Angiotensin II type 2 receptor (AT2 R) localization and antagonist-mediated inhibition of capsaicin responses and neurite outgrowth in human and rat sensory neurons.

BACKGROUND: The angiotensin II (AngII) receptor subtype 2 (AT2 R) is expressed in sensory neurons and may play a role in nociception and neuronal regeneration. METHODS: We used immunostaining with characterized antibodies to study the localization of AT2 R in cultured human and rat dorsal root ganglion (DRG) neurons and a range of human tissues. The effects of AngII and AT2 R antagonist EMA401 on capsaicin responses in cultured human and rat (DRG) neurons were measured with calcium imaging, on neurite length and density with Gap43 immunostaining, and on cyclic adenosine monophosphate (cAMP) expression using immunofluorescence. RESULTS: AT2 R expression was localized in small-/medium-sized cultured neurons of human and rat DRG. Treatment with the AT2 R antagonist EMA401 resulted in dose-related functional inhibition of capsaicin responses (IC50 = 10 nmol/L), which was reversed by 8-bromo-cAMP, and reduced neurite length and density; AngII treatment significantly enhanced capsaicin responses, cAMP levels and neurite outgrowth. The AT1 R antagonist losartan had no effect on capsaicin responses. AT2 R was localized in sensory neurons of human DRG, and nerve fibres in peripheral nerves, skin, urinary bladder and bowel. A majority sub-population (60%) of small-/medium-diameter neuronal cells were immunopositive in both control post-mortem and avulsion-injured human DRG; some very small neurons appeared to be intensely immunoreactive, with TRPV1 co-localization. While AT2 R levels were reduced in human limb peripheral nerve segments proximal to injury, they were preserved in painful neuromas. CONCLUSIONS: AT2 R antagonists could be particularly useful in the treatment of chronic pain and hypersensitivity associated with abnormal nerve sprouting.

A longitudinal study of sensory biomarkers of progression in patients with diabetic peripheral neuropathy using skin biopsies.

We aimed to identify biomarkers in skin punch biopsies that could be used to monitor progression of diabetic peripheral neuropathy (DPN), and, in future studies, to assess the efficacy of agents that may reduce progression. Patients with DPN were studied with clinical assessments, skin biopsies, quantitative sensory testing (QST), histamine-induced skin flare, nerve conduction studies and contact heat-evoked potentials (CHEPS). Skin biopsies were performed on two visits with a 6 month interval (n=29 patients) to quantify intraepidermal (IENF) and subepidermal (SENF) nerve fibres immunoreactive for: protein gene product 9.5 (PGP9.5), a pan-neuronal marker; transient receptor potential cation channel vanilloid 1 (TRPV1), the heat and capsaicin receptor; and growth associated protein-43 (GAP-43), a marker of regenerating fibres. The IENF were counted along the length of four non-consecutive sections, and results were expressed as fibres per millimetre length of section. SENF were measured by image analysis, and the area of highlighted immunoreactivity was obtained as a percentage (% area) of the field scanned. QST, skin flare and CHEPS were also performed at the two visits. We found that IENF and SENF were significantly reduced for both PGP9.5 and TRPV1 between the first and second skin biopsy over 6months. The annual rate ± standard error of the mean of IENF loss was 3.76 ± 1.46 fibres/mm for PGP9.5, and 3.13 ± 0.58 fibres/mm for TRPV1. The other tests did not show significant changes. Strongly positive GAP-43 nerve fibres were found in deep dermis in the patients with diabetes, even in those with an absence of IENF. We conclude that PGP9.5 and TRPV1 IENF and SENF in skin biopsies are useful markers of progression in DPN, whereas GAP-43 SENF could potentially help detect nerve regeneration in severe neuropathy.

Increased capsaicin receptor TRPV1-expressing sensory fibres in irritable bowel syndrome and their correlation with abdominal pain.

