Dopamine D1 and D3 receptor modulators restore morphine analgesia and prevent opioid preference in a model of neuropathic pain.

A secondary consequence of spinal cord injury (SCI) is debilitating chronic neuropathic pain, which is commonly morphine resistant and inadequately managed by current treatment options. Consequently, new pain management therapies are desperately needed. We previously reported that dopamine D3 receptor (D3R) dysfunction was associated with opioid resistance and increases in D1 receptor (D1R) protein expression in the spinal cord. Here, we demonstrate that in a model of SCI neuropathic pain, adjuvant therapy with a D3R agonist (pramipexole) or D1R antagonist (SCH 39166) can restore the analgesic effects of morphine and reduce reward potential. Prior to surgery thermal and mechanical thresholds were tested in three groups of female rats (naïve, sham, SCI). After surgery, testing was repeated under the following drug conditions: 1) saline, 2) morphine, 3) pramipexole, 4) SCH 39166, 5) morphine + pramipexole, and 6) morphine + SCH 39166. Reward potential of morphine and both combinations was assessed using conditioned place preference. Following SCI, morphine + pramipexole and morphine + SCH 39166 significantly increased both thermal and mechanical thresholds. Morphine alone induced conditioned place preference, but when combined with either the D3R agonist or D1R antagonist preference was not induced. The data suggest that adjunct therapy with receptor-specific dopamine modulators can restore morphine analgesia and decrease reward potential and thus, represents a new target for pain management therapy after SCI.

Morphometric and gene expression analyses of stromal expansion during development of the bovine fetal ovary.

During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n=27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P<0.05). The proliferation index and numerical density of proliferating cells in the stroma significantly decreased (P<0.05), whereas the numerical density of cells in the stroma did not change (P>0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P<0.05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.

The role of the Rx homeobox gene in retinal progenitor proliferation and cell fate specification.

The Retinal homeobox gene (Rx; also Rax) plays a crucial role in the early development of the vertebrate eye. Germline deletion of Rx in mice results in the failure of optic vesicle formation, leading to anophthalmia. Recent research using conditional mouse knockout models provides some clues to the role of Rx in eye development following optic vesicle formation. However, the functions of Rx in embryonic retinogenesis are still not fully understood. We investigated the function of Rx in the mouse neural retina using a conditional knockout where the Pax6α-Cre driver deletes Rx activity in early retinal progenitors. The deletion of Rx activity causes a loss of retinal lamination, a depletion of retinal progenitors, and a change in retinal cell fate in our conditional knockout model. The deletion of Rx leads to an absence of late-born retinal neurons (rods and bipolar cells) and Müller glia at postnatal ages, as well as a loss of the early-born cone photoreceptors. Decreased BrdU labeling in the Rx-deleted portion of the retina suggests a loss of retinal progenitors via early cell cycle exit, which likely prevents the formation of late-born cells. As early-born cells, cone photoreceptors should not be as affected by early cell cycle exit of retinal progenitors. However, embryonic cone photoreceptor labeling is also markedly reduced in Rx-deleted retinas. Together these data demonstrate the importance of Rx for retinal progenitor proliferation and a specific requirement of Rx for cone formation in mice.

Mirror-Image Packing Provides a Molecular Basis for the Nanomolar Equipotency of Enantiomers of an Experimental Herbicide.

Programs of drug discovery generally exploit one enantiomer of a chiral compound for lead development following the principle that enantiomer recognition is central to biological specificity. However, chiral promiscuity has been identified for a number of enzyme families, which have shown that mirror-image packing can enable opposite enantiomers to be accommodated in an enzyme's active site. Reported here is a series of crystallographic studies of complexes between an enzyme and a potent experimental herbicide whose chiral center forms an essential part of the inhibitor pharmacophore. Initial studies with a racemate at 1.85 Šresolution failed to identify the chirality of the bound inhibitor, however, by extending the resolution to 1.1 Šand by analyzing high-resolution complexes with the enantiopure compounds, we determined that both enantiomers make equivalent pseudosymmetric interactions in the active site, thus mimicking an achiral reaction intermediate.

