Tau filaments with the Alzheimer fold in cases with MAPT mutations V337M and R406W.

Frontotemporal dementia (FTD) and Alzheimer's disease are the most common forms of early-onset dementia. Dominantly inherited mutations in MAPT, the microtubule-associated protein tau gene, cause FTD and parkinsonism linked to chromosome 17 (FTDP-17). Individuals with FTDP-17 develop abundant filamentous tau inclusions in brain cells. Here we used electron cryo-microscopy to determine the structures of tau filaments from the brains of individuals with MAPT mutations V337M and R406W. Both mutations gave rise to tau filaments with the Alzheimer fold, which consisted of paired helical filaments in all V337M and R406W cases and of straight filaments in two V337M cases. We also identified a new assembly of the Alzheimer fold into triple tau filaments in a V337M case. Filaments assembled from recombinant tau(297-391) with mutation V337M had the Alzheimer fold and showed an increased rate of assembly.

Inhibition of hyaluronan synthesis prevents ß-cell loss in obesity-associated type 2 diabetes.

Pancreatic ß-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low concentrations of HA were present in healthy pancreatic islets. However, HA substantially accumulated in cadaveric islets of T2D patients and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the main HA receptor CD44, preserved glycemic control and insulin concentrations in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserved glycemic control in other settings of ß-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we found that 4-MU increased the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on ß-cells such that caspase 3 activation did not result in ß-cell apoptosis. These data indicated a role for HA accumulation in diabetes pathogenesis and suggested that it may be a viable target to ameliorate ß-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

Correction: Heterogeneous expression of CFTR in insulin-secreting ß-cells of the normal human islet.

[This corrects the article DOI: 10.1371/journal.pone.0242749.].

Association of Traumatic Brain Injury with Late Life Neuropathological Outcomes in a Community-Based Cohort.

BACKGROUND: Prior studies into the association of head trauma with neuropathology have been limited by incomplete lifetime neurotrauma exposure characterization. OBJECTIVE: To investigate the neuropathological sequelae of traumatic brain injury (TBI) in an autopsy sample using three sources of TBI ascertainment, weighting findings to reflect associations in the larger, community-based cohort. METHODS: Self-reported head trauma with loss of consciousness (LOC) exposure was collected in biennial clinic visits from 780 older adults from the Adult Changes in Thought study who later died and donated their brain for research. Self-report data were supplemented with medical record abstraction, and, for 244 people, structured interviews on lifetime head trauma. Neuropathology outcomes included Braak stage, CERAD neuritic plaque density, Lewy body distribution, vascular pathology, hippocampal sclerosis, and cerebral/cortical atrophy. Exposures were TBI with or without LOC. Modified Poisson regressions adjusting for age, sex, education, and APOE ɛ4 genotype were weighted back to the full cohort of 5,546 participants. RESULTS: TBI with LOC was associated with the presence of cerebral cortical atrophy (Relative Risk 1.22, 95% CI 1.02, 1.42). None of the other outcomes was associated with TBI with or without LOC. CONCLUSION: TBI with LOC was associated with increased risk of cerebral cortical atrophy. Despite our enhanced TBI ascertainment, we found no association with the Alzheimer's disease-related neuropathologic outcomes among people who survived to at least age 65 without dementia. This suggests the pathophysiological processes underlying post-traumatic neurodegeneration are distinct from the hallmark pathologies of Alzheimer's disease.

Inhibition of hyaluronan synthesis prevents ß-cell loss in obesity-associated type 2 diabetes.

Pancreatic ß-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We have identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low levels of HA are present in healthy pancreatic islets. However, HA substantially accumulates in cadaveric islets of human T2D and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the major HA receptor CD44, preserve glycemic control and insulin levels in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserve glycemic control in other settings of ß-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we find that 4-MU increases the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on ß-cells such that caspase 3 activation does not result in ß-cell apoptosis. These data indicate a role for HA accumulation in diabetes pathogenesis and suggest that it may be a viable target to ameliorate ß-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

Detection of Glycosaminoglycans in Pancreatic Islets and Lymphoid Tissues.

