Chronic pain in inflammatory bowel disease: characteristics and associations of a hospital-based cohort.

BACKGROUND: Chronic pain (CP) is a common symptom in patients with inflammatory bowel disease. This study aimed to examine its prevalence, severity, clinical associations, and impact on psychological well-being and to identify patient factors that independently predict the presence of severe/disabling pain. METHODS: One hundred and twenty consecutive patients with inflammatory bowel disease attending a hospital-based clinic provided information through questionnaires on quality of life, mood disturbance, and functional gut symptoms. Those who had CP (pain occurring every day for 3 months within the past 6 months) provided additional information on the pain's intensity and associated disability and management and coping strategies. RESULTS: Forty-six patients (38%) had CP, most commonly in the abdomen (91%), and they had higher disease activity, reduced quality of life, and more depression and anxiety and took more paracetamol and opiates than those without. These indices were worse in the subgroup of 23 with moderate-severe pain/disability. Criteria for irritable bowel syndrome were met in 70% of those with pain irrespective of its severity. Multivariate analysis identified 4 independent associations with moderate-severe pain/disability: active disease (odds ratio, 49 [95% confidence intervals, 1.6-1455]), catastrophizing tendency (35 [3-228]), medication belief score (0.05 [0.005-0.55], and depression score (1.80 [1.02-3.17]). CONCLUSIONS: CP has major effects on quality of life and functional and social outcomes. Active disease and maladaptive coping strategies and negative attitudes and beliefs toward symptoms are independently associated with more severe pain. Management strategies should move the focus away from analgesic dependence toward psychosocial intervention and nonpharmacologic therapy.

Cytomegalovirus disease, haemophagocytic syndrome, immunosuppression in patients with IBD: 'a cocktail best avoided, not stirred'.

We report two cases of cytomegalovirus (CMV) viraemia resulting in severe pneumonitis and associated haemophagocytic syndrome manifesting in patients with inflammatory bowel disease, on stable doses of azathioprine in clinical remission. In both cases, azathioprine was withdrawn at time of hospital presentation and after delays in diagnosis; intravenous ganciclovir was then administered, with resultant rapid improvement of haematological and clinical parameters. Following recovery, immunomodulators were not recommenced given patient aversion and the theoretical risk of CMV reactivation, albeit the evidence for this approach is limited. CMV-related haemophagocytic syndrome and organ dysfunction, in the context of immunomodulator therapy in IBD are rare but life-threatening, and thus requires further investigation and discussion.

Update in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a common disease in Australia and frequently encountered in primary care. Major developments in investigation and management have taken place. OBJECTIVE: This article aims to review some recent breakthroughs in IBD investigation and management. DISCUSSION: Diagnosis involves a changing combination of enhanced traditional techniques with new diagnostic tools, typically blood and stool testing with improved endoscopy and new radiological tests. Management has seen the introduction of new powerful biologic therapies, greater understanding in the way we use older therapies, and a focus on preventing complications such as malignancy or infection. Treatment philosophy now attempts to alter the natural history of the disease and prevent long term complications. The importance of associated, previously overlooked factors is being increasingly recognised. Only by taking a long term, patient centred multidisciplinary approach will an optimal outlook for the majority of patients with IBD be achieved.

Ectopic c-kit expression affects the fate of melanocyte precursors in Patch mutant embryos.

The Patch (Ph) mutation in the mouse, a deletion that includes the gene for PDGFR alpha, is a recessive lethal that exhibits a dominant pigment phenotype in heterozygotes. To assess whether the Ph mutation acts cell-autonomously or non-autonomously on melanocyte development, we have examined the melanogenic potential of neural crest populations from normal and mutant crest cells in vitro and the pattern of dispersal and survival of melanocyte precursors (MPs) in vivo. We report that trunk neural crest cells from homozygous Ph embryos give rise to pigmented melanocytes in vitro in response to Steel factor (SlF). In vivo, homozygous Ph embryos contain a subpopulation of crest-derived cells that express c-kit and tyrosinase-related protein-2 characteristic of MPs. These cells begin to migrate normally on the lateral crest migration pathway, but then fail to disperse in the dermal mesenchyme and subsequently disappear. Although dermal mesenchyme is adversely affected in Ph homozygotes, SlF mRNA expression by the cells of the dermatome is normal in Ph embryos when neural crest-derived MPs start to migrate on the lateral pathway. In contrast, mRNA for the SlF receptor, c-kit, was observed to be ectopically expressed in somites and lateral mesenchyme in embryos carrying the Ph mutation. Based on this ectopic expression of c-kit in Ph mutant embryos, and the observed distribution of SlF protein in normal and mutant embryos, we suggest that competition for limited amounts of SlF localized on the lateral neural crest migration pathway alters melanocyte dispersal and survival.