The effect of augmenting early nutritional energy delivery on quality of life and employment status one year after ICU admission.

Augmenting energy delivery during the acute phase of critical illness may reduce mortality and improve functional outcomes. The objective of this sub-study was to evaluate the effect of early augmented enteral nutrition (EN) during critical illness, on outcomes one year later. We performed prospective longitudinal evaluation of study participants, initially enrolled in The Augmented versus Routine approach to Giving Energy Trial (TARGET), a feasibility study that randomised critically ill patients to 1.5 kcal/ml (augmented) or 1.0 kcal/ml (routine) EN administered at the same rate for up to ten days, who were alive at one year. One year after randomisation Short Form-36 version 2 (SF-36v2) and EuroQol-5D-5L quality of life surveys, and employment status were assessed via telephone survey. At one year there were 71 survivors (1.5 kcal/ml 38 versus 1.0 kcal/ml 33; P=0.55). Thirty-nine (55%) patients consented to this follow-up study and completed the surveys (n = 23 and 16, respectively). The SF-36v2 physical and mental component summary scores were below normal population means but were similar in 1.5 kcal/ml and 1.0 kcal/ml groups (P=0.90 and P=0.71). EuroQol-5D-5L data were also comparable between groups (P=0.70). However, at one-year follow-up, more patients who received 1.5 kcal/ml were employed (7 versus 2; P=0.022). The delivery of 1.5 kcal/ml for a maximum of ten days did not affect self-rated quality of life one year later.

Crystal Structures Reveal that the Reaction Mechanism of Imidazoleglycerol-Phosphate Dehydratase Is Controlled by Switching Mn(II) Coordination.

Imidazoleglycerol-phosphate dehydratase (IGPD) catalyzes the Mn(II)-dependent dehydration of imidazoleglycerol phosphate (IGP) to 3-(1H-imidazol-4-yl)-2-oxopropyl dihydrogen phosphate during biosynthesis of histidine. As part of a program of herbicide design, we have determined a series of high-resolution crystal structures of an inactive mutant of IGPD2 from Arabidopsis thaliana in complex with IGP. The structures represent snapshots of the enzyme trapped at different stages of the catalytic cycle and show how substrate binding triggers a switch in the coordination state of an active site Mn(II) between six- and five-coordinate species. This switch is critical to prime the active site for catalysis, by facilitating the formation of a high-energy imidazolate intermediate. This work not only provides evidence for the molecular processes that dominate catalysis in IGPD, but also describes how the manipulation of metal coordination can be linked to discrete steps in catalysis, demonstrating one way that metalloenzymes exploit the unique properties of metal ions to diversify their chemistry.

Crystallization and preliminary crystallographic analysis of the putative sugar-binding protein Msmeg_0515 (AgaE) from Mycobacterium smegmatis.

Msmeg_0515, a gene from Mycobacterium smegmatis strain 155 encoding the ligand-binding domain, AgaE, of a putative ABC sugar transporter system, has been cloned into a pET-28a vector system, overexpressed in Escherichia coli and purified. The truncated protein lacking the first 27 residues, which correspond to a N-terminal signal sequence, was crystallized using the sitting-drop vapour-diffusion technique. The crystals of this protein diffracted to 1.48 Å resolution and belonged to space group P212121, with unit-cell parameters a = 64.06, b = 69.26, c = 100.74 Å, α = ß = γ = 90° and with one molecule in the asymmetric unit.

Pancreatic islet basement membrane loss and remodeling after mouse islet isolation and transplantation: impact for allograft rejection.

The isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3-10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet ß-cells. The islet BM, consisting of collagen type IV and components of Engelbreth-Holm-Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1(+) vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores ß-cell-matrix attachment, a recognized requirement for ß-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.

Cystic fibrosis.

There are now more adult than paediatric cystic fibrosis (CF) patients and their life expectancy continues to improve. This means that CF patients will be more commonly encountered in a variety of hospital settings including fertility services, gastrointestinal (GI) clinics, diabetes clinics, surgical wards, and acute admissions. Cystic fibrosis units welcome early contact when patients are admitted to other units and it is important to have a structured approach to their assessment and management.

