The correlation between pain-related behaviour and spinal microgliosis in four distinct models of peripheral neuropathy.

BACKGROUND: Peripheral nerve injury is associated with a spinal microglial response that has been correlated with the development of behaviours reflective of neuropathic pain. METHODS: To examine whether this phenomenon is generalizable to neuropathic pain of non-traumatic aetiology, this study investigated the association between spinal microgliosis and behavioural measures of neuropathic hypersensitivity and pain-related anxiety behaviour in four distinct rat models of peripheral neuropathic pain. These were traumatic neuropathy [L5 spinal nerve transection (SNT)], HIV-related neuropathies (either treatment with the antiretroviral drug Zalcitabine (ddC) or combination of perineural exposure to the HIV-gp120 protein and ddC treatment) and varicella zoster virus (VZV) infection. RESULTS AND CONCLUSION: Persistent mechanical hypersensitivity developed in all 'neuropathic' rats. However, spinal microgliosis, as measured by increased CD11b/c immunohistochemical staining and increased numbers of cells expressing CD11b measured by flow cytometry, was evident in the SNT and to a lesser extent in the HIV neuropathy models but not the VZV model. These results suggest that behavioural hypersensitivity and thigmotaxis can only be linked to a microglial response in certain models of neuropathy.

High glucose induced endothelial cell growth inhibition is associated with an increase in TGFbeta1 secretion and inhibition of Ras prenylation via suppression of the mevalonate pathway.

OBJECTIVE: Ras proteins are known to affect cellular growth and function. The influence of the prenylation status of Ras on the observed changes in endothelial cell growth under high glucose conditions has not previously been examined. METHODS: Human umbilical vein endothelial cells were exposed to normal or high glucose conditions for 72 h. They were then examined for proliferative and hypertrophic effects, transforming growth factor beta(1) (TGFbeta(1)) release, and phosphorylated p38 expression. The importance of prenylation was explored by the addition of mevalonate, isoprenoids or farnesyltransferase inhibitors to control the high glucose media and by measuring changes induced by high glucose and exogenous TGFbeta(1) in Ras prenylation and farnesyltransferase activity. Kidneys from diabetic rats treated with atorvastatin were also compared to specimens from untreated animals and the expression of the Ras effector p-Akt examined. RESULTS: High glucose conditions caused a reduction in cell number. This was reversed in the presence of mevalonate or farnesylpyrophosphate (FPP), suggesting that the cell growth abnormalities observed are due to high glucose induced inhibition of the mevalonate pathway and subsequent prenylation of proteins. Endothelial cells exposed to high glucose increased their secretion of TGFbeta(1) and the phosphorylation of p38 both of which were reversed by concurrent exposure to FPP. A reduction in farnesyltransferase activity was observed after exposure to both high glucose and TGFbeta(1). Exposure to a farnesyltransferase inhibitor in control conditions mimicked the growth response observed with high glucose exposure and prenylated Ras was reduced by exposure to both high glucose and TGFbeta(1). Finally, interruption of the mevalonate pathway with a statin reduced the expression of p-Akt in diabetic rat kidneys. CONCLUSION: This study demonstrates that high glucose induced significant alterations in endothelial cell growth by inhibition of the mevalonate pathway, which subsequently mediates the increase in TGFbeta(1) and inhibition of Ras prenylation.

Quantification of the rat spinal microglial response to peripheral nerve injury as revealed by immunohistochemical image analysis and flow cytometry.

Microgliosis is implicated in the pathophysiology of several neurological disorders, including neuropathic pain. Consequently, perturbation of microgliosis is a mechanistic and drug development target in neuropathic pain, which highlights the requirement for specific, sensitive and reproducible methods of microgliosis measurement. In this study, we used the spinal microgliosis associated with L5 spinal nerve transection and minocycline-induced attenuation thereof to: (1) evaluate novel software based semi-quantitative image analysis paradigms for the assessment of immunohistochemical images. Microgliosis was revealed by immunoreactivity to OX42. Several image analysis paradigms were assessed and compared to a previously validated subjective categorical rating scale. This comparison revealed that grey scale measurement of the proportion of a defined area of spinal cord occupied by OX42 immunoreactive cells is a robust image analysis paradigm. (2) Develop and validate a flow cytometric approach for quantification of spinal microgliosis. The flow cytometric technique reliably quantified microgliosis in spinal cord cell suspensions, using OX42 and ED9 immunoreactivity to identify microglia. The results suggest that image analysis of immunohistochemical revelation of microgliosis reliably detects the spinal microgliosis in response to peripheral nerve injury and pharmacological attenuation thereof. In addition, flow cytometry provides an alternative approach for quantitative analysis of spinal microgliosis elicited by nerve injury.

