contributed to wound healing in this study requires further investigation.

The amyloid-beta (Abeta) peptide has been linked to the pathology of Alzheimer's disease (AD). There is now evidence to support a vasoconstrictive effect of Abeta protein that could be detected in peripheral skin microvasculature. In this study we investigated the ability of acetylcholinesterase (AChE) inhibitors, Donepezil and Rivastigmine, to modulate the vasoconstrictor activity of Abeta25-35 and Abeta1-40. The ability of these drugs to improve endothelial mediated vascular responses to acetylcholine and bradykinin subsequent to perfusion of Abeta peptides was also investigated. The vascular responses to Abeta peptides, acetylcholine, bradykinin and sodium nitroprusside and their modulation by acetylcholinesterase inhibitors were examined in the base of a vacuum induced blister raised on the rat hind footpad using laser Doppler flowmetry. Abeta25-35 (1 microM) and Abeta1-40 (0.1 microM) induced a vasoconstrictor effect and significantly reduced the vasodilator response to acetylcholine (100 microM) and bradykinin (1 microM). Donepezil (100 microM) and Rivastigmine (100 microM) both reduced the vasoconstrictor effect of Abeta peptides, and significantly restored the endothelial vascular response to acetylcholine. Similarly, Donepezil significantly restored the endothelial vascular response to bradykinin. The results also showed that the actions of acetylcholinesterase inhibitors are independent of a direct action on smooth muscle cell reactivity or on endothelial cell function in the absence of Abeta. The current study provides the first evidence in vivo to suggest that acetylcholinesterase inhibitors modulate the vasoconstrictive effects of Abeta peptides at the level of skin microvasculature. We raise the notion that Donepezil and Rivastigmine mediate these vascular modulatory effects via an action on Abeta-mediated vasoconstrictor mechanisms rather than an independent action on endothelial or smooth muscle cell mediated responses.

This paper demonstrates the capacity of the neuronal nicotinic acetylcholine receptor (nAChR) antagonist alpha-conotoxin Vc1.1 to inhibit pain responses in vivo. Vc1.1 suppressed pain behaviors when tested in two models of peripheral neuropathy of the rat sciatic nerve, the chronic constriction injury (CCI) and partial nerve ligation (PNL) models. Mechanical hyperalgesia was assessed using an Ugo Basile Analgesymeter. Vc1.1 was administered by intramuscular bolus injection near the site of injury at doses of 0.036 microg, 0.36 microg and 3.6 microg in CCI rats and at a dose of 0.36 microg in PNL rats. Vc1.1 was also administered contralaterally in CCI rats at doses of 0.36 microg and 3.6 microg. Treatment started after the development of hyperalgesia and continued for 7 days. Vc1.1 significantly attenuated mechanical hyperalgesia in both CCI and PNL rats for up to a week following cessation of treatment. Vc1.1 also accelerated functional recovery of injured neurones. A blister was raised over the footpad innervated by the peripheral terminals of the injured nerve. The ability of these terminals to mount an inflammatory vascular response upon perfusion of the blister base with substance P provided a measure of functional recovery. This study shows that alpha-conotoxin Vc1.1, a neuronal nAChR antagonist, suppressed mechanical pain responses associated with peripheral neuropathy in rats in vivo and accelerated functional recovery of the injured neurones. A role for neuronal nAChRs in the analgesic activity of Vc1.1 is proposed.

This study aims to seek new drugs from the marine environment, with a particular focus on venomous animals, in particular the marine cone snail, Conus, a genus of predatory animal with a highly evolved venom and delivery apparatus. These remarkable creatures provide an example of the rich diversity of therapeutic molecules still to be discovered in marine species. In this article, a description of the value of these animals for medical research and the methods used to realize the therapeutic potential of their venom are presented. The study showed that DNA- or gene-based mining can, when coupled with sensitive bioassays, be an environmentally friendly method for discovering novel therapeutic peptides from marine sources. Simple peptide synthesis combined with a sensitive bioassay is the primary screen. This detects those peptides that form a functional 3-D structure spontaneously under the conditions of primary amino acid chain synthesis and oxidation. The screening process therefore selects lead compounds that are simple to manufacture. Spontaneity of folding may also indicate greater stability of the structure under physiological conditions, and the selection procedure, therefore, may pick those peptides with greater inherent stability in vivo, a further bonus that may be related to the success of delivery and survival of the lead compounds when used therapeutically.

When applied 20 min after injection of histamine into human forearm skin, tea tree oil (TTO) reduces the developing cutaneous vascular response. In this study, the effect of TTO on inflammatory microvascular changes was dissected at the base of an experimental blister on rat skin. 1,8-Cineole, representing 2% of TTO, reduced vascular changes induced by sensory neuropeptides released when the distal portion of a cut sciatic nerve was electrically stimulated. The pre-terminal modulatory effect of 1,8-Cineole was confirmed in tests in sensory-denervated rats. Terpinen-4-ol (approximately 40% TTO) reduced substance P-induced microvascular changes and protein extravasation by a direct nitric oxide-mediated effect on the microvasculature, without sensory nerve involvement. alpha-Terpineol (3% of TTO) regulated both pre- and post-sensory nerve terminals. In human skin, terpinen-4-ol applied 10 min after histamine injection, but not alpha-terpineol or 1,8-cineole, regulated the developing wheal and flare suggesting that the histamine-induced responses in humans (at the dose used in this study, 50 microL of 330 microM histamine) are in large part determined by histamine directly affecting the vasculature via post-terminal-mediated events. The underlying strength of these studies is the use of a well-established rat physiologic model to differentiate the mechanism of regulation of microvascular changes by modulatory agents.

Marine cone snails from the genus Conus are estimated to consist of up to 700 species. These predatory molluscs have devised an efficient venom apparatus that allows them to successfully capture polychaete worms, other molluscs or in some cases fish as their primary food sources. The toxic venom used by the cone shells contains up to 50 different peptides that selectively inhibit the function of ion channels involved in the transmission of nerve signals in animals. Each of the 700 Conus species contains a unique set of peptides in their venom. Across the genus Conus, the conotoxins represent an extensive array of ion channel blockers each showing a high degree of selectivity for particular types of channels. We have undertaken a study of the conotoxins from Australian species of Conus that have the capacity to inhibit specifically the nicotinic acetylcholine receptors in higher animals. These conotoxins have been identified by mass spectroscopy and their peptide sequences in some cases deduced by the application of modern molecular biology to the RNA extracted from venom ducts. The molecular biological approach has proven more powerful than earlier protein/peptide based technique tor the detection of novel conotoxins [1,2]. Novel conotoxins detected in this way have been further screened for their abilities to modify the responses of tissues to pain stimuli as a first step in describing their potential as lead compounds for novel drugs. This review describes the progress made by several research groups to characterise the properties of conopeptides and to use them as drug leads for the development of novel therapeutics for the treatment of a range of neurological conditions.