BoTULS: a multicentre randomised controlled trial to evaluate the clinical effectiveness and cost-effectiveness of treating upper limb spasticity due to stroke with botulinum toxin type A.

OBJECTIVE: To compare the clinical effectiveness and cost-effectiveness of treating upper limb spasticity due to stroke with botulinum toxin type A plus an upper limb therapy programme with the upper limb therapy programme alone. DESIGN: A multicentre open-label parallel-group randomised controlled trial and economic evaluation. SETTING: Twelve stroke services in the north of England, UK. PARTICIPANTS: Three hundred and thirty-three adults with upper limb spasticity at the shoulder, elbow, wrist or hand and reduced upper limb function due to stroke more than 1 month previously. INTERVENTIONS: The intervention group received botulinum toxin type A injection(s) plus a 4-week programme of upper limb therapy. The control group received the upper limb therapy programme alone. Participants were clinically reassessed at 3, 6 and 9 months to determine the need for repeat botulinum toxin type A injection(s) and/or therapy. MAIN OUTCOME MEASURES: The primary outcome was upper limb function 1 month after study entry measured by the Action Research Arm Test (ARAT). A successful outcome was defined as: (1) a change of three or more points on the ARAT scale for a participant whose baseline ARAT score was between 0 and 3, (2) a change of six or more points on the ARAT scale for a participant whose baseline ARAT score was between 4 and 51, or (3) a final ARAT score of 57 for a participant whose baseline ARAT score was 52-56. Outcome assessments were undertaken at 1, 3 and 12 months by an assessor who was blinded to the study group allocation. Upper limb impairment and activity limitation were assessed by: Modified Ashworth Scale; Motricity Index; grip strength; ARAT; Nine-Hole Peg Test; upper limb basic functional activity questions and the Barthel Activities of Daily Living (ADL) Index. Stroke-related quality of life/participation restriction was measured using the Stroke Impact Scale, European Quality of Life-5 Dimensions (EQ-5D) and the Oxford Handicap Scale. Upper limb pain was assessed using numerical rating scales. Participant-selected upper limb goal achievement (1 month only) was measured using the Canadian Occupational Performance Measure. Adverse events were compared. Health-care and social services resource use was compared during the first 3 months postrandomisation. EQ-5D data were used to calculate the quality-adjusted life-years (QALYs) associated with intervention and control treatments, and the incremental cost per QALY gained of botulinum toxin type A plus therapy compared with therapy alone was estimated. The sensitivity of the base-case results to alternative assumptions was investigated, and cost-effectiveness acceptability curves, which summarise the evidence of botulinum toxin type A plus therapy being cost-effective for a range of societal willingness to pay for a QALY values, are presented. RESULTS: Randomisation groups were well matched at baseline. There was no significant difference between the groups for the primary outcome of improved arm function at 1 month. This was achieved by 30/154 (19.5%) in the control group and 42/167 (25.1%) in the intervention group (p = 0.232). The relative risk of having a 'successful treatment' in the intervention group compared with the control group was 1.3 [95% confidence interval (CI) 0.9 to 2.0]. No significant differences in improved arm function were seen at 3 or 12 months. In terms of secondary outcomes, muscle tone/spasticity at the elbow was decreased in the intervention group compared with the control group at 1 month. The median change in the Modified Ashworth Scale was - 1 in the intervention group compared with zero in the control group (p < 0.001). No difference in spasticity was seen at 3 or 12 months. Participants treated with botulinum toxin type A showed improvement in upper limb muscle strength at 3 months. The mean change in strength from baseline (upper limb component of the Motricity Index) was 3.5 (95% CI 0.1 to 6.8) points greater in the intervention group compared with the control group. No differences were seen at 1 or 12 months. Participants in the intervention group were more likely to be able to undertake specific basic functional activities, e.g. dress a sleeve, clean the palm and open the hand for cutting fingernails. At 1 month, 109/144 (75.7%) of the intervention group and 79/125 (63.2%) of the control group had improved by at least one point on a five-point Likert scale for at least one of these tasks (p = 0.033). At 3 months the corresponding proportions were 102/142 (71.8%) of the intervention group and 71/122 (58.2%) of the control group (p = 0.027). Improvement was sustained at 12 months for opening the hand for cleaning the palm and opening the hand for cutting the nails but not for other activities. Pain rating improved by two points on a 10-point severity rating scale in the intervention group compared with zero points in the control group (p = 0.004) at 12 months, but no significant differences were seen at 1 or 3 months. There were a number of occasions when there were statistically significant differences in favour of the intervention group; however, these differences were small and of uncertain clinical relevance. These differences were: 3 months - upper limb function (change in ARAT score from baseline), pain (EQ-5D) and participation restriction (Oxford Handicap Scale); 12 months - anxiety/depression (EQ-5D) and participation restriction (Oxford Handicap Scale). No differences in grip strength, dexterity or the Barthel ADL Index were found at any time point. There were no differences between the groups for achievement of patient-selected goals. There was a higher incidence of general malaise/flu-like/cold symptoms in participants treated with botulinum toxin type A with a relative risk of 7.6 (95% CI 1.8 to 32.3). Only one serious adverse event (dysphagia) was potentially related to botulinum toxin type A. Time since stroke and severity of initial upper limb function were preplanned subgroup analyses. There was no significant difference in either subgroup for achievement of ARAT 'success' following treatment with botulinum toxin type A. The base-case incremental cost-effectiveness ratio was 93,500 pounds per QALY gained and estimation of the cost-effectiveness acceptability curve for botulinum toxin type A plus the upper limb therapy programme indicated that there was only a 0.36 probability of it being cost-effective at a threshold ceiling ratio of 20,000 pounds per QALY. CONCLUSIONS: The addition of botulinum toxin type A to an upper limb therapy programme to treat spasticity due to stroke did not enhance improvement in upper limb function when assessed by the prespecified primary outcome measure at 1 month. However, improvements were seen in muscle tone at 1 month, upper limb strength at 3 months, upper limb functional activities related to undertaking specific basic functional tasks at 1, 3 and 12 months, and upper limb pain at 12 months. Botulinum toxin was well tolerated and side effects were minor. The addition of botulinum toxin type A to an upper limb therapy programme for the treatment of upper limb spasticity due to stroke was not estimated to be cost-effective at levels of willingness to pay for a QALY set by NHS decision-makers. TRIAL REGISTRATION: ISRCTN78533119; EudraCT 2004-002427-40; CTA 17136/0230/001.