Engineering strontium binding affinity in an EF-hand motif: a quantum chemical and molecular dynamics study.

Proteins with the ability to specifically bind strontium would potentially be of great use in the field of nuclear waste management. Unfortunately, no such peptides or proteins are known -- indeed, it is uncertain whether they exist under natural conditions due to low environmental concentrations of strontium. To investigate the possibility of devising such molecules, one of us (CV), in a previous experimental study, proposed starting from an EF-hand motif of the protein calmodulin and mutating some residues to change the motif's specificity for calcium into one for strontium. In this paper, which represents a theoretical complement to the experimental work, we analyzed small-molecule crystallographic structures and performed quantum chemical calculations to identify possible mutations. We then constructed seven mutant sequences of the EF-hand motif and analyzed their dynamical and binding behaviors using molecular dynamics simulations and free-energy calculations (using the MM/PBSA method). As a result of these analyzes we were able to isolate some characteristics that could lead to mutant peptides with enhanced strontium affinity.

Kousseff syndrome: a causally heterogeneous disorder.

The existence of Kousseff syndrome as a distinct entity has been thrown into doubt by a recent study conducted on the family originally reported by Kousseff. In all cases where chromosome 22q11.2 FISH testing has been undertaken, including the original sibship, a chromosome 22q11.2-microdeletion has been identified. We report two cases of sacral myelomeningocele associated with a conotruncal cardiac anomaly or "Kousseff syndrome." The first case, a 4-year-old girl, had a sacral myelomeningocele, tetralogy of Fallot, microcephaly, hydrocephalus, hypoplasia of the corpus callosum and mild-moderate developmental delay. Chromosome 22q11.2 FISH was normal and the facial phenotype was not that of velocardiofacial syndrome. Sequencing of the entire coding region of CITED2 did not reveal a mutation. The second case, a male infant, was found to have a 22q11.2-microdeletion. These cases confirm Kousseff syndrome to be a causally heterogeneous disorder.

PEHO and PEHO-like syndromes: report of five Australian cases.

PEHO syndrome is a rare progressive infantile encephalopathy with onset within the first few months of life. Few patients fulfilling the diagnostic criteria for PEHO syndrome have been reported outside Finland. Affected infants have facial dysmorphism and suffer from severe hypotonia, profound mental retardation, convulsions (often with a hypsarrhythmic EEG pattern), transient or persistent peripheral oedema, and optic atrophy. Cerebellar and brainstem atrophy are usually present on neuroimaging. A PEHO-like syndrome has been described, in which the affected individuals have neither optic atrophy nor the typical neuroradiological findings. We report five Australian patients, the first with classical features of PEHO syndrome, and four who have a PEHO-like disorder. We compare their features with other published cases. We suggest that PEHO or a PEHO-like syndrome may affect more patients than are currently identified, based on the original diagnostic criteria for this disorder.

Theoretical study of the conformation of the lipoamide arm in a mutant H protein.

The lipoamide arm of the H protein plays a pivotal role in the catalytic cycle of the glycine decarboxylase complex (GDC) by being successively methylamine loaded (Hmet), reduced (Hred), and oxidized (Hox). In a previous study, we calculated free-energy surfaces as a function of the lipoamide arm position of the three forms of the wild-type protein and found close agreement with the available experimental data. Our simulations, together with crystallographic and NMR data, showed that the methylamine-loaded arm is locked in a cavity by interaction with Ser12, Glu14, and Asp67. In this work, we investigate the behavior of the methylamine-loaded form of a mutant H protein (HEA) where Glu14 has been replaced by Ala. We find that the arm can still be held in the cavity but that the energy barrier to release of the arm is halved from approximately 40 kcal mol(-1) for Hmet to approximately 12 kcal mol(-1) for HEA. To compensate for the loss of Glu14, the methylamine group shifts toward Ser66 in the mutant form. These results provide a structural basis for the equilibrium between the loaded and the unloaded forms of the arm observed by Gueguen et al. (Gueguen et al., J Biol Chem 1999;274:26344-26352) in HEA.