OBJECTIVE: The capsaicin receptor TRPV1 (transient receptor potential vanilloid type-1) may play an important role in visceral pain and hypersensitivity states. In irritable bowel syndrome (IBS), abdominal pain is a common and distressing symptom where the pathophysiology is still not clearly defined. TRPV1-immunoreactive nerve fibres were investigated in colonic biopsies from patients with IBS, and this was related to abdominal pain. METHODS: Rectosigmoid biopsies were collected from 23 IBS patients fulfilling Rome II criteria, and from 22 controls. Abdominal pain scores were recorded using a validated questionnaire. TRPV1-, substance P- and neuronal marker protein gene product (PGP) 9.5-expressing nerve fibres, mast cells (c-kit) and lymphocytes (CD3 and CD4) were quantified, following immunohistochemistry with specific antibodies. The biopsy findings were related to the abdominal pain scores. RESULTS: A significant 3.5-fold increase in median numbers of TRPV1-immunoreactive fibres was found in biopsies from IBS patients compared with controls (p<0.0001). Substance P-immunoreactive fibres (p = 0.01), total nerve fibres (PGP9.5) (p = 0.002), mast cells (c-kit) (p = 0.02) and lymphocytes (CD3) (p = 0.03) were also significantly increased in the IBS group. In multivariate regression analysis, only TRPV1-immuno-reactive fibres (p = 0.005) and mast cells (p = 0.008) were significantly related to the abdominal pain score. CONCLUSIONS: Increased TRPV1 nerve fibres are observed in IBS, together with a low-grade inflammatory response. The increased TRPV1 nerve fibres may contribute to visceral hypersensitivity and pain in IBS, and provide a novel therapeutic target.

Normalization of substance P levels in rectal mucosa of patients with faecal incontinence treated successfully by sacral nerve stimulation.

BACKGROUND: Sacral nerve stimulation (SNS) may improve faecal incontinence by modulating rectal sensation. This study measured changes in the peripheral expression of various neural epitopes in response to SNS. METHODS: Rectal mucosal biopsies were taken from 12 patients before and after temporary SNS, and from ten responders at 90 days after permanent stimulation. Sections were immunostained for substance P, transient receptor potential vanilloid (TRPV) 1, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP). Levels were compared with those in nine continent controls. RESULTS: Baseline levels of percentage area immunoreactivities of substance P (median 0.51 (95 per cent confidence interval 0.31 to 0.73) versus 0.13 (0.07 to 0.27) per cent; P < 0.001) and TRPV1 (0.76 (0.41 to 1.11) versus 0.09 (0.04 to 0.14) per cent; P < 0.001), but not of VIP (1.26 (0.37 to 2.15) versus 1.28 (0.39 to 2.17); P = 0.943), were significantly greater than in controls. Successful SNS resulted in a significant decrease in substance P immunostaining after temporary (0.15 (0.06 to 0.51) per cent; P = 0.051) and permanent (0.17 (0 to 0.46) per cent; P = 0.051) stimulation. Immunoreactivity of TRPV1, VIP, CGRP and neural markers showed no qualitative change. CONCLUSION: Patients with faecal incontinence demonstrate normalization of raised rectal mucosal substance P levels following successful SNS.

The potential role of nerve growth factor (NGF) in painful neuromas and the mechanism of pain relief by their relocation to muscle.

Painful neuromas have been successfully treated by surgical procedures including relocation to muscle, but the underlying molecular mechanism remains unclear. Nerve growth factor (NGF) is secreted by tissues and promotes the expression of ion channels and neuropeptides in sensory neurons involved in pain transmission. We hypothesised that excess of NGF may lead to pain in neuromas and that the efficacy of surgical relocation results from deprivation of NGF, i.e. translocation from NGF-rich regions, particularly sub-cutaneous structures associated with injury or inflammation, to NGF-poor structures such as muscle or bone. Using immunohistological methods with primary antibodies to rhNGF, we report that NGF levels were elevated in 13 painful neuromas in comparison with six control nerves. However, in four painful neuromata re-located into muscle with pain relief, the NGF level was similar to that of controls. NGF levels suggest an explanation for the development of painful neuromas and the efficacy of relocation.

A histological study of the hph-1 mouse mutant: an animal model of phenylketonuria and infantile hypertrophic pyloric stenosis.