In this chapter, we describe the detection of the glycosaminoglycans hyaluronan and heparan sulfate in pancreatic islets and lymphoid tissues. The identification of hyaluronan in tissues is achieved by utilizing a highly specific hyaluronan binding protein (HABP) probe that interacts with hyaluronan in tissue sections. The HABP probe is prepared by enzymatic digestion of the chondroitin sulfate proteoglycan aggrecan which is present in bovine nasal cartilage and is then biotinylated in the presence of bound hyaluronan and the link protein. Hyaluronan is then removed by gel filtration chromatography. The biotinylated HABP-link protein complex is applied to tissue sections, and binding of the complex to tissue hyaluronan is visualized by enzymatic precipitation of chromogenic substrates.To determine hyaluronan content in tissues, tissues are first proteolytically digested to release hyaluronan from the macromolecular complexes that this molecule forms with other extracellular matrix constituents. Digested tissue is then incubated with HABP . The hyaluronan-HABP complexes are extracted, and the hyaluronan concentration in the tissue is determined using an ELISA-like assay.Historically, heparan sulfate was identified in tissue sections using the cationic dye Alcian blue and histochemistry based on the critical electrolyte concentration principle of differential staining of glycosaminoglycans using salt solutions. For both human and mouse pancreas sections, the current optimal method for detecting heparan sulfate is by indirect immunohistochemistry using a specific anti-heparan sulfate monoclonal antibody. A peroxidase-conjugated secondary antibody is then applied, and its binding to the anti-heparan sulfate antibody is visualized by oxidation and precipitation of a chromogenic substrate.

The Vbeta13 T Cell Receptor Monoclonal Antibody Reduces Hyaluronan and CD68+, CD3+, and CD8+ Cell Infiltrations to Delay Diabetes in Congenic BB DRLyp/Lyp Rats.

The depleting Vß13a T cell receptor monoclonal antibody (mAb) 17D5 prevents both induced and spontaneous autoimmune diabetes in BB rats. Here it was tested in congenic DRLyp/Lyp rats, all of which spontaneously developed diabetes. Starting at 40 days of age, rats were injected once weekly with either saline, His42 Vß16 mAb, or 17D5 mAb and monitored for hyperglycemia. Diabetes occurred in 100% (n = 5/5) of saline-treated rats (median age, 66 days; range 55-73), and in 100% (n = 6/6) of His42-treated rats (median age, 69 days; range 59-69). Diabetes occurred in fewer (n = 8/11, 73%) 17D5-treated rats at a later age (median 76 days, range 60-92). Three (27%) of the 17D5-treated rats were killed at 101-103 days of age without diabetes (17D5 no-diabetes rats). Survival analysis demonstrated that 17D5 mAb delayed diabetes onset. Saline- and His42-treated rats had severely distorted islets with substantial loss of insulin-positive cells. These rats exhibited prominent hyaluronan (HA) staining, with the intra-islet HA+ accumulations measuring 5,000 ± 2,400 µm2 and occupying 36 ± 12% of islet area, and severe (grade 4) insulitis with abundant infiltration by CD68+, CD3+, and CD8+ cells. The 17D5 mAb-treated rats with delayed diabetes onset exhibited less severe insulitis (predominantly grade 3). In contrast, the 17D5 no-diabetes rats had mostly normal islets, with insulin+ cells representing 76 ± 3% of islet cells. In these rats, the islet HA deposits were significantly smaller than in the diabetic rats; the intra-islet HA+ areas were 1,200 ± 300 µm2 and accounted for 8 ± 1% of islet area. Also, islet-associated CD68+ and CD3+ cells occurred less frequently (on average in 60 and 3% of the islets, respectively) than in the diabetes rats (present in >95% of the islets). No CD8+ cells were detected in islets in all 17D5 no-diabetes rats. We conclude that mAb 17D5 delayed diabetes in DRLyp/Lyp rats and markedly reduced expression of HA and concomitant infiltration of CD68+, CD3+, and CD8+ cells. Our findings underscore the importance of refining immune suppression in prevention or intervention clinical trials to use mAb reagents that are directed against specific T cell receptors.

Identification and characterisation of tertiary lymphoid organs in human type 1 diabetes.