Extending Medicare coverage to medically necessary dental care.

BACKGROUND: Periodically, Congress considers expanding Medicare coverage to include some currently excluded health care services. In 1999 and 2000, an Institute of Medicine committee studied the issues related to coverage for certain services, including "medically necessary dental services." METHODS: The committee conducted a literature search for dental care studies in five areas: head and neck cancer, leukemia, lymphoma, organ transplantation, and heart valve repair or replacement. The committee examined evidence to support Medicare coverage for dental services related to these conditions and estimated the cost to Medicare of such coverage. RESULTS: Evidence supported Medicare coverage for preventive dental care before jaw radiation therapy for head or neck cancer and coverage for treatment to prevent or eliminate acute oral infections for patients with leukemia before chemotherapy. Insufficient evidence supported dental coverage for patients with lymphoma or organ transplants and for patients who had undergone heart valve repair or replacement. CONCLUSIONS: The committee suggested that Congress update statutory language to permit Medicare coverage of effective dental services needed in conjunction with surgery, chemotherapy, radiation therapy or pharmacological treatment for life-threatening medical conditions. PRACTICE IMPLICATIONS: Dental care is important for members of all age groups. More direct, research-based evidence on the efficacy of medically necessary dental care is needed both to guide treatment and to support Medicare payment policy.

State of the evidence base for medically necessary oral health care.

OBJECTIVE: The purpose of this article was to highlight the recent call for an evidence-based approach to public policy decision making with respect to funding dental services and the need this creates for enhanced clinical research activities. STUDY DESIGN: Systematic reviews on topics of importance to oral health care practitioners are being conducted and published by various national and international groups. Recent activities to assess evidence to support medically necessary dental services were reviewed. RESULTS: An Institute of Medicine Committee on Medicare Coverage Extensions found little published scientific evidence that directly assessed the effectiveness of dental services in preventing or managing systemic health outcomes for patients with head and neck cancer, lymphoma, leukemia, organ transplantation, and heart valve repair or replacement. CONCLUSIONS: The scientific community must strive to meet the challenge of conducting well-designed randomized, controlled trials that test the impact of dental treatment interventions on systemic health to meet the growing need for evidence to support or refute widely accepted dental treatment protocols for medically complex patients.

Ligand diffusion in the catalase from Proteus mirabilis: a molecular dynamics study.

The role of the channels and cavities present in the catalase from Proteus mirabilis (PMC) was investigated using molecular dynamics (MD) simulations. The reactant and products of the reaction, H(2)O(2) -->1/2 O(2) + H(2)O, catalyzed by the enzyme were allowed to diffuse to and from the active site. Dynamic fluctuations in the structure are found necessary for the opening of the major channel, identified in the X-ray model, which allows access to the active site. This channel is the only pathway to the active site observed during the dynamics, and both the products and reactant use it. H(2)O and O(2) are also detected in a cavity defined by the heme and Ser196, which could play an important role during the reaction. Free energy profiles of the ligands diffusing through the major channel indicate that the barriers to ligand diffusion are less than 20 kJ mol(-1) for each of the species. It is not clear from our study that minor channels play a role for access to the protein active site or to the protein surface.

A hybrid potential reaction path and free energy study of the chorismate mutase reaction.

We present a combination of two techniques--QM/MM statistical simulation methods and QM/MM internal energy minimizations--to get a deeper insight into the reaction catalyzed by the enzyme chorismate mutase. Structures, internal energies and free energies, taken from the paths of the reaction in solution and in the enzyme have been analyzed in order to estimate the relative importance of the reorganization and preorganization effects. The results we obtain for this reaction are in good agreement with experiment and show that chorismate mutase achieves its catalytic efficiency in two ways; first, it preferentially binds the active conformer of the substrate and, second, it reduces the free energy of activation for the reaction relative to that in solution by providing an environment which stabilizes the transition state.