The roles of the ovarian extracellular matrix in fertility.

In the mammalian ovary there is considerable and continuous remodelling of tissue during both fetal and adult life, necessitating changes in extracellular matrix. Matrix is a diverse group of molecules varying in its composition and roles, which include regulation of growth factor activity and cell behaviour. Here we discuss four topical aspects of matrices in ovaries. (1) Our current state of knowledge of latent TGFFbeta binding proteins that can bind the extracellular matrix fibrillins. Fibrillins and latent TGFbeta binding proteins may be very important given the genetic linkage data implicating a role for fibrillin 3 in polycystic ovarian syndrome. They will almost certainly be important in the stromal compartments of the ovary by regulating TGFbeta bioactivity. (2) Follicles which have an unusual ultrastructural follicular basal lamina and poor quality oocytes. The results suggest that the use of oocytes from these follicles should be avoided in assisted reproductive technologies. (3) Evidence that expression of components of focimatrix correlates with expression of aromatase and cholesterol side-chain cleavage in granulosa cells. Focimatrix is a novel type of basal lamina associated with granulosa cells with expression beginning before deviation and continuing until ovulation. It may be involved in maturation of granulosa cells and selection of the dominant follicle. (4) Evidence is presented in support of a hypothesis that follicular fluid accumulates in follicles due to the osmotic potential of hyaluronan and versican, which are matrices produced by granulosa cells and too large to traverse the follicular antrum. These examples illustrate the diversity of matrix and foreshadow potential important discoveries involving extracellular matrix in ovaries.