AIM: To quantify the chronological sequence of changes in the morphology and immunoreactivity for neurotransmitters in the pylorus of an animal model of infantile hypertrophic pyloric stenosis and phenylketonuria. METHOD: Thirty specimens of pylorus from hph-1 mice and age/sex matched controls (age range: 10-180 days) were examined using conventional histology and immunohistochemistry for a variety of antigens: protein gene product 9.5, a pan neuronal marker; vasoactive intestinal polypeptide; nitric oxide synthase two antigens coalesced to the same inhibitory neurons in humans; substance P, a potent excitatory neurotransmitter; and calcitonin gene related peptide, a neurotransmitter implicated in the somatic afferent innervation of the stomach. The changes in the morphology of the muscle layers were quantified and statistically analysed for each age group (10, 20, 40, 90 and 180 days). RESULTS: Between 10 and 90 days of age, all muscle layers of the hph-1 mice were hypertrophied, for example, 10 days, hph-1 longitudinal muscle mean diameter = 3.4, control = 1.8; hph-1 circular muscle width = 11.5, control = 4.7. The hph-1 mice were significantly smaller during this period (40 days, hph-1 weight = 10 g, control = 25 g). There was no change in the pattern of expression of the antigens examined within the hph-1 mice compared with the controls. CONCLUSION: Hph-1 mice develop a transient smooth muscle hypertrophy of the pylorus attended by gastric distension and failure to gain weight. These changes resolve as the pyloric muscle hypertrophy resolves.

Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency.

BACKGROUND: Faecal urgency and incontinence with rectal hypersensitivity is a distressing, unexplained disorder that is inadequately treated. We aimed to determine whether expression of the heat and capsaicin receptor vanilloid receptor 1 (TRPV1 or VR1) was changed in rectal sensory fibres, and to correlate nerve fibre density with sensory abnormalities. METHODS: We compared full-thickness rectal biopsy samples from nine patients with physiologically characterised rectal hypersensitivity with tissue samples from 12 controls. Sensory thresholds to rectal balloon distension and heating the rectal mucosa were measured before biopsy. We assessed specimens with immunohistochemistry and image analysis using specific antibodies to TRPV1; nerve growth factor (NGF) receptor tyrosine kinase A; glial cell line-derived neurotrophic factor (GDNF); neuropeptides calcitonin gene-related peptide (CGRP) and substance P; the related vanilloid receptor-like protein (VRL) 2; glial markers S-100 and glial fibrillary acid protein (GFAP); and the nerve structural marker peripherin. FINDINGS: In rectal hypersensitivity, nerve fibres immunoreactive to TRPV1 were increased in muscle, submucosal, and mucosal layers: in the mucosal layer, the median% area positive was 0.44 (range 0.30-0.59) in patients who were hypersensitive and 0.11 (0.00-0.21) in controls (p=0.0005). The numbers of peripherin-positive fibres also increased in the mucosal layer (hypersensitive 3.00 [1.80-6.50], controls 1.20 [0.39-2.10]: (p=0.0002). The increase in TRVP1 correlated significantly with the decrease in rectal heat (p=0.03) and the distension (p=0.02) sensory thresholds. The thresholds for heat and distension were also significantly correlated (p=0.0028). Expression of nerve fibres positive for GDNF (p=0.001) and tyrosine kinase A (p=0.002) was also increased, as were cell bodies of the submucosal ganglia immunoreactive to CGRP (p=0.0009). INTERPRETATION: Faecal urgency and rectal hypersensitivity could result from increased numbers of polymodal sensory nerve fibres expressing TRPV1. The triggering factor or factors remain uncertain, but drugs that target nerve terminals that express this receptor, such as topical resiniferatoxin, deserve consideration.

A quantitative study of the neural changes underlying pyloric stenosis in dogs.

This study aimed to quantify the neural changes in congenital pyloric stenosis in dogs and to study the comparative anatomy between this condition in dogs and that in infantile hypertrophic pyloric stenosis. Eight specimens from the pylorus of dogs with pyloric stenosis and six control specimens were examined using conventional histology and immunohistochemistry for a range of neural antigens. The changes in the proportion of nerves immunoreactive for each antigen were quantified and analysed statistically. The morphology of the nerves in the diseased dogs was similar to that in controls. Only vasoactive intestinal peptide was reduced in expression in dogs (median proportion in control dogs 0.57, in diseased dogs 0.17; P = 0.065). This study demonstrates both morphological similarities and significant differences between closely related conditions in dogs, humans and other species.

Novel capsaicin (VR1) and purinergic (P2X3) receptors in Hirschsprung's intestine.