Utilization of an intramolecular hydrogen bond to increase the CNS penetration of an NK(1) receptor antagonist.

This paper describes the synthesis and physical and biological effects of introducing different substituents at the alpha-position of the tryptophan containing neurokinin-1 receptor antagonist [(R)-2-(1H-indol-3-yl)-1-methyl-1-((S)-1-phenyl-ethylcarbamoyl)-ethyl]-carbamic acid benzofuran-2-ylmethyl ester (CI 1021). The described compounds all exhibit less than 5 nM binding affinities for the human neurokinin-1 receptor and selectivity over the tachykinin NK(2) and NK(3) receptor subtypes. Application of variable temperature nuclear magnetic resonance spectroscopy studies of the amide and urethane protons was utilized to determine the existence of an intramolecular hydrogen bond. This intramolecular hydrogen bond increases the apparent lipophilicity to allow increased central nervous system penetration and pharmacological activity (gerbil foot tap test) in the case of the highest affinity compound [(S)-1-dimethylaminomethyl-2-(1H-indol-3-yl)-1-((S)-1-phenyl-ethylcarbamoyl)-ethyl]-carbamic acid benzofuran-2-ylmethyl ester (PD 174424) over those analogues that could not form an intramolecular hydrogen bond.

Communication and information technology in medical education.

The past few years have seen rapid advances in communication and information technology (C&IT), and the pervasion of the worldwide web into everyday life has important implications for education. Most medical schools provide extensive computer networks for their students, and these are increasingly becoming a central component of the learning and teaching environment. Such advances bring new opportunities and challenges to medical education, and are having an impact on the way that we teach and on the way that students learn, and on the very design and delivery of the curriculum. The plethora of information available on the web is overwhelming, and both students and staff need to be taught how to manage it effectively. Medical schools must develop clear strategies to address the issues raised by these technologies. We describe how medical schools are rising to this challenge, look at some of the ways in which communication and information technology can be used to enhance the learning and teaching environment, and discuss the potential impact of future developments on medical education.

Pregabalin may represent a novel class of anxiolytic agents with a broad spectrum of activity.

The present study examines the effect of pregabalin (previously S-Isobutylgaba and CI-1008) in two distinct rat models of anxiety. Pregabalin binds with high affinity and selectivity to the alpha(2)delta subunit of voltage dependent calcium channels (VDCC). Its corresponding R-enantiomer (R-isobutylgaba) is approximately 10 fold weaker. Pregabalin dose-dependently induced anxiolytic-like effects in both the rat conflict test and elevated X-maze with respective minimum effective doses (MED) of 3 and 10 mg kg(-1). In contrast, R-isobutylgaba only showed activity at the highest dose of 100 mg kg(-1) in the conflict test. These data indicate that pregabalin may possess clinical utility as a novel anxiolytic agent and demonstrates the importance of the alpha(2)delta subunit of VDCC in the mediation of anxiety related behaviours.

Enalaprilat directly ameliorates in vitro cyclosporin nephrotoxicity in human tubulo-interstitial cells.