We describe the identification of a conopeptide sequence in venom duct mRNA from Conus victoriae that suppresses a vascular response to pain in the rat. PCR-RACE was used to screen venom duct cDNAs for those transcripts that encode specific antagonists of vertebrate neuronal nicotinic acetylcholine receptors (nAChRs). One of these peptides, Vc1.1, was active as an antagonist of neuronal nAChRs in receptor binding and functional studies in bovine chromaffin cells. It also suppressed the vascular responses to unmyelinated sensory nerve C-fiber activation in rats. Such vascular responses are involved in pain transmission. Furthermore, its ability to suppress C-fiber function was greater than that of MVIIA, an omega-conotoxin with known analgesic activity in rats and humans. Vc1.1 has a high degree of sequence similarity to the alpha-conotoxin family of peptides and has the 4,7 loop structure characteristic of the subfamily of peptides that act on neuronal-type nAChRs. The results suggest that neuronal alpha-conotoxins should be further investigated with respect to their potential to suppress pain.

Recently there has been recognition in Australia of the importance of experimental gerontological research. To date, the major areas of research into the biology of ageing have been oxidative stress and mitochondrial dysfunction. Future directions for gerontological research in Australia are likely to place more emphasis on comprehensive studies that integrate basic biological research, with clinical studies and the behavioural and social domains of ageing. Priority will be given to studies incorporating biological markers that are population based and longitudinal. The growing interest in experimental gerontological research complements Australian research strengths in other domains such as disease-based research, social gerontology, population studies and health policy research.

There is a direct correlation between dermal mast cell prevalence in dorsal skin of different mouse strains and susceptibility to UVB-induced systemic immunosuppression; highly UV-susceptible C57BL/6 mice have a high dermal mast cell prevalence while BALB/c mice, which require considerable UV radiation for 50% immunosuppression, have a low mast cell prevalence. There is also a functional link between the prevalence of dermal mast cells and susceptibility to UVB- and cis-urocanic acid (UCA)-induced systemic immunosuppression. Mast cell-depleted mice are unresponsive to UVB or cis-UCA for systemic immunosuppression unless they are previously reconstituted at the irradiated or cis-UCA-administered site with bone marrow-derived mast cell precursors. cis-UCA does not stimulate mast cell degranulation directly. Instead, in support of studies showing that neither UVB nor cis-UCA was immunosuppressive in capsaicin-treated, neuropeptide-depleted mice, cis-UCA-stimulated neuropeptide release from sensory c-fibers which, in turn, could efficiently degranulate mast cells. Studies in mice suggested that histamine, and not tumor necrosis factor alpha (TNF-alpha), was the product from mast cells that stimulated downstream immunosuppression. Histamine receptor antagonists reduced by approximately 60% UVB and cis-UCA-induced systemic immunosuppression. Indomethacin administration to mice had a similar effect which was not cumulative with the histamine receptor antagonists. Histamine can stimulate keratinocyte prostanoid production. We propose that both histamine and prostaglandin E(2) are important in downstream immunosuppression; both are regulatory molecules supporting the development of T helper 2 cells and reduced expression of type 1 immune responses such as a contact hypersensitivity reaction.

Previous studies using an antibody to cis-urocanic acid and mast-cell-depleted mice implicated both cis-urocanic acid and mast cells in the mechanisms by which ultraviolet B light suppresses systemic contact hypersensitivity responses in mice. In the absence of a direct stimulatory effect of cis-urocanic acid on connective tissue mast cells, an indirect association was investigated. A blister induced in the rat hind footpad was used to examine the effects of slowly perfused cis-urocanic acid on cutaneous blood flow. cis-Urocanic acid but not trans-urocanic acid increased microvascular flow by a mechanism largely dependent on the combined activity of the neuropeptides, substance P and calcitonin gene-related peptide. Perfusion of cis-urocanic acid over the base of blisters induced in sensory-neuropeptide-depleted rats did not have any stimulatory effect above that seen with perfusion of cis-urocanic acid together with neuropeptide receptor antagonists in control rats. There was a small direct effect of cis-urocanic acid on microvascular blood flow. As both substance P and calcitonin gene-related peptide could directly degranulate connective tissue mast cells, this study suggests that cis-urocanic acid indirectly activates mast cells via its effects on peripheral terminals of unmyelinated primary afferent sensory nerves. cis-Urocanic-acid-induced neuropeptides may also contribute to ultraviolet-B-induced cutaneous inflammation and alterations to Langerhans cell activity.

During aging there is a decline in sensory nerve function that is associated with reduced neurogenic inflammation and poor wound repair. The cellular mechanism(s) responsible for this decline in function with age is not well understood. We previously reported that sensory nerves in aged rats release sensory neuropeptides preferentially in response to low-frequency (5 Hz) as compared with higher-frequency (15 Hz) antidromic electrical stimulation, and that low-frequency transcutaneous electrical nerve stimulation accelerates wound healing. The present study investigates possible mechanisms for this preferential response. Using laser Doppler techniques, we have measured changes in blood flow in the base of vacuum-induced blisters induced in the rat hind footpad of young and old animals in response to low-frequency (5 Hz) or high-frequency (15 Hz) electrical stimulation (20 V, 2 ms for 1 minute) of the sciatic nerve. The relative contributions of the sensory neuropeptides, substance P and calcitonin gene-related peptide (CGRP), and of N -type voltage-gated calcium channels to the vascular responses were assessed by using the specific receptor antagonists RP67580, which is 2-(1-imino-2-(2 methoxy phyenyl) ethyl)-7,7 diphenyl-4 perhydroisoindolone-(3aR, 7aR); CGRP 8-37 ; and � -conotoxin GVIA ( Conus geographus ), respectively. The results showed a greater involvement of substance P at highfrequency electrical stimulation and of CGRP at low-frequency stimulation. Our finding that � -conotoxin-sensitive N -type calcium channel function was preserved with age and was only involved in the vascular response to low-frequency electrical stimulation could explain our previous report demonstrating beneficial effects of low-frequency transcutaneous electrical nerve stimulation to wound repair in aged animals. The current results have important practical implications for improving tissue repair in the aged. EUROGENIC vascular responses to antidromic electrical stimulation (ES) of the saphenous and sciatic