Transient steel factor dependence by neural crest-derived melanocyte precursors.

Growth/trophic factors, encountered by dispersing neural crest cells, affect the development of different crest-derived subpopulations. Here, we address the dependence by cells of the crest-derived melanogenic lineage on Steel factor (SLF). Specifically, we have asked when SLF-responsive cells first appear in cultured neural crest cell populations and whether there is a defined period during which this melanogenic subpopulation requires the presence of the SLF. We demonstrate that survival of a melanogenic subpopulation of cultured murine crest cells requires SLF for a critical period, which begins only after the second day of dispersal in vitro. We also conclude that the critical period of SLF dependence lasts about 4 days, ending about the time that melanocytes differentiate, as indicated by the presence of functional melanosomes, but before their overt melanization. Finally, we demonstrate that although the final stages of melanogenesis (i.e., melanization) do not appear to require SLF, this process may be enhanced by extended exposure to high levels of SLF in vitro.

Steel factor and c-kit receptor: from mutants to a growth factor system.

Mutations within the Steel and Dominant Spotting loci of mice have led to the recent identification of a growth factor/receptor system required for the normal development of germ cells, pigment cells and hematopoietic cells. Interactions between the products of these genes, Steel factor and c-Kit respectively, have now been demonstrated to influence various developmental processes, including survival, proliferation, and/or differentiation of cells in a tissue specific manner. In addition, our current understanding of the molecular basis of various Steel and Dominant Spotting alleles coupled with the emerging information on the expression pattern of steel factor and c-kit transcripts during development, is now beginning to explain the pleiotropic affects of these mutations.

Regulation and role of PDGF receptor alpha-subunit expression during embryogenesis.

The platelet-derived growth factor receptor alpha-subunit (PDGFR alpha) is the form of the PDGF receptor that is required for binding of PDGF A-chain. Expression of PDGFR alpha within the early embryo is first detected as the mesoderm forms, and remains characteristic of many mesodermal derivatives during later development. By 9.5 days of development, embryos homozygous for the Patch mutation (a deletion of the PDGFR alpha) display obvious growth retardation and deficiencies in mesodermal structures, resulting in the death of more than half of these embryos. Mutant embryos that survive this first critical period are viable until a new set of defects become apparent in most connective tissues. For example, the skin is missing the dermis and connective tissue components are reduced in many organs. By this stage, expression of PDGFR alpha mRNA is also found in neural crest-derived mesenchyme, and late embryonic defects are associated with both mesodermal and neural crest derivatives. Except for the neural crest, the lens and choroid plexus, PDGFR alpha mRNA is not detected in ectodermal derivatives until late in development in the central nervous system. Expression is not detected in any embryonic endodermal derivative at any stage of development. These results demonstrate that PDGFR alpha is differentially expressed during development and that this expression is necessary for the development of specific tissues.

A PDGF receptor mutation in the mouse (Patch) perturbs the development of a non-neuronal subset of neural crest-derived cells.