BACKGROUND/PURPOSE: Studies of Hirschsprung's disease (HSCR) have shown that hypertrophic nerves in aganglionic bowel are mainly of extrinsic origin and may contain sensory elements. Recent advances have shown a specific capsaicin receptor VR1 (vanilloid receptor-1), and an ATP-gated ion channel P2X(3), which are expressed by sensory neurons. METHODS: This study investigated, for the first time, the distribution of VR1- and P2X(3)-immunoreactivity in normal adult, infant, and HSCR large intestine, using specific antibodies for immunohistochemistry. RESULTS: VR1-immunoreactive fibers and nerve fascicles, but not somata, were detected in all regions of the bowel in controls with few weakly immunostained fibers in the mucosa/lamina propria. Hypertrophic nerve bundles in hypoganglionic and aganglionic bowel showed intense VR1-immunoreactivity, whereas normoganglionic regions of HSCR were similar to controls. P2X(3)-immunoreactive neuronal cell bodies, in some instances with long axonal processes, were detected in the myenteric and submucous plexuses in control infant, adult, and ganglionic HSCR samples. Aganglionic samples showed weak P2X(3)-immunoreactivity in hypertrophic nerve fasciculi in the submucous and myenteric plexuses. CONCLUSIONS: The presence of VR1- and P2X(3)-immunoreactivities in aganglionic HSCR bowel indicates that sensory nerves may form a significant proportion of its hypertrophic innervation. The functional significance of P2X(3) and VR1 receptors in enteric nerves deserves further investigation.

ATP-gated ion channel P2X(3) is increased in human inflammatory bowel disease.

P2X(3) is a novel ATP-gated cation channel that is selectively expressed by small-diameter sensory neurones in rodents, and may play a role in nociception by binding ATP released from damaged or inflamed tissues. We have studied, for the first time, P2X(3) immunoreactivity in human inflammatory bowel disease, using Western blotting and immunohistochemistry. A major 66-kDa specific protein was found by Western blotting in all colon extracts. In the inflamed group there was a significant two-fold increase in the relative optical density of the 66-kDa band (21.2 +/- 3.1; n=8) compared to controls (11.4 +/- 3.7; n=8; P=0.009). In the control colon, P2X(3)-immunoreactive neurones were scattered throughout the myenteric and submucosal plexuses, with some neurones showing immunopositive axons/dendrites. The pattern of immunostaining was similar to the neuronal marker peripherin. In general, the intensity of the staining was greater in myenteric than submucosal neurones. The number of P2X(3)-immunoreactive neurones was significantly increased in the myenteric plexus of inflamed colon compared to controls (n=13; P=0.01). In humans, unlike rodents, P2X(3) is thus not restricted to sensory neurones. Increased P2X(3) in inflamed intestine suggests a potential role in dysmotility and pain, for which it represents a new therapeutic target.

Increased acid-sensing ion channel ASIC-3 in inflamed human intestine.

OBJECTIVES: Acid-sensing ion channels (ASICs) are expressed by rat sensory neurons and may mediate pain associated with tissue acidosis after inflammation or injury. Our aim was to examine the molecular forms and localization of ASICs in human intestine and dorsal root ganglia using immunochemical techniques, and to measure the effects of inflammation and injury. DESIGN AND METHODS: Inflamed Crohn's disease intestine and injured human dorsal root ganglia, with appropriate controls, were studied by Western blotting and immunohistochemistry, using specific affinity-purified ASIC antibodies. RESULTS: In the Western blot, there was a significant three-fold increase in the mean relative optical density of the ASIC-3 55-kDa band (but not ASIC-1 or ASIC-2) in full-thickness inflamed intestine, as well as in separated muscle and mucosal layers. There was a corresponding trend for an increased immunoreactive density and increased number of ASIC-3-positive neurons in the myenteric and sub-mucous plexus of inflamed intestine. In dorsal root ganglia, immunoreactivity for all ASICs was restricted to a sub-population (about 50%) of small-diameter (nociceptor) sensory neurons, and was generally less intense after injury. CONCLUSIONS: Increased ASIC-3 in inflamed intestine suggests a role in pain or dysmotility, for which ASICs represent new therapeutic targets.

Capsaicin receptor VR1 and ATP-gated ion channel P2X3 in human urinary bladder.