Morphological classification of bovine ovarian follicles.

Follicle classification is an important aid to the understanding of follicular development and atresia. Some bovine primordial follicles have the classical primordial shape, but ellipsoidal shaped follicles with some cuboidal granulosa cells at the poles are far more common. Preantral follicles have one of two basal lamina phenotypes, either a single aligned layer or one with additional layers. In antral follicles <5 mm diameter, half of the healthy follicles have columnar shaped basal granulosa cells and additional layers of basal lamina, which appear as loops in cross section ('loopy'). The remainder have aligned single-layered follicular basal laminas with rounded basal cells, and contain better quality oocytes than the loopy/columnar follicles. In sizes >5 mm, only aligned/rounded phenotypes are present. Dominant and subordinate follicles can be identified by ultrasound and/or histological examination of pairs of ovaries. Atretic follicles <5 mm are either basal atretic or antral atretic, named on the basis of the location in the membrana granulosa where cells die first. Basal atretic follicles have considerable biological differences to antral atretic follicles. In follicles >5 mm, only antral atresia is observed. The concentrations of follicular fluid steroid hormones can be used to classify atresia and distinguish some of the different types of atresia; however, this method is unlikely to identify follicles early in atresia, and hence misclassify them as healthy. Other biochemical and histological methods can be used, but since cell death is a part of normal homoeostatis, deciding when a follicle has entered atresia remains somewhat subjective.

What defines a pulmonary exacerbation? The perceptions of adults with cystic fibrosis.

BACKGROUND: There is no standardised definition of a pulmonary exacerbation in cystic fibrosis (CF). In attempting to achieve standardised criteria it is important to identify patient-reported indicators. METHODS: Interviews were undertaken with 47 adults with CF. Participants were asked to report symptoms experienced during a pulmonary exacerbation in two ways: the first symptoms they become aware of, and how they subsequently recognised when they were improving. RESULTS: A range of systemic and respiratory symptoms were reported. Their relative importance varied by severity of disease. The severity and subsequent improvement of an exacerbation was often described as limitations on their activities. CONCLUSION: These preliminary data suggest that patient-reported indicators of a pulmonary exacerbation may not be the same for all adults with CF. Whether different indicators are associated with specific demographic or clinical variables remains to be evaluated.

Dietary manipulation of Bos indicus x heifers during gestation affects the reproductive development of their heifer calves.

The effect of nutrition during the first and second trimesters of pregnancy in composite beef heifers on reproductive parameters of their female calves was determined in the present study. At artificial insemination, heifers were assigned to one of four treatment groups (i.e. HH, HL, LowH and LL) depending on the level of crude protein intake (H = high; L = low) for first and second trimesters of pregnancy. Gonadotrophin concentrations and ovarian parameters were measured in their female calves at 5 and 23 months of age. Crude protein intake was positively associated with dam plasma urea (P < 0.001). The density of healthy follicles in heifers at the time of death was negatively correlated with dam plasma urea at Day 179 (P = 0.009). Heifers from LowH dams had a smaller-sized prepubertal largest ovarian follicle (P = 0.03) and lower densities of primordial and primary follicles (P = 0.02) and healthy antral follicles (P = 0.009) when they were killed. There was a positive correlation between plasma FSH concentrations at 5 and 23 months of age (P = 0.02), as well as between the sizes of the largest ovarian follicles at 6 and 23 months of age (P = 0.01). In conclusion, the reproductive development of heifers may be affected by prenatal nutrition during early and mid-gestation.

Crystallization and preliminary X-ray analysis of D-2-hydroxyacid dehydrogenase from Haloferax mediterranei.