BACKGROUND/AIMS: Several recent studies have suggested that angiotensin-converting enzyme (ACE) inhibitors ameliorate chronic cyclosporin A (CyA) tubulo-interstitial disease by mechanisms independent of their antihypertensive effects. The aim of the present study was to determine whether ACE inhibition exerts a direct beneficial effect on the tubulo-interstitium in an in vitro model of chronic CyA nephropathy. METHODS: Primary cultures of human proximal tubular cells (PTC) and renal cortical fibroblasts (CF) were exposed for 24 h to CyA in the presence or absence of enalaprilat. Parameters of tubulo-interstitial nephrotoxicity were then measured including collagen synthesis (proline incorporation), tubular viability and function (thymidine incorporation, lactate dehydrogenase release, and apical sodium-hydrogen exchange), and secretion of insulin-like growth factor I, transforming growth factor beta 1 (TGFbeta1), and platelet-derived growth factor. RESULTS: CyA promoted CF collagen synthesis, PTC cytotoxicity (suppressed viability, growth and sodium transport), and tubulo-interstitial fibrogenic cytokine release (CF secretion of insulin-like growth factor I and PTC secretion of TGFbeta1 and platelet-derived growth factor). Enalaprilat completely reversed the stimulatory effects of CyA on CF collagen synthesis (CyA + enalaprilat 6.40 +/- 0.50% vs. CyA alone 8.33 +/- 0.56% vs. control 6.57 +/- 0.62% vs. enalaprilat alone 5.55 +/- 0.93%, p < 0.05) and PTC secretion of TGFbeta1 (0.71 +/- 0.11, 1.13 +/- 0.09, 0.89 +/- 0.07, and 0.67 +/- 0.09 ng/mg protein/day, respectively, p < 0.05). However, the other manifestations of CyA toxicity were not significantly reversed by concomitant enalaprilat administration. CONCLUSIONS: ACE inhibition directly prevents CyA-induced interstitial fibrosis, but not proximal tubule cytotoxicity, independently of haemodynamic and systemic renin-angiotensin system effects. Renoprotection may be partially afforded by directly preventing the tubular secretion of TGFbeta1.

Further evidence for the role of the alpha(2)delta subunit of voltage dependent calcium channels in models of neuropathic pain.

1. Current analgesic therapy is dominated by NSAIDs and opiates, however these agents have limited efficacy in the treatment of neuropathic pain. The novel anticonvulsant agent gabapentin (Neurontin) has been shown to be an effective treatment for neuropathic pain in the clinic. Recent studies have demonstrated that gabapentin selectively interacts with the alpha(2)delta subunit of voltage dependent calcium channels (VDCCs) which may be important in its mechanism of action. 2. Previous studies have identified a gabapentin analogue, 3-methyl gabapentin, that stereoselectively interacts with the alpha(2)delta subunit of VDCCs. Thus, whilst (1S, 3R) 3-methyl gabapentin binds to the alpha(2)delta protein with high affinity (IC(50)=42 nM), the corresponding (1R,3R) isomer is 300 times weaker (Bryans et al., 1998: J. Med. Chem., 41, 1838 - 1845). The present study examines the activity of diastereoisomers of 3-methyl gabapentin in two rat models of neuropathic pain to assess the importance of an interaction with the alpha(2)delta subunit of VDCCs. 3. (1S,3R) 3-methyl-gabapentin dose-dependently (10 - 100 mg kg(-1), p.o.) blocked the maintenance of static allodynia in the rat streptozocin and Chung models of neuropathic pain with MEDs of 30 mg kg(-1). This isomer also dose-dependently blocked the maintenance of dynamic allodynia in both models with respective MEDs of 30 and 100 mg kg(-1). In contrast, (1R,3R) 3-methyl gabapentin (100 mg kg(-1), p.o.) failed to block either static or dynamic allodynia in the streptozocin model. 4. It is concluded that these data further support the hypothesis that the alpha(2)delta subunit of VDCCs plays an important role in the maintenance of mechanical hypersensitivity in models of neuropathic pain.

Evaluation of selective NK(1) receptor antagonist CI-1021 in animal models of inflammatory and neuropathic pain.