OBJECTIVES: To determine the presence, distribution and molecular forms of the vanilloid receptor VR1, and confirm the presence and distribution of the ATP-gated ion channel P2X3 in the human urinary bladder. Materials and methods Normal urinary bladder tissues were obtained at postmortem from four subjects. Eight urinary bladder biopsies were also taken from patients with detrusor hyper-reflexia treated with intravesical resiniferatoxin. The specimens were studied using affinity-purified specific antibodies to VR1 and P2X3 by Western blotting and immunocytochemistry, and compared with immunostaining using antibodies to the pan-neuronal marker PGP 9.5 and Schwann cell marker S-100. RESULTS: VR1- and P2X3-immunoreactive fine nerve fibres were scattered throughout the suburothelium of the normal bladder and cystoscopic biopsies, and traversed the muscle layer. They had a similar distribution to PGP 9.5-immunoreactive fibres, but there were fewer, suggesting localization in subsets of axons. Western blot studies showed an expected 100-kDa VR1 protein and a P2X3-immunoreactive 66-kDa protein. Conclusion VR1 and P2X3 are present in the human urinary bladder and may contribute to distinct pathophysiological states of bladder overactivity, in accord with their differential expression in sensory neurones. Intravesical vanilloids act via VR1 and are effective in the treatment of detrusor hyper-reflexia. P2X3 may represent a selective therapeutic target for other causes of overactive bladder.

Decreased tyrosine kinase C expression may reflect developmental abnormalities in Hirschsprung's disease and idiopathic slow-transit constipation.

BACKGROUND: Some patients with Hirschsprung's disease have refractory constipation following excision of aganglionic bowel, as do patients with idiopathic slow-transit constipation (STC). Gut motility depends on enteric neuronal development in response to expression of trophic factors and their receptors. Recent studies indicate the importance of neurotrophin 3 (NT-3) and its high-affinity receptor tyrosine kinase C (trk C) in enteric neuronal development. METHODS: Blinded quantitative immunohistochemical analysis of colon from patients with Hirschsprung's disease (aganglionic, hypoganglionic and normoganglionic) (n = 5), STC (n = 6) and appropriate age-matched control tissues (n = 5) was performed for NT-3 and trk C. Sural nerve morphometry and immunostaining were undertaken in three patients with STC who had abnormalities on limb autonomic and sensory testing. RESULTS: A significantly higher proportion of submucous plexus neurones was trk C immunoreactive in control infant than adult colon (mean(s.e.m.) 73(9) versus 16(3) per cent of the total; P < 0.001), in accord with a role in development. The proportion of submucous plexus trk C-immunoreactive neurones was reduced in colon from patients with Hirschsprung's disease (28(7) per cent of total in normoganglionic Hirschsprung's disease; P < 0.007 versus infant controls) and STC (10(1) per cent of total; P = 0.053 versus adult controls). No abnormalities of STC sural nerves were detected by morphometry or immunostaining. CONCLUSION: Decreased trk C expression may reflect developmental abnormalities in Hirschsprung's disease and idiopathic STC. Trk C activation by NT-3 or drugs may provide novel treatments. Presented in abstract form to the Pacific Association of Pediatric Surgeons, Las Vegas, Nevada, USA, May 2000

Immunolocalization of SNS/PN3 and NaN/SNS2 sodium channels in human pain states.

The tetrodotoxin-resistant (TTX-R) voltage-gated sodium channel SNS/PN3 and the newly discovered NaN/SNS2 are expressed in sensory neurones, particularly in nociceptors. Using specific antibodies, we have studied, for the first time in humans, the presence of SNS/PN3 and NaN/SNS2 in peripheral nerves, including tissues from patients with chronic neurogenic pain. In brachial plexus injury patients, there was an acute decrease of SNS/PN3- and NaN/SNS2-like immunoreactivity in sensory cell bodies of cervical dorsal root ganglia (DRG) whose central axons had been avulsed from spinal cord, with gradual return of the immunoreactivity to control levels over months. In contrast, there was increased intensity of immunoreactivity to both channels in some peripheral nerve fibers just proximal to the site of injury in brachial plexus trunks, and in neuromas. These findings suggest that the expression of these sodium channels in neuronal cell bodies is reduced after spinal cord root avulsion injury in man, but that pre-synthesized channel proteins may undergo translocation with accumulation at sites of nerve injury, as in animal models of peripheral axotomy. The latter may contribute to positive symptoms, as our patients all showed a positive Tinel's sign. Nerve terminals in distal limb neuromas and skin from patients with chronic local hyperalgesia and allodynia all showed marked increases of SNS/PN3-immunoreactive fibers, but little or no NaN/SNS2-immunoreactivity, suggesting that the former may be related to the persistent hypersensitive state. Axonal immunoreactivity to both channels was similar to control nerves in sural nerve biopsies in a selection of neuropathies, irrespective of nerve inflammation, demyelination or spontaneous pain, including a patient with congenital insensitivity to pain. Our studies suggest that the best target for SNS/PN3 blocking agents is likely to be chronic local hypersensitivity.