D-2-hydroxyacid dehydrogenase (D2-HDH) from Haloferax mediterranei has been overexpressed in Escherichia coli, solubilized in 8 M urea and refolded by rapid dilution. The protein was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate or PEG 3350 as precipitant. Two crystal forms representing the free enzyme and the nonproductive ternary complex with alpha-ketohexanoic acid and NAD(+) grew under these conditions. Crystals of form I diffracted to beyond 3.0 A resolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 66.0, b = 119.6, c = 86.2 A, beta = 96.3 degrees . Crystals of form II diffracted to beyond 2.0 A resolution and belonged to the triclinic space group P1, with unit-cell parameters a = 66.5, b = 75.2, c = 77.6 A, alpha = 109.1, beta = 107.5, gamma = 95.9 degrees. The calculated values for V(M) and analysis of the self-rotation and self-Patterson functions suggest that the asymmetric unit in both crystal forms contains two dimers related by pseudo-translational symmetry.

Studies of granulosa cell maturation in dominant and subordinate bovine follicles: novel extracellular matrix focimatrix is co-ordinately regulated with cholesterol side-chain cleavage CYP11A1.

During growth of antral ovarian follicles granulosa cells first become associated with a novel type of extracellular matrix, focimatrix, and at larger sizes follicles become either subordinate or dominant. To examine this, bovine subordinate (9.0+/-S.E.M. 0.4 mm; n=16), partially dominant (12.0+/-0.6 mm; n=18) and fully dominant (15.0+/-0.4 mm; n=14) follicles were examined by real time RT-PCR analyses of granulosa cells and by immunohistochemistry of focimatrix. Changes in the expression of FSH receptor, LH receptor, cholesterol side-chain cleavage (CYP11A1), 3beta-hydroxysteroid dehydrogenase, aromatase (CYP19A1) and inhibin-alpha and beta-B were observed as expected for follicle sizes examined. After adjusting for size differences, only CYP11A1 was significantly different between the groups, and elevated in dominant follicles. Also after adjusting for differences in size there were no significant differences in expression of focimatrix components collagen type IV alpha-1 (COL4A1), laminin beta-2, nidogen 1 (NID1), and perlecan (HSPG2) or the volume density of NID1 and -2 and HSPG2. The volume density of focimatrix components in laminin 111 was significantly elevated in dominant follicles. Adjusting for analysis of more than one follicle per animal and for multiple correlations, CYP11A1 mRNA levels were highly correlated with the focimatrix genes COL4A1, NID1 and -2 and HSPG2. Thus, focimatrix may potentially regulate CYP11A1 expression, and the regulation of both could be important in follicular dominance.

Molecular composition of the peri-islet basement membrane in NOD mice: a barrier against destructive insulitis.

AIMS/HYPOTHESIS: This study examined whether the capsule which encases islets of Langerhans in the NOD mouse pancreas represents a specialised extracellular matrix (ECM) or basement membrane that protects islets from autoimmune attack. METHODS: Immunofluorescence microscopy using a panel of antibodies to collagens type IV, laminins, nidogens and perlecan was performed to localise matrix components in NOD mouse pancreas before diabetes onset, at onset of diabetes and after clinical diabetes was established (2-8.5 weeks post-onset). RESULTS: Perlecan, a heparan sulphate proteoglycan that is characteristic of basement membranes and has not previously been investigated in islets, was localised in the peri-islet capsule and surrounding intra-islet capillaries. Other components present in the peri-islet capsule included laminin chains alpha2, beta1 and gamma1, collagen type IV alpha1 and alpha2, and nidogen 1 and 2. Collagen type IV alpha3-alpha6 were not detected. These findings confirm that the peri-islet capsule represents a specialised ECM or conventional basement membrane. The islet basement membrane was destroyed in islets where intra-islet infiltration of leucocytes marked the progression from non-destructive to destructive insulitis. No changes in basement membrane composition were observed before leucocyte infiltration. CONCLUSIONS/INTERPRETATION: These findings suggest that the islet basement membrane functions as a physical barrier to leucocyte migration into islets and that degradation of the islet basement membrane marks the onset of destructive autoimmune insulitis and diabetes development in NOD mice. The components of the islet basement membrane that we identified predict that specialised degradative enzymes are likely to function in autoimmune islet damage.