We have previously shown an age-related decline in the modulation of skin vascular reactivity by sensory nerves that correlates with a decline in wound repair efficacy. This study was designed to examine the possibility that improving the functional ability of aged sensory nerves using noninvasive transcutaneous electrical nerve stimulation (TENS) could also accelerate tissue repair. TENS of the sciatic nerve, combined with measuring blood flow responses in the rat hind-footpad using laser Doppler flowmetry, was used to establish the vascular effects. Following TENS (using parameters 20V, 5 Hz for 1 min), similar increases in vascular responses were obtained in both young (13.2+/-0.9 cm2) and old rats (11.6+/-2.3 cm2). In contrast, capsaicin-pretreated rats showed markedly diminished responses. Sympathetic fibers did not appear to modulate these sensory nerve responses. In the second part, a thermal wound was induced (using a CO2 laser) in the interscapular region of old rats (under anesthesia). In the active treatment group, TENS was applied twice daily for the initial 5 days, and the sham group received inactive TENS. Using the healing endpoint as the time when full wound contraction occurred, the active group required 14.7+/-0.2 days for complete healing, a significant improvement over the sham group (21.8+/-0.3 days). We contend that low-frequency TENS can improve the vascular response of old rats. In addition, wound healing in aged rats can be accelerated by peripheral activation of sensory nerves at low-frequency electrical stimulation parameters.

Transcutaneous electrical nerve stimulation (TENS) has been the subject of extensive research, yet relatively little consensus exists regarding optimal analgesic stimulation parameters. A high degree of TENS response variability has led to the institution of different modes of treatment, which vary in their featured operating parameters and efficacy. Much attention has focused on the frequency of stimulation and, to date, little attempt has been made to compare alternative modes of TENS systematically, especially high-intensity applications using experimental pain protocols and healthy, pain-free subjects. Moreover, the lack of a well-circumscribed set of effective analgesic parameters has made it difficult to devise suitable placebo protocols. This review attempts to explore the possibility that stimulation intensity may be the most important parameter consideration for TENS modulation of experimentally induced pain and the implication this may have for the creation of more appropriate TENS plac...

Six fungal species (Fusarium sp, Exophiala jeanselmei, Penicillium spp, Aspergillus niger, Paecilomyces spp, and Alternaria spp) were used to inoculate soft contact lenses. Four types of soft lenses were used: high-water (58%) and low-water (38%) content lenses and lenses that were unworn or worn for 1 day. The fungi displayed a range of macroscopic and microscopic features that allowed differentiation of species. There was no statistically significant effect of lens water content on growth rate and only Penicillium spp showed significantly higher growth for worn versus unworn lenses. A number of the fungi showed secretions, thought to be enzymes, which potentially aid in the process of lens penetration. © Elsevier Science Inc. 2000

We previously reported that the time frame and the extent of the changes in the peripheral neurogenic inflammatory response in the skin area, innervated by an injured nerve, coincide with those of pain behaviours. We raised the possibility that common factors might operate to modulate neuropathic pain and peripheral neurogenic processes in rats with chronic constriction nerve injury (CCI). In the present study we examined the role of free radicals in modulating the neurogenic vascular response and thermal hyperalgesia in rats with CCI of the sciatic nerve. Free radicals, via an interaction with nitric oxide (NO) to form peroxynitrite, have previously been implicated in the maintenance of thermal hyperalgesia in CCI rats. In this study, we induced CCI of the sciatic nerve and the activity of xanthine oxidase (XO), which catalyzes the formation of superoxide anions, was measured in the injured nerve. In addition, we examined the effect of antioxidants on thermal hyperalgesia and on the neurogenic vascular response to substance P (SP) perfused over the base of a blister induced on the hind footpad skin which is innervated by the injured sciatic nerve. Compared with the sham operated group, CCI rats had a significantly higher XO activity in the injured sciatic nerve and significantly reduced thermal threshold and peripheral neurogenic vascular response to SP. Treatment with antioxidants, superoxide dismutase (SOD) or tirilazad significantly improved the neurogenic vascular response while tirilazad treatment significantly alleviated thermal hyperalgesia. The results therefore, suggest that free radicals are elevated in CCI animals and that they contribute to the maintenance of thermal hyperalgesia and the reduction in peripheral microvascular blood flow in the area innervated by the injured nerve. We raise the possibility that common mechanisms may govern the changes in neuropathic pain and in the peripheral neurogenic vascular responses in tissues innervated by the injured nerve.

1. Sensory nerves are important for the initiation of neurogenic inflammation and tissue repair. Both calcitonin gene-related peptide (CGRP) and nitric oxide (NO) have been implicated in neurogenic vasodilatation and inflammatory responses. 2. A blister model in the rat hind footpad was used as a site to induce neurogenic vasodilatation in response to antidromic electrical stimulation of the sciatic nerve. Blood flux was monitored with a laser Doppler flow monitor. 3. The quantitative contributions of CGRP and NO to vasodilatation were examined by use of the CGRP receptor antagonist CGRP8-37 and NO synthase inhibitors 7-nitroindazole (7-NI), 3-bromo 7-NI and N(G)-nitro L-arginine methyl ester (L-NAME). The potential modulatory role of endothelin was examined by use of the ET(A) receptor antagonist BQ-123. 4. CGRP8-37 (10 microM) was perfused over the blister base before nerve stimulation and continuously throughout the post-stimulation period, resulting in a significant reduction (41%) in the blood flux vascular response. 5. Pretreatment with the specific neuronal NO synthase inhibitors, 7-NI and 3-bromo 7-NI (10 mg kg(-1), i.v.), and of the non-specific L-NAME (100 microM), resulted in significant inhibition of the blood flux response (36%, 72% and 57% decrease, respectively). In contrast, 7-NI treatment in young rats pretreated with capsaicin had no further effect on the vascular response, suggesting that the source of NO is the sensory nerves. 6. BQ-123 (10 microM) significantly enhanced the stimulation-induced blood flux response (61% increase). When 7-NI was co-administered with either CGRP8-37 or BQ-123, the drug actions were additive, suggesting that there was no interaction between NO and CGRP or endothelin. 7. These data suggest that both NO and CGRP participate in neurogenic vasodilatation in rat skin microvasculature and that this response is modulated by endogenous endothelin.