Neurotrophin-3 is increased in skin in human diabetic neuropathy.

Neurotrophin-3 (NT-3), a member of the neurotrophin family, has been shown to be necessary for the development of muscle spindle and Merkel cell afferent nerve fibres in animal models. The presence of NT-3 in the suprabasal epidermis, where many unmyelinated sensory fibres terminate, has been shown for the first time. As these fibres are affected in early diabetic neuropathy and a clinical trial of recombinant human NT-3 in diabetic neuropathy is in progress, the concentrations of endogenous NT-3 in skin of 24 patients at different stages of diabetic polyneuropathy have been investigated. NT-3 concentrations, measured with a specific immunoassay, were significantly higher in affected skin biopsies from patients with diabetic neuropathy than matched control skin (diabetic skin 6.32 (1.18) pg/mg v control skin 1.28 (0.05) (mean (SEM)); p<0.004, Mann-Whitney U test), particularly in the later stages. The optical density of NT-3-immunostaining was also significantly greater in the epidermis in diabetic patients (diabetic epidermis 0.30 (0.06) v controls 0.24 (0.01); p<0.02). No correlation was found between individual quantitative sensory tests and the increase of NT-3 concentration. The increase of NT-3 seems to reflect the degree of skin denervation in diabetic neuropathy, and may represent a compensatory mechanism. The concentrations of NT-3 in other peripheral targets deserve study in diabetic neuropathy.

GDNF and its receptor component Ret in injured human nerves and dorsal root ganglia.

Glial cell line-derived neurotrophic factor (GDNF) is trophic to motor and sensory neurones in animal models. GDNF mRNA is up-regulated in Schwann cells after peripheral nerve injury in rats. We have quantified and localized GDNF and its receptor component Ret, for the first time in any species, in injured human peripheral nerves and dorsal root ganglia (DRG) avulsed from the spinal cord. Significantly higher levels of GDNF were found in nerve distal to the site of the injury than in proximal or intact nerve, and in avulsed DRG than in post-mortem control DRG. GDNF immunostaining was seen in Schwann cells and in DRG neurones, especially of small and medium size, with significantly increased numbers of medium sized sensory neurones immunoreactive for GDNF after avulsion. Ret immunoreactivity was restricted to DRG neurones and axons, with no significant changes in numbers of positive DRG cells after injury. Our findings suggest that GDNF may play a role in injured human nerves and sensory ganglia, particularly in medium sized sensory neurones.

Correlation of quantitative tests of nerve and target organ dysfunction with skin immunohistology in leprosy.

Loss of nociception and hypohidrosis in skin are hallmarks of leprosy, attributed to early invasion by Mycobacterium leprae of Schwann cells related to unmyelinated nerve fibres. We have studied skin lesions and contralateral clinically unaffected skin in 28 patients across the leprosy spectrum with a range of selective quantitative sensory and autonomic tests, prior to biopsy of both sites. Unaffected sites showed normal skin innervation, when antibodies to the pan-neuronal marker PGP (protein gene product) 9.5 were used, with the exception of intraepidermal fibres which were not detected in the majority of cases. Elevation of thermal thresholds and reduced sensory axon-reflex flare responses in affected skin correlated with decreased nerve fibres in the subepidermis, e.g. axon-reflex flux units (means+/-SEM) for no detectable innervation; decreased innervation; and clinically unaffected skin, were 23+/-3.1; 41.2+/-7.3; and 84.5+/-4.0, respectively. Reduced nicotine-induced axon-reflex sweating was correlated with decreased innervation of sweat glands. Where methacholine-induced direct activation of sweat glands was affected, there was inflammatory infiltrate and loss of sweat gland structure. This study demonstrates a correlation between selective nerve dysfunction on clinical tests and morphological changes in skin, irrespective of the type of leprosy, and is the first to show that loss of sweating in leprosy may result either from decreased innervation and/or involvement of the sweat glands. The findings have implications for the selection and monitoring of patients with leprosy in clinical trials which aim to restore cutaneous function.