The Patch (Ph) mutation in mice is a deletion of the gene encoding the platelet-derived growth factor receptor alpha subunit (PDGFR alpha). Patch is a recessive lethal recognized in heterozygotes by its effect on the pattern of neural crest-derived pigment cells, and in homozygous mutant embryos by visible defects in craniofacial structures. Since both pigment cells and craniofacial structures are derived from the neural crest, we have examined the differentiation of other crest cell-derived structures in Ph/Ph mutants to assess which crest cell populations are adversely affected by this mutation. Defects were found in many structures populated by non-neuronal derivatives of cranial crest cells including the thymus, the outflow tract of the heart, cornea, and teeth. In contrast, crest-derived neurons in both the head and trunk appeared normal. The expression pattern of PDGFR alpha mRNA was determined in normal embryos and was compared with the defects present in Ph/Ph embryos. PDGFR alpha mRNA was expressed at high levels in the non-neuronal derivatives of the cranial neural crest but was not detected in the crest cell neuronal derivatives. These results suggest that functional PDGF alpha is required for the normal development of many non-neuronal crest-derived structures but not for the development of crest-derived neuronal structures. Abnormal development of the non-neuronal crest cells in Ph/Ph embryos was also correlated with an increase in the diameter of the proteoglycan-containing granules within the crest cell migratory spaces. This change in matrix structure was observed both before and after crest cells had entered these spaces. Taken together, these observations suggest that functional PDGFR alpha can affect crest development both directly, by acting as a cell growth and/or survival stimulus for populations of non-neurogenic crest cells, and indirectly, by affecting the structure of the matrix environment through which such cells move.

Association between collagen and glycosaminoglycans is altered in dermal extracellular matrix of fetal Steel (Sld/Sld) mice.

Altered extracellular matrix produced by Steel mutant fetuses affects the pigmentation of neural crest cells in vitro (K. Morrison-Graham, L. West-Johnsrud, and J.A. Weston, 1990, Dev. Biol. 139). Here, we demonstrate that collagen bundle morphology and hyaluronidase sensitivity of the glycosaminoglycans associated with the collagen fibrils differ between normal and mutant dermis. Although no differences were detected in the amounts of collagen or glycosaminoglycans produced in vitro or present in vivo, hyaluronic acid was more readily extracted from Sld/Sld than from normal skin. We suggest that the Steel mutation alters the organization of collagen bundles and associated hyaluronic acid within the extracellular matrix.

Extracellular matrix from normal but not Steel mutant mice enhances melanogenesis in cultured mouse neural crest cells.

The Steel mutation is a non-cell-autonomous defect in mice that affects the development of several stem cell populations, including germ cells, hematopoietic cells, and neural crest-derived pigment cells. To characterize the environmental lesion caused by the Steel mutation, we have compared the ability of normal and mutant extracellular matrix material to support the differentiation of normal mouse neural crest cells in vitro. Extracellular matrix deposited by cultured skin cells isolated from normal fetuses enhanced melanogenesis by crest cells over that observed on plastic substrata. In contrast, matrix material produced by Steel-Dickie (Sld) fetal skin cells failed to enhance melanogenesis. Adrenergic differentiation by neural crest-derived cells was promoted equally by both normal and mutant extracellular matrix compared to control substrata. We conclude that the environmental defect in mutant embryos selectively affects a melanogenic subpopulation of neural crest cells and resides, at least in part, in the extracellular matrix.

Mouse mutants provide new insights into the role of extracellular matrix in cell migration and differentiation.

Migratory cell populations in the developing embryo disperse, localize and eventually differentiate in environments rich in extracellular matrix material. The extracellular matrix provides both a substratum for migration and a source of differentiative cues for the developing cells. Mutations in mice and other animals that alter embryonic interstitial environments are now providing information about the role of the extracellular matrix in these early developmental processes.

Morphological properties and membrane channels of the growth cones induced in PC12 cells by nerve growth factor.

Large growth cones were produced in vitro by nerve growth factor (NGF) treatment of multinucleate cells produced by chemical fusion of cells of the neuron-like clone PC12. These endings were studied both at the light microscopic and ultrastructural levels. The activity of ionic channels at growth cones was recorded with intracellular microelectrodes, patch recording of single channels, and whole cone recording from mechanically isolated growth cones. Morphologically, these large growth cones were characterized by the presence of microspikes and filopodia, by the presence of actin demonstrated immunohistochemically, and by the presence of catecholamine fluorescence. At the ultrastructural level they contained a broad spectrum of organelles with a distribution characteristic of neuronal growth cones, including dense core vesicles, abundant smooth membrane cisternae, microtubules, and a filamentous network. The presence of channels capable of generating action potentials was revealed by intracellular microelectrode recording from the growth cone in the presence of locally applied tetraethylammonium (TEA). TEA appeared to block outward current channels that could effectively shunt inward current activated by depolarization. Action potentials elicited by depolarizing current in the presence of TEA could be blocked reversibly by Cd2+, a specific blocker of Ca channels. These action potentials were often followed by a long after-hyperpolarization lasting hundreds of milliseconds. This after-hyperpolarization was similar to that recorded in the cell body of PC12 cells where it appears to be mediated by Ca-activated K current. Single channel recording from outside-out excised patches of membrane from the growth cones perfused with KF revealed the presence of voltage sensitive Na channels, Ca-activated K channels, and K channels resembling delayed rectifier K channels. Macroscopic currents recorded from mechanically isolated growth cones in the "whole cone" configuration showed rapid inward currents at potentials greater than or equal to -40 mV, followed by delayed outward currents at more positive potentials, a finding providing additional evidence for the presence of Na and K channels in growth cones.