AIMS/HYPOTHESIS: We and others previously reported the presence of tertiary lymphoid organs (TLOs) in the pancreas of NOD mice, where they play a role in the development of type 1 diabetes. Our aims here are to investigate whether TLOs are present in the pancreas of individuals with type 1 diabetes and to characterise their distinctive features, in comparison with TLOs present in NOD mouse pancreases, in order to interpret their functional significance. METHODS: Using immunofluorescence confocal microscopy, we examined the extracellular matrix (ECM) and cellular constituents of pancreatic TLOs from individuals with ongoing islet autoimmunity in three distinct clinical settings of type 1 diabetes: at risk of diabetes; at/after diagnosis; and in the transplanted pancreas with recurrent diabetes. Comparisons were made with TLOs from 14-week-old NOD mice, which contain islets exhibiting mild to heavy leucocyte infiltration. We determined the frequency of the TLOs in human type 1diabetes with insulitis and investigated the presence of TLOs in relation to age of onset, disease duration and disease severity. RESULTS: TLOs were identified in preclinical and clinical settings of human type 1 diabetes. The main characteristics of these TLOs, including the cellular and ECM composition of reticular fibres (RFs), the presence of high endothelial venules and immune cell subtypes detected, were similar to those observed for TLOs from NOD mouse pancreases. Among 21 donors with clinical type 1 diabetes who exhibited insulitis, 12 had TLOs and had developed disease at younger age compared with those lacking TLOs. Compartmentalised TLOs with distinct T cell and B cell zones were detected in donors with short disease duration. Overall, TLOs were mainly associated with insulin-containing islets and their frequency decreased with increasing severity of beta cell loss. Parallel studies in NOD mice further revealed some differences in so far as regulatory T cells were essentially absent from human pancreatic TLOs and CCL21 was not associated with RFs. CONCLUSIONS/INTERPRETATION: We demonstrate a novel feature of pancreas pathology in type 1 diabetes. TLOs represent a potential site of autoreactive effector T cell generation in islet autoimmunity and our data from mouse and human tissues suggest that they disappear once the destructive process has run its course. Thus, TLOs may be important for type 1 diabetes progression.

The extracellular matrix molecules versican and hyaluronan in urethral and vaginal tissues in stress urinary incontinence.

PURPOSE: Abnormal extracellular matrix (ECM) changes are correlated with stress urinary incontinence (SUI). The ECM components versican (Vcan) and hyaluronan (HA) play key roles in regulating tissue inflammation and maintaining connective tissue homeostasis. We analyzed the localization and expression of these ECM components in urethral and vaginal tissues from a rat model of urinary incontinence and from human clinical specimens. METHODS: Nulliparous rats underwent vaginal distension (VD), a rodent model of SUI, or a sham procedure. Tissues were harvested from six rats per group at days 1, 4, and 21 for immunohistochemistry and RNA expression analysis of ECM components. Periurethral vaginal samples from female patients with SUI were also examined. RESULTS: High-intensity staining for Vcan was observed 1 day after procedure in both control and VD animals. This level of abundance persisted at day 4 in VD compared to control, with concurrent reduced messenger RNA (mRNA) expression of the Vcan-degrading enzymes ADAMTS5 and ADAMTS9 and reduced staining for the Vcan cleavage epitope DPEAAE. Abundance of HA was not different between VD and control, however mRNA expression of the HA synthase Has2 was significantly reduced in VD tissues at day 4. Abundant Vcan staining was observed in 60% of SUI patient samples, which was strongest in regions of disrupted elastin. CONCLUSION: Reduction of Vcan-degrading enzymes and HA synthases at day 4 postsurgery indicates a potential delay in ECM turnover associated with SUI. Abundant Vcan is associated with inflammation and elastin fiber network disruption, warranting further investigation to determine its role in SUI pathogenesis.

Heterogeneous expression of CFTR in insulin-secreting ß-cells of the normal human islet.