A supersensitivity to the neuropeptide substance P (SP) has been shown to develop in post-terminal membranes of many denervated tissues. This study examined changes in the sensitivity of post-terminal vascular receptors to SP and calcitonin gene-related peptide (CGRP) in rat skin microvasculature following sciatic nerve section. In anaesthetised rats, 0.5 cm of sciatic nerve in the right mid-thigh region was removed. Two weeks later, SP (100 microM) and sodium nitroprusside (SNP, 1 mM), a direct smooth muscle vasodilator, were introduced into denervated intact footpad skin, via the electrophoresis technique. Laser doppler flowmeter was used to record changes in relative blood flow in the rat hind footpad. The results showed a significant increase in SP response over controls and slight increase in smooth muscle reactivity as determined by an increase in the vascular response to SNP. In another set of experiments, the sensitivity of post-terminal receptors was examined over a 4 weeks period in an acutely injured footpad skin of sciatic nerve lesioned rats. A vacuum-induced blister was raised on the hind footpad and SP, CGRP (each at 1 microM) or SNP (100 microM) were superfused over the blister base. In nerve lesioned rats, using the acutely injured footpad skin model, the results showed a reduction in the vascular responses to SP, CGRP and SNP. The response to SP continued to decrease over time reaching 22% of control values by 4 weeks. Responses to SNP and CGRP were reduced to 53% and 45% respectively by 2 weeks and then improved to 75% of control values by 4 weeks. Possible contributions of sympathetic efferents and the saphenous nerve to these reduced responses in acutely injured skin of nerve lesioned rats were examined using guanethidine (50 mg/kg i.p.) or sectioned saphenous nerve respectively. These procedures did not significantly modify the reduced vascular responses in the blister base of lesioned rats. Possible activation of endogenous opioids and/or the release of endothelin due to blister induction in nerve lesioned rats was examined using naloxone and the endothelin receptor antagonist, BQ-123, respectively. Treatment with naloxone increased SP response in lesioned rats to 41% of control value with no change in smooth muscle reactivity. BQ-123 significantly increased the responses to SP and SNP to 51% and 100% of their own control values respectively. It is concluded that supersensitivity of post-terminal vascular receptors develops in intact skin following chronic nerve lesion. On the other hand, acute injury of the denervated skin area induces activation of endogenous inhibitory modulatory mechanisms that masks this supersensitivity.

The role of nitric oxide in inflammatory responses to substance P and other mediators of inflammation was examined in rat skin microvasculature in a blister base raised on the hind footpad. Superfusion of substance P (1 microM) over the blister base caused an increase in plasma extravasation and a vasodilator response which was not maintained. N(G)-Nitro-L-arginine (100 microM), an inhibitor of nitric oxide biosynthesis, attenuated vasodilatation and plasma extravasation due to substance P. The inactive isomer N(G)-nitro-D-arginine was without effect. Neurokinin A (1 microM), 5-hydroxytryptamine (1 microM), ATP (50 microM) and vasoactive intestinal polypeptide (1 microM) elicited vasodilation, which for vasoactive intestinal polypeptide was maintained even after washout. 5-Hydroxytryptamine and neurokinin A, but not ATP or vasoactive intestinal polypeptide, significantly increased plasma extravasation. Vasodilatation to neurokinin A, 5-hydroxytryptamine and ATP, and the increase in plasma extravasation due to neurokinin A and 5-hydroxytryptamine were unaffected by N(G)-nitro-L-arginine (100 microM), whereas vasodilation due to vasoactive intestinal polypeptide was significantly attenuated. These findings suggest that in rat skin microvasculature in vivo, nitric oxide is involved in vasodilator responses due to substance P and vasoactive intestinal polypeptide, and plasma extravasation due to substance P, but does not contribute significantly to vasodilatation induced by neurokinin A, 5-hydroxytryptamine or ATP, or the plasma extravasation induced by neurokinin A or 5-hydroxytryptamine.

The oceans are a source of a large group of structurally unique natural products that are mainly found in invertebrates such as sponges, tunicates, bryozoans, and molluscs. It is interesting to note that the majority of marine compounds currently in clinical trials or under preclinical evaluation are produced by these species rather than as secondary metabolites by marine algae [1]. Through the combined efforts of marine natural products chemists and pharmacologists a number of promising compounds have been identified that are either already at advanced stages of clinical trials such as the new anti-cancer drug marine alkaloid ecteinascidin 743 [2], or have been selected as promising candidates for extended preclinical evaluation [3]. This is the case for conotoxins, (Table 1) where a number of conopeptides are currently being developed as analgesics for the treatment of neuropathic pain.

The primary constituent of the senile plaque core in Alzheimer's disease (AD) is the β-amyloid protein (βA4). A discrete 11 amino acid fragment of the βA4, βA425–35, has been implicated in mediating in vitro neurotoxicity and an inflammatory response surrounding senile plaques in AD via interaction with the Serpin Enzyme Complex (SEC) receptor. Substance P (SP), a neuropeptide of the tachykinin family and a major mediator of neurogenic inflammation, shows sequence homology to βA425–35 and has been shown to protect against the neurotoxicity of β-amyloid. SP also competes with βA425–35 for binding to the SEC-receptor. SP neurons have also been found to be depleted in AD. Using a blister model of inflammation in the rat hind footpad, we have examined the effect of βA425–35 and its interaction with SP in rat skin microvasculature and determined age-related changes to these phenomena. In addition, pharmacological manipulation of these responses using SEC-receptor ligands (peptide 105Y and 105C) was also undertaken. Because of the evidence for co-existence and co-release of SP and calcitonin gene-related peptide (CGRP) from the peripheral terminals of sensory nerves, it was of interest to examine the interaction of CGRP with βA425–35 on rat skin microvasculature. βA425–35 (10 μM) was perfused over the base of a blister raised on the hind footpad of anaesthetised young and old rats. This was followed by perfusion of SP (1 μM) or CGRP (1 μM) after Ringer's solution. Relative blood flow was monitored using a Laser-Doppler Flowmeter. Peptide 105Y or Peptide 105C (100 μM) was perfused over the blister base prior to perfusion of βA425–35 and SP. The results showed that βA425–35 induced a vasoconstrictor effect on rat skin microvasculature that was significantly greater in old than in young rats. Furthermore, βA425–35 prevented a subsequent SP-, but not CGRP-induced vasodilator response in old rats. Perfusion of SEC receptor ligands (peptide 105Y and 105C) or brief perfusion of SP prior to βA425–35 resulted in a reduction in the vasoconstrictor effect of βA425–35 and allowed a subsequent SP perfusion to induce a small vasodilator response. This pharmacological manipulation was most evident using peptide 105C. This study demonstrates that skin can provide an index of the effect of βA425–35 on the microvasculature in vivo and suggests that βA425–35 prevents SP from inducing a vasodilator effect in old rats through an interaction with the SEC receptor. These modulatory effects can be pharmacologically manipulated using the SEC receptor ligands peptide 105Y, 105C and SP. This blister model of inflammation should be useful for examining further the nature of these interactions and their significance in AD.