A simple and reliable method for the localization in cell culture of single identifiable cells for ultrastructural analyses.

A simple method for relocating single cells in monolayer cultures for subsequent morphological or ultrastructural analysis is reported. This consists of producing, on the culture dish surface, a nontoxic carbon grid that is preserved during processing for either transmission (TEM) or scanning (SEM) electron microscopy. For TEM studies these grids are readily transferred along with the cells into the embedding plastic, and thus individual grid squares containing a cell(s) of interest can be quickly located, remounted, and sectioned. These grids may be useful for ultrastructural analyses of single cells previously studied electrophysiologically or after microinjection of macromolecules.

An anatomical and electrophysiological study of synapse elimination at the developing frog neuromuscular junction.

Synapse elimination was examined in the developing frog cutaneous pectoris muscle using histological and electrophysiological techniques. Morphological synapse elimination occurred in two phases. The first phase, which began at the time of metamorphosis and continued until the second to third postmetamorphic week, was characterized by a rapid decline in the number of endplates receiving greater than or equal to 3 synaptic inputs. However, 50% of the muscle fibers still remained dually innervated. This dual innervation decreased with a much slower time course; approximately 20% of the muscle fibers were dually innervated in 1- to 2-year-old frogs. During the first phase of synapse elimination no difference was noted between the distribution of acetylcholine receptors or acetylcholinesterase activity associated with the terminal arborizations formed by separate axons at one synaptic site. However, terminal arborizations formed by small diameter axons and consisting of varicosities separated by thin interconnectives became apparent during this period. Such varicose arborizations responded to nerve stimulation and released acetylcholine in proportion to their terminal length as did the nonvaricose arborizations. In addition, the number of morphological and physiological inputs at one endplate site was well correlated throughout the first phase of synapse elimination.

Sprouting and regeneration of frog motoneurons during synapse elimination.

The ability of frog axons to sprout and reinnervate during the period of synapse elimination was examined in the cutaneous pectoris muscle from young postmetamorphic frogs using histological staining of nerve terminals and postsynaptic acetylcholinesterase. Partial denervation of the cutaneous pectoris muscle during the period of synapse elimination produced rapid sprouting of the intact axons. The majority of denervated endplates were being reinnervated by sprouts within 3 days. In addition, total denervation performed by either sectioning or crushing the nerve was followed by functional reinnervation. Approximately 98% of the endplates were being reinnervated within 7 days after a nerve crush and 10 days after sectioning the nerve.

Structure of developing frog neuromuscular junctions.

Developing neuromuscular junctions in the cutaneous pectoris muscle from tadpoles and and postmetamorphic frogs were studied in the light microscope. Presynaptic nerve terminals and postsynaptic acetylcholinesterase (AChE) activity simultaneously demonstrated at these developing junctions using the NBT-AChE method. The earliest nerve contacts studied were small enlargements at the ends of unmyelinated axons. As development progressed, single nerve contacts, often with growth cones, grew in length, generally parallel to the long axis of the myotube. Further endplate maturation was characterized by individual terminal processes developing secondary and tertiary branches, ultimately leading to highly complex terminal arborizations. The initial synaptic contact at developing neuromuscular junctions was made by a single axon, but with further development these same synaptic sites became multiply innervated. The occurrence of multiple innervation was a transient phenomena; the multiple synaptic inputs were eliminated during further maturation. The time course of synapse elimination was protracted, with some multiple innervation even persisting in relatively large adult frogs. The first nerve contacts were generally devoid of observable endplate AChE activity. Early appearance of AChE activity was sometimes graded and in some cases portions of the nerve terminal processes were associated with AChE while other regions of the same terminal arborization were not.