Cystic fibrosis (CF) is due to mutations in the CF-transmembrane conductance regulator (CFTR) and CF-related diabetes (CFRD) is its most common co-morbidity, affecting ~50% of all CF patients, significantly influencing pulmonary function and longevity. Yet, the complex pathogenesis of CFRD remains unclear. Two non-mutually exclusive underlying mechanisms have been proposed in CFRD: i) damage of the endocrine cells secondary to the severe exocrine pancreatic pathology and ii) intrinsic ß-cell impairment of the secretory response in combination with other factors. The later has proven difficult to determine due to low expression of CFTR in ß-cells, which results in the general perception that this Cl-channel does not participate in the modulation of insulin secretion or the development of CFRD. The objective of the present work is to demonstrate CFTR expression at the molecular and functional levels in insulin-secreting ß-cells in normal human islets, where it seems to play a role. Towards this end, we have used immunofluorescence confocal and immunofluorescence microscopy, immunohistochemistry, RT-qPCR, Western blotting, pharmacology, electrophysiology and insulin secretory studies in normal human, rat and mouse islets. Our results demonstrate heterogeneous CFTR expression in human, mouse and rat ß-cells and provide evidence that pharmacological inhibition of CFTR influences basal and stimulated insulin secretion in normal mouse islets but not in islets lacking this channel, despite being detected by electrophysiological means in ~30% of ß-cells. Therefore, our results demonstrate a potential role for CFTR in the pancreatic ß-cell secretory response suggesting that intrinsic ß-cell dysfunction may also participate in the pathogenesis of CFRD.

An IL-2 mutein engineered to promote expansion of regulatory T cells arrests ongoing autoimmunity in mice.

Interleukin-2 (IL-2) controls the homeostasis and function of regulatory T (Treg) cells, and defects in the IL-2 pathway contribute to multiple autoimmune diseases. Although recombinant IL-2 therapy has been efficacious in certain inflammatory conditions, the capacity for IL-2 to also activate inflammatory effector responses highlights the need for IL-2-based therapeutics with improved Treg cell specificity. From a panel of rationally designed murine IL-2 variants, we identified IL-2 muteins with reduced potency and enhanced Treg cell selectivity due to increased dependence on the IL-2 receptor component CD25. As an Fc-fused homodimer, the optimal Fc.IL-2 mutein induced selective Treg cell enrichment and reduced agonism of effector cells across a wide dose range. Furthermore, despite being a weaker agonist, overall Treg cell growth was greater and more sustained due to reduced receptor-mediated clearance of the Fc.IL-2 mutein compared with Fc-fused wild-type IL-2. Preferential Treg cell enrichment was also observed in the presence of activated pathogenic T cells in the pancreas of nonobese diabetic (NOD) mice, despite a loss of Treg cell selectivity in an IL-2R proximal response. These properties facilitated potent and extended resolution of NOD diabetes with infrequent dosing schedules.

Hyaluronan deposition in islets may precede and direct the location of islet immune-cell infiltrates.