Chemical analysis of a marine-derived Streptomyces sp. (CMB-M0423) isolated from beach sand off Heron Island, Australia, yielded three new members of the rare pyrroloterpene biosynthetic structure class. Identified by detailed spectroscopic analysis as the first reported examples of naturally occurring 2-nitropyrroles, heronapyrroles A-C (1-3) displayed promising biological activity-with low to submicromolar IC(50) activity against Gram-positive bacteria but no cytotoxicity toward mammalian cell lines.

This study experimentally evaluates the effect of fiber-reinforced polymer (FRP) confinement on the seismic response of circular reinforced concrete columns with spliced reinforcement at the critical hinging regions. A general expression previously was derived and verified for designing the thickness of external FRP jackets required for bond strengthening of spliced reinforcement within the critical hinging region of rectangular column sections. The current study applies the corresponding design expression for circular column sections. Full-scale circular columns with spliced reinforcement were tested under lateral load reversals. Findings show that all of the unconfined columns suffered premature splitting bond failure leading to almost total strength and stiffness degradation. Confining the splice zone with external FRP jackets within the range of thicknesses predicted by the proposed design expression substantially improved seismic performance. The mechanism by which the FRP confinement improves the bond strength of spliced bars in circular columns is similar to that in rectangular columns. Compared with other proposed design methods, the equation under consideration predicts a realistic and conservative jacket thickness and accounts for all parameters that are known to influence the splice strength of steel bars in tension.

Screening for glucose intolerance during pregnancy provides an opportunity to offer management to those women diagnosed with gestational diabetes mellitus. However, there is a need to diagnose gestational diabetes early to minimize exposure of the developing fetus to suboptimal conditions and prevent perinatal complications and their sequelae. The purpose of this study was to identify potential biomarkers for impending gestational diabetes that appear in the plasma before impaired glucose tolerance. Pregnant women were prospectively recruited to the study and blood was collected at the first antenatal visit and at the time of routine oral glucose tolerance test. Women diagnosed with gestational diabetes were matched with an equal number of normal pregnant (control) women. Biomarkers under investigation included endocrine and metabolic hormones, cytokines and chemokines, and surrogate markers of oxidative stress. Compared to controls, women with gestational diabetes exhibited elevated plasma insulin and reduced plasma adiponectin concentrations at 28 weeks gestation. Significant differences in insulin and adiponectin concentrations were also observed in plasma at 11 weeks gestation. Bivariate logistic regression analysis showed that both insulin and adiponectin are associated with subsequent development of gestational diabetes. Plasma insulin and adiponectin concentrations, when measured at 11 weeks, may be predictive of impending gestational diabetes. Further studies are warranted to determine the reliability of these biomarkers.

Pain therapeutics discovered by molecular mining of the expressed genome of Australian predatory cone snails are providing lead compounds for the treatment of neurological diseases such as multiple sclerosis, shingles, diabetic neuropathy and other painful neurological conditions. The high specificity exhibited by these novel compounds for neuronal receptors and ion channels in the brain and nervous system indicates the high degree of selectivity that this class of neuropeptides can be expected to show when used therapeutically in humans. A lead compound, ACV1 (conotoxin Vc1.1 from Conus victoriae), has entered Phase II clinical trials and is being developed for the treatment for neuropathic pain. ACV1 will be targeted initially for the treatment of sciatica, shingles and diabetic neuropathy. The compound is a 16 amino acid peptide [Sandall et al., 2003. A novel alpha-conotoxin identified by gene sequencing is active in suppressing the vascular response to selective stimulation of sensory nerves in vivo. Biochemistry 42, 6904-6911], an antagonist of neuronal nicotinic acetylcholine receptors. It has potent analgesic activity following subcutaneous or intramuscular administration in several preclinical animal models of human neuropathic pain [Satkunanathan et al., 2005. Alpha conotoxin Vc1.1 alleviates neuropathic pain and accelerates functional recovery of injured neurons. Brain. Res. 1059, 149-158]. ACV1 may act as an analgesic by decreasing ectopic excitation in sensory nerves. In addition ACV1 appears to accelerate the recovery of injured nerves and tissues.

The amyloid-beta (Abeta) peptide has been linked to the pathology of Alzheimer's disease (AD). There is now evidence to support a vasoconstrictive effect of Abeta protein that could be detected in peripheral skin microvasculature. In this study we investigated the ability of acetylcholinesterase (AChE) inhibitors, Donepezil and Rivastigmine, to modulate the vasoconstrictor activity of Abeta25-35 and Abeta1-40. The ability of these drugs to improve endothelial mediated vascular responses to acetylcholine and bradykinin subsequent to perfusion of Abeta peptides was also investigated. The vascular responses to Abeta peptides, acetylcholine, bradykinin and sodium nitroprusside and their modulation by acetylcholinesterase inhibitors were examined in the base of a vacuum induced blister raised on the rat hind footpad using laser Doppler flowmetry. Abeta25-35 (1 microM) and Abeta1-40 (0.1 microM) induced a vasoconstrictor effect and significantly reduced the vasodilator response to acetylcholine (100 microM) and bradykinin (1 microM). Donepezil (100 microM) and Rivastigmine (100 microM) both reduced the vasoconstrictor effect of Abeta peptides, and significantly restored the endothelial vascular response to acetylcholine. Similarly, Donepezil significantly restored the endothelial vascular response to bradykinin. The results also showed that the actions of acetylcholinesterase inhibitors are independent of a direct action on smooth muscle cell reactivity or on endothelial cell function in the absence of Abeta. The current study provides the first evidence in vivo to suggest that acetylcholinesterase inhibitors modulate the vasoconstrictive effects of Abeta peptides at the level of skin microvasculature. We raise the notion that Donepezil and Rivastigmine mediate these vascular modulatory effects via an action on Abeta-mediated vasoconstrictor mechanisms rather than an independent action on endothelial or smooth muscle cell mediated responses.

The N-terminal metabolite of the undecapeptide substance P (SP), substance P1-7 (SP1-7), is known to modulate nociception in the central nervous system (CNS) and often has opposite effects from SP. This study investigated the ability of SP(1-7) to modulate the vasodilatation response to SP in anaesthetized rats under different injury conditions using a blister model of inflammation on the hind footpad. The results indicated that SP1-7 inhibited the vascular response to SP in a dose-dependent manner. The putative antagonists naloxone and D-Pro2-D-Phe7-SP1-7 (D-SP1-7) reversed the effect of SP1-7. D-SP1-7 improved the responsiveness to SP under chronic nerve injury, which suggests a role for endogenous SP1-7 in this model. SP1-7 did not inhibit the response to electrical stimulation of the sciatic nerve, which indicates that the heptapeptide interacts at a post-terminal binding site. The current results suggest that SP1-7 may have inhibitory properties in inflammation, analogous to its antinociceptive role in the central nervous system.

Previous studies from our laboratory demonstrated an age-related functional decline in sensory neurones and their modulation of microvascular blood flow (primary ageing processes) that correlated with a deficiency in tissue repair (a secondary ageing process). We also raised the notion of a possible role for free radicals in these age-related changes. The aim of this study was to investigate the impact of antioxidant therapy on modulating sensory neurovascular function and tissue repair with age. Twenty-four-month-old Sprague-Dawley rats were treated with vitamin E for short-term (40 mg/kg, i.p., every other day for 2 weeks) or long-term (for 12 months in advance, 10 g/kg, incorporated in food). These treated rats were assessed for the effectiveness of treatment and tested for their sensory neurovascular function, repair of full-thickness burn, and recovery from hyperalgesia following nerve injury. The results indicate that both short- and long-term vitamin E treatments are effective in improving sensory neurovascular function and in reducing the time required for complete wound closure of full-thickness burn injury. Short-term vitamin E treatment was more effective in protecting against the development of hyperalgesia following nerve injury. An initial increase in wound size and in hyperalgesia was observed in the treated animals, and could reflect possible side effects of the antioxidant therapy and support the importance of free radicals in early stages of the repair process. The data, overall, support the notion that oxidative damage contributes to both primary and secondary ageing processes.

Freshly prepared soluble amyloid (Abeta) peptide has been reported to have vascular actions both in vitro and in vivo. This study was designed to examine the in vivo microvascular effects of beta in two skin microvascular model systems that might reflect possible short and long-term vascular effects of this peptide. Short-term vascular effects were examined using freshly prepared soluble Abeta(1-40) peptide superfused over naive rat skin microvasculature for 15 min. Peripheral microvascular functional changes in 9-months-old transgenic (Tg) mice overexpressing soluble beta in the brain, peripheral circulation and other tissues, were also examined. Microvascular responses were monitored using laser Doppler flowmetry from the base of a blister raised on the hind footpad of the animals. Endothelial-dependent and independent vasodilatation responses (VD) were examined using acetylcholine (ACh) and sodium nitroprusside (SNP) respectively. The exposure of naive rat skin microvasculature to Abeta(1-40) resulted in an immediate vasoconstriction (VC) that prevented ACh but not SNP from inducing a subsequent VD response. The vascular effects of Abeta(1-40) were reversed by antioxidants (superoxide dismutase and catalase) and an endothelin A (ETA) receptor antagonist (BQ-123). Tg mice overexpressing soluble Abeta and C100 showed significant reductions in both endothelial-dependent and endothelial-independent VD that were also reversed by antioxidants and BQ-123. In conclusion, this study provided evidence to support the notion of peripheral vascular effects of Abeta in vivo and present novel evidence for alterations in endothelial and smooth muscle cell function in peripheral skin microvasculature in Tg mice overexpressing Abeta and C100. We suggest that skin microvasculature is a useful model to examine the mechanisms underlying the vascular actions of the Abeta protein.

To explore the mechanism underlying changes in microvascular reactivity in single- and double-transgenic mice.Peripheral vascular reactivity to the vasodilators, acetylcholine and sodium nitroprusside, on perfused microvasculature of the hind footpad was investigated using nontransgenic mice, single-transgenic mice expressing the human APP-C100 (TgC100. WT or TgC100. V717F) and double-transgenic mice coexpressing human APP-C100 and human SOD(1) (G93A) genes.Single TgC100 and double Tg mice C100/SOD(1) (G93A) at 2 - 3 months old showed a statistical decrease of 28% in blood flux compared to nontransgenic control mice. In addition, vasodilative responsiveness was markedly reduced to 34% in 8 - 9 months old TgC100 mice compared to control mice. There was no significant difference in the profile of vasodilative reaction between TgC100. WT and TgC100. V717F mice. TgC100 and double Tg mice also had higher levels of A beta peptide in plasma than nontransgenic mice (P0.01).The present study suggests that the altered reactivity of the microvasculature may be mediated by circulating soluble A beta peptides. The mechanisms underlying the vasoactivity of circulating A beta in TgC100 and double Tg mice may involve both the endothelium and nonendothelium.

Objective and design: The aim of the present study was to examine the contribution of the two kinin receptors B1 and B2 to the increased blood flow observed in response to bradykinin (BK) in a blister model under different injury conditions.¶Material: Young male Sprague-Dawley rats weighing 250-350 g were used.¶Methods: A vacuum-induced blister was raised in the rat hind paw and blood flow measured in the superfused blister base under four different conditions including, early phase acute injury; late phase acute injury; recurrent injury and early phase acute injury in the setting of chronic nerve damage. BK (10 μM) was superfused alone, or in the presence of the B1 antagonist DesArg9Leu8BK (DALBK), (10 nM) and/or the B2 antagonist [D-Arg,Hyp3,Thi5 D-Tic7,Oic8] Bradykinin (HOE 140) (10 nM).¶Results: HOE 140 significantly inhibited the BK response in all models. Significant inhibition of BK-induced vasodilatation by DALBK was only observed in the late phase acute and recurrent injury models.¶Conclusions: The results suggest that the involvement of the inducible B1 receptor in skin inflammation site is related to the site, duration and recurrence of the injury condition.