CI-1021 ([(2-benzofuran)-CH(2)OCO]-(R)-alpha-MeTrp-(S)-NHCH(CH (3))Ph) is a selective and competitive neurokinin-1 (NK(1)) receptor antagonist. This study examines its activity in animal models of inflammatory and neuropathic pain. In mice, CI-1021 (1-30 mg/kg, s.c.) dose dependently blocked the development of the late phase of the formalin response with a minimum effective dose (MED) of 3 mg/kg. Two chemically unrelated NK(1) receptor antagonists, CP-99,994 (3-30 mg/kg) and SR 140333 (1-100 mg/kg), also dose dependently blocked the late phase, with respective MEDs of 3 and 10 mg/kg. PD 156982, a NK(1) receptor antagonist with poor central nervous system penetration, failed to have any effect. However, when administered i. c.v., it selectively blocked the late phase of the formalin response. Chronic constrictive injury (CCI) to a sciatic nerve in the rat induced spontaneous pain, thermal and mechanical hyperalgesia, and cold, dynamic, and static allodynia. CI-1021 (10-100 mg/kg) and morphine (3 mg/kg) blocked all the responses except dynamic allodynia. Carbamazepine (100 mg/kg) was weakly effective against all the responses. Once daily administration of morphine (3 mg/kg, s. c.) in CCI rats led to the development of tolerance within 6 days. Similar administration of CI-1021 (100 mg/kg, s.c.) for up to 10 days did not induce tolerance. Moreover, the morphine tolerance failed to cross-generalize to CI-1021. CI-1021 blocked the CCI-induced hypersensitivity in the guinea pig, with a MED of 0.1 mg/kg, p.o. CI-1021 (10-100 mg/kg, s.c.) did not show sedative/ataxic action in the rat rota-rod test. It is suggested that NK(1) receptor antagonists possess a superior side effect profile to carbamazepine and morphine and may have a therapeutic use for the treatment of inflammatory and neuropathic pain.

Fibrogenic effects of cyclosporin A on the tubulointerstitium: role of cytokines and growth factors.

The clinical utility of cyclosporin A (CyA) as an immunosuppressive agent has been significantly limited by the frequent occurrence of chronic nephrotoxicity, characterised by tubular atrophy, interstitial fibrosis and progressive renal impairment. The pathogenesis of this condition remains poorly understood, but has been postulated to be due to either direct cytotoxicity or indirect injury secondary to chronic renal vasoconstriction. Using primary cultures of human proximal tubule cells (PTCs) and renal cortical fibroblasts (CFs) as an in vitro model of the tubulointerstitium, we have been able to demonstrate that clinically relevant concentrations of CyA are directly toxic to these cells and promote fibrogenesis by a combination of suppressed matrix metalloproteinase activity and augmented fibroblast collagen synthesis. The latter effect occurs secondary to the ability of CyA to stimulate autocrine secretion of insulin-like growth factor-I by CFs and paracrine secretion of transforming growth factor-beta(1) by PTCs. Many of these pro-fibrotic mechanisms are completely reversed by concurrent administration of the angiotensin-converting enzyme inhibitor, enalaprilat, which has proven efficacy in preventing chronic CyA nephropathy in vivo. These studies highlight the unique potential that human renal cell cultures offer for studying the role of local cytokine networks in tubulointerstitial disease and for developing more effective treatment strategies which specifically target fibrogenic growth factor activity following nephrotoxic injuries.

Detection of static and dynamic components of mechanical allodynia in rat models of neuropathic pain: are they signalled by distinct primary sensory neurones?

In the present study, chronic constrictive injury (CCI model) of the sciatic nerve or tight ligation of L5 and L6 spinal nerves (Chung model) produced both dynamic and static components of mechanical allodynia in rats. The two responses were detected, respectively, by lightly stroking the hind paw with cotton wool or application of pressure using von Frey hairs. Animals with spinal nerve ligation developed both types of responses at a faster rate compared to animals with the CCI. Morphine (1-3 mg/kg, s.c.) dose-dependently blocked static but not dynamic allodynia. In contrast, pregabalin (previously S-isobutylgaba and CI-1008) dose-dependently (3-30 mg/kg, p.o.) blocked both types of allodynia. In CCI animals, two administrations of capsaicin (100 microg/50 microl) into the plantar surface of the ipsilateral paw at 1-h intervals blocked the maintenance of thermal hyperalgesia without affecting either static or dynamic allodynia. The similar administration of a further two doses of capsaicin into the same animals blocked the maintenance of static allodynia without affecting the dynamic response. These data indicate that thermal hyperalgesia, static and dynamic allodynia are respectively signalled by C-, Adelta- and Abeta/capsaicin insensitive Adelta- primary sensory neurones. It is suggested that pregabalin possesses a superior antiallodynic profile than morphine and may represent a novel class of therapeutic agents for the treatment of neuropathic pain.