AIMS/HYPOTHESIS: Substantial deposition of the extracellular matrix component hyaluronan (HA) is characteristic of insulitis in overt type 1 diabetes. We investigated whether HA accumulation is detectable in islets early in disease pathogenesis and how this affects the development of insulitis and beta cell mass. METHODS: Pancreas tissue from 15 non-diabetic organ donors who were positive for islet autoantibodies (aAbs) and from 14 similarly aged aAb- control donors were examined for the amount of islet HA staining and the presence of insulitis. The kinetics of HA deposition in islets, along with the onset and progression of insulitis and changes in beta cell mass, were investigated in BioBreeding DRLyp/Lyp rats (a model of spontaneous autoimmune diabetes) from 40 days of age until diabetes onset. RESULTS: Abundant islet HA deposits were observed in pancreas tissues from n = 3 single- and n = 4 double-aAb+ donors (aAb+HAhigh). In these seven tissues, the HA-stained areas in islets measured 1000 ± 240 µm2 (mean ± SEM) and were fourfold larger than those from aAb- control tissues. The aAb+HAhigh tissues also had a greater prevalence of islets that were highly rich in HA (21% of the islets in these tissues contained the largest HA-stained areas [>2000 µm2] vs less than 1% in tissues from aAb- control donors). The amount of HA staining in islets was associated with the number of aAbs (i.e. single- or double-aAb positivity) but not with HLA genotype or changes in beta cell mass. Among the seven aAb+HAhigh tissues, three from single- and one from double-aAb+ donors did not show any islet immune-cell infiltrates, indicating that HA accumulates in aAb+ donors independently of insulitis. The three aAb+HAhigh tissues that exhibited insulitis had the largest HA-stained areas and, in these tissues, islet-infiltrating immune cells co-localised with the most prominent HA deposits (i.e. with HA-stained areas >2000 µm2). Accumulation of HA in islets was evident prior to insulitis in 7-8-week-old presymptomatic DRLyp/Lyp rats, in which the islet HA-stained area measured 2370 ± 170 µm2 (mean ± SEM), which was threefold larger than in 6-week-old rats. This initial islet HA deposition was not concurrent with beta cell loss. Insulitis was first detected in 9-10-week-old rats, in which the HA-stained areas were 4980 ± 500 µm2. At this age, the rats also exhibited a 44% reduction in beta cell mass. Further enlargement of the HA-positive areas (mean ± SEM: 7220 ± 880 µm2) was associated with invasive insulitis. HA deposits remained abundant in the islets of rats with destructive insulitis, which had lost 85% of their beta cells. CONCLUSIONS/INTERPRETATION: This study indicates that HA deposition in islets occurs early in type 1 diabetes and prior to insulitis, and points to a potential role of HA in triggering islet immune-cell infiltration and the promotion of insulitis.

Structural abnormalities in islets from very young children with cystic fibrosis may contribute to cystic fibrosis-related diabetes.

Cystic fibrosis (CF)-related diabetes (CFRD) is thought to result from beta-cell injury due in part to pancreas exocrine damage and lipofibrosis. CFRD pancreata exhibit reduced islet density and altered cellular composition. To investigate a possible etiology, we tested the hypothesis that such changes are present in CF pancreata before the development of lipofibrosis. We evaluated pancreas and islet morphology in tissues from very young CF children (<4 years of age), and adult patients with CF and CFRD. The relative number of beta-cells in young CF tissues was reduced by 50% or more when compared to age-matched controls. Furthermore, young CF tissues displayed significantly smaller insulin-positive areas, lower proportion of beta-cells positive for the proliferation marker Ki67 or the ductal marker CK19 vs. control subjects, and islet inflammatory cell infiltrates, independently of the severity of the exocrine lesion and in the absence of amyloid deposits. CFRD pancreata exhibited greater islet injury with further reduction in islet density, decreased relative beta-cell number, and presence of amyloid deposits. Together, these results strongly suggest that an early deficiency in beta-cell number in infants with CF may contribute to the development of glucose intolerance in the CF pediatric population, and to CFRD, later in life.

Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes.

CD44 is a multi-functional receptor with multiple of isoforms engaged in modulation of cell trafficking and transmission of apoptotic signals. We have previously shown that injection of anti-CD44 antibody into NOD mice induced resistance to type 1 diabetes (T1D). In this communication we describe our efforts to understand the mechanism underlying this effect. We found that CD44-deficient NOD mice develop stronger resistance to T1D than wild-type littermates. This effect is not explained by the involvement of CD44 in cell migration, because CD44-deficient inflammatory cells surprisingly had greater invasive potential than the corresponding wild type cells, probably owing to molecular redundancy. We have previously reported and we show here again that CD44 expression and hyaluronic acid (HA, the principal ligand for CD44) accumulation are detected in pancreatic islets of diabetic NOD mice, but not of non-diabetic DBA/1 mice. Expression of CD44 on insulin-secreting ß cells renders them susceptible to the autoimmune attack, and is associated with a diminution in ß-cells function (e.g., less insulin production and/or insulin secretion) and possibly also with an enhanced apoptosis rate. The diabetes-supportive effect of CD44 expression on ß cells was assessed by the TUNEL assay and further strengthened by functional assays exhibiting increased nitric oxide release, reduced insulin secretion after glucose stimulation and decreased insulin content in ß cells. All these parameters could not be detected in CD44-deficient islets. We further suggest that HA-binding to CD44-expressing ß cells is implicated in ß-cell demise. Altogether, these data agree with the concept that CD44 is a receptor capable of modulating cell fate. This finding is important for other pathologies (e.g., cancer, neurodegenerative diseases) in which CD44 and HA appear to be implicated.