Using a reversible chronic constriction injury (CCI) model of neuropathic pain, we previously demonstrated that changes in thermal hyperalgesia correlate with the changes in peripheral microvascular blood flow in the affected paw, and that recovery can be assessed by normalization of both behavioral and vascular responses. Using the same model, this study examined age-related changes in recovery after nerve injury and the involvement of free radicals and nitric oxide (NO) in these changes. Four loose, nonconstrictive ligatures were applied to the sciatic nerve in the right, mid-thigh region of young and old (3 and 24 months) Sprague Dawley rats. All rats were monitored weekly (for 8-10 weeks) for their thermal threshold using a 46 degrees C water bath and some groups were used to examine endothelial and smooth muscle-dependent microvascular responses to substance P (SP) and sodium nitroprusside (SNP), respectively. These substances were perfused over the base of blisters raised on the footpad innervated by the injured nerve. Free radical activity in the sciatic nerve was assessed by measuring the activity of xanthine oxidase (XO) and lipid hydroperoxides (LPO). Young rats showed signs of recovery (reduction in thermal hyperalgesia and improvement of peripheral microvascular blood flow) from the fifth week. No signs of recovery were observed in old rats for 8 weeks, with some reduction in thermal hyperalgesia observed by weeks 9 and 10. XO activity was significantly higher in young injured nerves compared to sham (400%) and was even significantly greater in old injured nerves (680%). Similarly, old injured nerves showed 300% increase in LPO levels compared to sham. The role of reactive oxygen species (ROS) in delayed recovery in old rats was examined using the antioxidant tirilazad mesylate. Tirilazad (20 mg/kg) was injected intramuscularly (im) in the mid-thigh region starting on day 1 post CCI, (early treatment) or day 7 (late treatment). Levels of LPO in the injured sciatic nerves were significantly reduced using either early or late treatment, however tirilazad had opposing effects on recovery, prolonging or alleviating thermal hyperalgesia, respectively. The role of neuronal nitric oxide (nNO) was then examined using the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-bromo-7-nitroindazole (3Br-7NI) (10 mg/kg). 3Br-7NI resulted in a significant alleviation of thermal hyperalgesia with improvement in the vascular responses from weeks 5 and 6 onwards. A combination of 3Br-7NI and tirilazad treatment was also used but did not show an additive effect. The results suggest that ROS and nNO contribute to delayed recovery of injured nerves in old rats and to the maintenance of thermal hyperalgesia and the reduction in microvascular blood flow in the area innervated by the injured nerve. The results also raise the notion that possible interaction of free radicals with NO to form peroxynitrite might be responsible for such delayed recovery. Ironically, this study also reveals a positive role for free radicals in tissue repair and raises the notion that early intervention with antioxidants could exert a negative effect on repair of injured nerves.

Following tissue injury, adequate inflammatory vascular responses are essential for subsequent tissue repair. The aims of this study were to investigate the role of reactive oxygen species (ROS, generated at the injury site) in modulating the inflammatory response under acute- and chronic-injury conditions. The effect of age and the implications of this modulation for tissue repair was investigated. Using laser Doppler flowmetry, inflammatory vascular responses were monitored in the base of vacuum-induced blisters in the hind footpad of anesthetized rats (65 mg/kg Nembutal). Inflammation was amplified by superfusion of substance P (SP) over the blister base. The inflammatory response was examined in acute blisters induced on either naive skin (acute-injury model) or on skin innervated by a chronically injured nerve (chronic-injury model). Furthermore, the acute-injury model was examined during early and late phases, 0 and 5 h after blister induction, respectively. The involvement of ROS was assessed by either combined superfusion of the antioxidants: superoxide dismutase and catalase over the blister base in acute-injury, or intramuscular injection of tirilazad in chronically injured rats. The results showed that antioxidant treatment had no effect on the response during early and late phases of acute inflammation in young rats. However in old rats, the vascular response was significantly attenuated (60%) or significantly increased (40%) during the early and late phases of acute inflammation, respectively. Under chronic-injury conditions, antioxidant treatment significantly enhanced the response in both young and old rats. We then examined the effect of antioxidant, tirilazad, on the healing of a full thickness thermal injury induced in the intrascapular region (using a CO(2) laser) of the rat. Following burn injury, tirilazad was injected around the wound site starting on day 1 (early treatment) or day 6 (late treatment). Tirilazad had opposing actions on wound closure with early and late treatments delaying (24.6 +/- 0.6 d) or accelerating (14.2 +/- 0.3 d) wound closure compared with the group of aged controls (20.3 +/- 0.8 d). The results suggest that ROS have a paradoxical role exerting either a positive or negative effect on the inflammatory response with age. We contend that the role of ROS in modulating inflammation should be considered when designing treatment protocols to accelerate tissue repair.

T-cell-derived antigen-binding molecules (TABMs) specific for benzoic acid were isolated from the serum of a toluene-sensitive patient. The resulting purified TABMs (BA-TABMs) did not contain immunoglobulin G and were associated with the cytokine transforming growth factor-beta (TGF-beta). BA-TABMs bound to benzoic acid conjugated to human serum albumin (BA-HSA), as well as to other chemicals conjugated to human serum albumin-including dinitrophenol and oxazolone. The binding of BA-TABMs to the conjugated chemicals increased the level of detectable TGF-beta, and a similar effect was observed with the unconjugated chemicals, benzoic acid and 2,4-dinitrophenol glycine. The increase in TGF-beta was critically dependent on the ratio between BA-TABMs and the conjugated or unconjugated chemicals; the increase was optimum at intermediate concentrations and absent at low and high concentrations. The authors used an established animal model in vivo and demonstrated that TGF-beta enhanced the inflammatory response induced by the release of neuropeptides from sensory nerves; this enhancement occurred in a dose-dependent manner. The BA-TABMs also enhanced this neurogenic inflammatory response in a dose-dependent manner, and this effect was blocked by anti-TGF-beta antibody. When the authors added either BA-HSA or benzoic acid, the effect of BA-TABMs on neurogenic inflammation was further enhanced at intermediate concentrations of antigen and was unaltered or reduced at higher concentrations. TABMs specific to particular chemicals, as a result of their association with cytokines (e.g., TGF-beta), may be implicated in symptom production in chemically sensitive patients.

The effect of age on hyperalgesia, one of the most common signs of injury, has not been previously examined in humans. A psychophysical study was conducted in 10 young (26.9+/-4.6 years) and 10 older (79. 0+/-5.7 years) healthy volunteers to investigate the effect of age on the development of hyperalgesia induced by topical application of capsaicin (0.1 ml, 5 mg/ml). The capsaicin patch (diameter 2 cm) was applied for 1 h. The intensity of capsaicin-induced spontaneous sensation, mechanical pain threshold, area of flare, heat and punctate hyperalgesia were measured hourly for 3 h after the application. Older adults took a longer period to report first pain. There was no age effect on the magnitude of spontaneous sensation, flare size and area of heat hyperalgesia. The area of heat hyperalgesia rapidly decreased over time in both age groups. In marked contrast, the area of punctate hyperalgesia and associated reduction in the mechanical pain threshold were maintained in older adults over the entire 3 h test period, but resolved rapidly in young adults. We conclude that, given the same intensity of noxious stimulation, older adults display a similar magnitude of hyperalgesia as younger persons. However, once initiated, punctate hyperalgesia appears to resolve more slowly in older people. This finding may indicate age differences in the plasticity of spinal cord neurons following an acute injury.

An intact nociceptor system of capsaicin-sensitive primary afferent sensory nerves is important for the initiation of the inflammatory process and successful tissue repair. Dysfunction of this system with age could contribute to delayed wound healing in the elderly. This chapter addresses age-related changes in the function of the small unmyelinated primary afferents (referred to as capsaicin-sensitive primary afferents) and their peptide neurotransmitters and the implication of these changes to tissue repair. Emphasis is focused on healing of skin wounds as skin is one of the most frequently injured tissues and its repair processes must be activated promptly to ensure survival of the organism.

Sensory nerves mediate peripheral inflammation via the release of sensory peptides at the site of tissue injury. Using a blister model of inflammation, we have previously documented that endogenous opioids modulate chronic but not acute inflammation. Hemorphins are nonclassical opioid peptides found in the region of the beta-chain of hemoglobin (Hb). The heptapeptide hemorphin-7 is identical with residues 35-41 of the beta-chain of the human Hb. The aim of this study was to examine the effect of hemorphin-7 on the inflammatory response in acute and chronic injury models.We have used a vacuum-induced blister model in the footpad of anaesthetized rats to induce an inflammatory response in naive skin by (a) electrical stimulation (ES) of the distal end of the cut sciatic nerve at 20 V, 5 Hz, 2 ms for 1 min or (b) superfusion of sensory peptides; substance P (SP) or calcitonin gene related peptide (CGRP) over the blister base. In addition, we examined the effect of hemorphin-7 on the inflammatory response to SP induced in a previously injured but healed skin site (recurrent injury model) and in denervated skin site due to chronic nerve lesion (chronic injury model).The results showed that prior and concomitant perfusion of hemorphin-7 over the blister base inhibited the acute inflammatory response to ES of the sciatic nerve at C-fibre strength in a dose-dependent manner. Significant inhibition was achieved at 20 and 200 microM concentration of hemorphin-7. When hemorphin-7 (20 microM) was perfused prior to and together with SP or CGRP (both at 1 microM), over the base of acutely induced blister in naive skin, it significantly reduced the inflammatory response to SP (both plasma extravasation and vasodilatation), but was without effect on the vasodilatation response to CGRP. Naloxone, the general opioid antagonist at (1 mg/kg i.v.) reversed the inhibitory effect of hemorphin-7 on the inflammatory response to SP. On the other hand, hemorphin-7 had no effect on the inflammatory response to SP in the recurrent injury or the chronic injury models.The results of this study suggest that hemorphins might play a role in inhibiting the inflammatory response in acute, but not in recurrent or chronic injury conditions. Evidence is also provided that the modulatory inhibitory effect of hemorphin-7 is mediated via activation of opioid receptor(s). The significance of this study in conjunction with our previous work, is that it raises the possibility that different endogenous inhibitory mechanisms may operate under different injury conditions - endogenous hemorphins and opioids may modulate acute and chronic inflammation, respectively.

Deposition of beta-amyloid protein (beta A4) in extracellular senile plaques is a pathologic hallmark of Alzheimer's disease (AD). The neurotoxic effect of beta A4 has been ascribed to a discrete 11-amino acid internal sequence (beta A(4)25-35). Substance P (SP) has been found to be depleted in the brain of AD patients while its presence was found to protect against the neurodegenerative effect of beta A(4)25-35. Our previous studies, in vivo, in aged rats showed that beta A(4)25-35 exhibits a potent vasoconstrictor (VC) effect in rat skin microvasculature and can prevent SP but not calcitonin gene-related peptide (CGRP) from inducing a vasodilator (VD) response. It was postulated that beta A(4)25-35 might be interacting with SP at the level of the second messenger system via the phosphoinositide pathway. Using a blister model of inflammation in the rat hind footpad, we examined the ability of beta A(4)25-35 to modulate the vascular activity of bradykinin (BK) and serotonin (5-HT) which also activate the phosphoinositide pathway. In addition, the role of nitric oxide (NO), endothelin (ET, an endothelium-derived constrictor factor) and protein kinase C (PKC) in the vascular effects of beta A(4)25-35 were examined using the NO synthase inhibitor, NG-nitro-L-arginine (L-NOARG), the ET-receptor antagonist, BQ-123, and the PKC inhibitor, bisindolylmaleimide (BIM) respectively. Changes in microvascular blood flow were monitored using laser Doppler flowmetry and the area within the response curve measured. The results showed that beta A(4)25-35 (10 microM) induced a VC effect and inhibited the subsequent VD response to BK (10 microM) and 5-HT (1 microM) in a similar fashion to its effect on SP (1 microM). In the presence of L-NOARG (100 microM), the VD effect of SP was reduced and further attenuated after perfusion of beta A(4)25-35. Superfusion of the blister base with BQ-123 (10 microM) or BIM (1 microM) prior to and during perfusion with beta A(4)25-35 abolished its VC effect and allowed SP to induce a normal VD response in both young and old rats. Based on these results, we suggest that the vascular activity of the active fragment, beta A(4)25-35, is mediated by ET via activation of PKC. This study provides new findings which may help to elucidate the signal transduction mechanisms involved in the vascular activity of beta A(4)25-35. The relevance of these mechanisms to those underlying the pathological effects of beta A4 and their significance in AD remains to be determined.

An intact nociceptor system of primary afferent sensory nerves is important for the initiation of the inflammatory process and successful tissue repair. Dysfunction of this system with age could be a contributing factor for delayed wound healing in the elderly. This study was designed to examine the role of sensory nerves (using capsaicin-pretreated rats) and the effect of aging on the healing of a thermal wound induced on the interscapular region (using a CO2 laser). The healing endpoint was the time when full wound contraction had occurred. The ability of the sensory peptides, substance P (SP) and calcitonin gene-related peptide (CGRP), in modulating wound healing in aged rats was examined by taking into account the modulatory interaction effects between these peptides. A blister model in the rat hind footpad combined with a laser Doppler flowmeter was used initially to establish these vascular interaction effects. The results showed a significant delay in thermal wound healing in both the capsaicin-treated and aged rats. Using the blister model, we demonstrated the ability of SP and CGRP to induce a limited and a prolonged vasodilator response, respectively. When tested together, SP attenuated the vascular effect of CGRP, an effect that was reversed using the tachykinin antagonist spantide II. When injected at the wound site, SP and CGRP were equally potent in promoting wound healing in aged rats. The beneficial effect of taking the interaction effects into account was most evident in a group of rats that received the tachykinin antagonist as part of their initial treatment with CGRP and 6 h later, an injection of SP; the time to complete wound closure was 11.3 +/- 0.6 days compared to 21.0 +/- 0.9 days in aged controls. We contend that would healing in aged rats can be accelerated by exogenous administration of sensory peptides. Furthermore, modulatory interaction effects between sensory peptides should be taken into consideration when designing any treatment protocol that purports to accelerate wound healing.