Glial-derived neurotrophic factor in human adult and fetal intestine and in Hirschsprung's disease.

BACKGROUND & AIMS: Glial cell line-derived neurotrophic factor (GDNF) signals through the product of the ret proto-oncogene, which is known to be mutated in Hirschsprung's disease and other conditions with gut dysmotility. The aim of this study was to determine the presence of GDNF and its receptor component (RET) in human adult and developing intestine and to evaluate their distribution pattern in Hirschsprung's disease. METHODS: GDNF and RET were detected immunohistochemically, and GDNF was quantified by immunoassay in specimens of human adult and fetal colon and Hirschsprung's disease intestine. RESULTS: GDNF-like immunoreactivity was detected in all specimens. Immunostaining of GDNF was restricted to neural fiber-like structures across the gut wall and was similar to staining with markers of glia and Schwann cells. In contrast, RET immunoreactivity was found only in neural cell bodies. GDNF levels determined by immunoassay were higher in muscle than mucosal gut layers, and there was no difference between affected and unaffected segments of Hirschsprung's disease. CONCLUSIONS: GDNF is present in adult and fetal human gut, where it may play a neurotrophic role. Its staining pattern suggests that it is localized in glia or Schwann cells. There seems to be no difference of GDNF levels between affected and unaffected intestinal segments in Hirschsprung's disease.

Increased expression of cell adhesion molecule P-selectin in active inflammatory bowel disease.

The pathogenic changes of inflammatory bowel disease (IBD) depend on migration of circulating leucocytes into intestinal tissues. Although leucocyte rolling and tenuous adhesion are probably regulated by inducible selectins on vascular endothelia, little is known about the expression of these molecules in Crohn's disease and ulcerative colitis. Using immunohistochemistry on surgically resected specimens, this study investigated endothelial P-selectin (CD62, granular membrane protein-140) in frozen sections of histologically uninvolved tissues adjacent to inflammation (Crohn's disease = 10; ulcerative colitis = 10), from highly inflamed areas (Crohn's disease = 20; ulcerative colitis = 13), and from normal bowel (n = 20). By light microscopy, two forms of P-selectin immunoreactivity were detected that apparently corresponded ultrastructurally to stored and released distributions. Compared with the normal gut, there was a 3.7-fold increase of P-selectin immunoreactivity on veins (p < 0.0001), venules (p < 0.0001), and capillaries (p < 0.05) in the highly inflamed gut, without differences between Crohn's disease and ulcerative colitis. In the uninvolved gut, P-selectin expression was similar to that seen in normal controls, except for a focal increase of P-selectin in the vicinity of small lymphocyte aggregates. The dramatic upregulation of P-selectin in the inflamed tissue and its potential role in leucocyte trafficking support the concept of P-selectin blocking therapy for the control of active IBD.

Altered expression of cell adhesion molecules in uninvolved gut in inflammatory bowel disease.

Adhesion of circulating cells to vascular endothelium occurs in the early phase of inflammation, and is mediated by specific cell adhesion molecules. Many such adhesion molecules are increased in inflamed regions of ulcerative colitis (UC) and Crohn's disease (CD) but there is limited knowledge of their expression in the uninvolved gut, adjacent to inflammation. We investigated immunohistochemically the expression of platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) on resected specimens taken at a distance of 2-4 cm from the inflamed area and without histological signs of inflammation. Compared with normal gut, we found (i) a significant increase of PECAM-1-positive vessels in the mucosa of uninvolved UC (149.0 +/- 24.1 vessels/mm2 (mean +/- s.d.); normal colon = 123.1 +/- 21.6; P = 0.004); (ii) a significant decrease of ICAM-1-positive vessels in uninvolved CD (111.9 +/- 22.6 vessels/mm2; normal ileum = 136.9 +/- 27.6; P = 0.04); and (iii) a moderate but statistically insignificant increase of LFA-1-positive cells in the mucosa of uninvolved UC and Crohn's ileitis. This altered expression of cell adhesion molecules may contribute to the early lesion in inflammatory bowel disease and provide new therapeutic opportunities.