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3+ Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals.

Hyaluronan: A Mediator of Islet Dysfunction and Destruction in Diabetes?

Hyaluronan (HA) is an extracellular matrix (ECM) component that is present in mouse and human islet ECM. HA is localized in peri-islet and intra-islet regions adjacent to microvessels. HA normally exists in a high molecular weight form, which is anti-inflammatory. However, under inflammatory conditions, HA is degraded into fragments that are proinflammatory. HA accumulates in islets of human subjects with recent onset type 1 diabetes (T1D), and is associated with myeloid and lymphocytic islet infiltration, suggesting a possible role for HA in insulitis. A similar accumulation of HA, in amount and location, occurs in non-obese diabetic (NOD) and DORmO mouse models of T1D. Furthermore, HA accumulates in follicular germinal centers and in T-cell areas in lymph nodes and spleen in both human and mouse models of T1D, as compared with control tissues. Whether HA accumulates in islets in type 2 diabetes (T2D) or models thereof has not been previously described. Here we show evidence that HA accumulates in a mouse model of islet amyloid deposition, a well-known component of islet pathology in T2D. In summary, islet HA accumulation is a feature of both T1D and a model of T2D, and may represent a novel inflammatory mediator of islet pathology.

The detection of glycosaminoglycans in pancreatic islets and lymphoid tissues.

In this chapter, we describe the detection of the glycosaminoglycans hyaluronan and heparan sulfate in pancreatic islets and lymphoid tissues. The identification of hyaluronan in tissues is achieved by utilizing a highly specific hyaluronan binding protein (HABP) probe that interacts with hyaluronan in tissue sections. The HABP probe is prepared by enzymatic digestion of the chondroitin sulfate proteoglycan aggrecan which is present in bovine nasal cartilage, and is then biotinylated in the presence of bound hyaluronan and the link protein. Hyaluronan is then removed by gel filtration chromatography. The biotinylated HABP-link protein complex is applied to tissue sections and binding of the complex to tissue hyaluronan is visualized by enzymatic precipitation of chromogenic substrates. To determine hyaluronan content in tissues, tissues are first proteolytically digested to release hyaluronan from the macromolecular complexes that this molecule forms with other extracellular matrix constituents. Digested tissue is then incubated with HABP. The hyaluronan-HABP complexes are extracted and the hyaluronan concentration in the tissue is determined using an ELISA-like assay. Heparan sulfate is identified in mouse tissues by Alcian blue histochemistry and indirect immunohistochemistry. In human tissues, heparan sulfate is best detected by indirect immunohistochemistry using a specific anti-heparan sulfate monoclonal antibody. A biotinylated secondary antibody is then applied in conjunction with streptavidin-peroxidase and its binding to the anti-heparan sulfate antibody is visualized by enzymatic precipitation of chromogenic substrates.

Extracellular matrix components in the pathogenesis of type 1 diabetes.

Type 1 diabetes (T1D) results from progressive immune cell-mediated destruction of pancreatic ß cells. As immune cells migrate into the islets, they pass through the extracellular matrix (ECM). This ECM is composed of different macromolecules localized to different compartments within and surrounding islets; however, the involvement of this ECM in the development of human T1D is not well understood. Here, we summarize our recent findings from human and mouse studies illustrating how specific components of the islet ECM that constitute basement membranes and interstitial matrix of the islets, and surprisingly, the intracellular composition of islet ß cells themselves, are significantly altered during the pathogenesis of T1D. Our focus is on the ECM molecules laminins, collagens, heparan sulfate/heparan sulfate proteoglycans, and hyaluronan, as well as on the enzymes that degrade these ECM components. We propose that islet and lymphoid tissue ECM composition and organization are critical to promoting immune cell activation, islet invasion, and destruction of islet ß cells in T1D.

Hyaluronan and hyaluronan-binding proteins accumulate in both human type 1 diabetic islets and lymphoid tissues and associate with inflammatory cells in insulitis.

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor-stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes.