Autoimmunity against type VII collagen in inflammatory bowel disease

Autoimmunity against type VII collagen, an adhesion molecule of the extracellular matrix in epithelial basement membranes, is causing the rare organ-specific epidermolysis bullosa acquisita (EBA). An intriguing association between EBA and inflammatory bowel disease (IBD) has been extensively documented over the last decades, but, because of the very low incidence of EBA, received little attention from physicians involved in the care of patients with IBD. More recently, autoantibodies against type VII collagen have been detected in up to 68% of IBD patients. Although these findings suggest that chronic intestinal inflammation in IBD predisposes for autoimmunity against type VII collagen, their relevance for the pathogenesis of both IBD and EBA is still unclear. In this review article, the main features of the association between IBD and EBA are presented and pathomechanistic hypotheses as well as future lines of investigation in this area are discussed. Future research should provide new pathomechanistic insights and will likely facilitate the development of more specific and effective immunotherapeutic strategies for both conditions.     

Laminin extraction and disorganization of collagen fibrils in snail muscles by EDTA treatment

Summary— Snail muscles were extracted by a solution of EDTA and electron microscopy showed that the extract contained dispersed, depolymerized collagen fibrils and cross-shaped laminin-like structures. The extracts were purified by ultracentrifugation followed by two different procedures which enriched the content of laminin-like structures. The laminin-related molecules displayed unique properties when analyzed by biochemical, immunological and morphological methods. Electrophoretic patterns of the molecular form purified primarily by ion exchange chromatography, resembled EHS-tumor laminin and displayed a cruciform shape when viewed by electron microscopy. Immunohistology, using antiserum obtained against the agarose gel-purified protein, showed that this laminin was primarily located in the extracellular matrix surrounding muscle fibers. Western blots using anti-EHS laminin antibody showed reaction of a 300 kDa subunit of this snail laminin. The protein obtained by another procedure, initially using gel filtration, followed by ion exchange chromatography, also appeared to be a laminin. It had a collapsed cruciform appearance when viewed by electron microscopy. It contained several different subunits, one of which, ca 300 kDa, reacted with anti-EHS-laminin antibody and with anti-snail laminin antibody. In contrast, EHS laminin did not react with the anti-snail laminin antibody. The composite results suggest that at least two different forms of laminin are extractable from snail muscle and that they share molecular properties and immune determinants with mouse tumor laminin.     

Importance of Balance between Extracellular Matrix Synthesis and Degradation in Basement Membrane Formation

The epidermal basement membrane (BM) plays important roles in adhesion between epidermis and dermis and in controlling epidermal differentiation. In a skin-equivalent (SE), components of the epidermal BM such as laminin 5 and type IV and VII collagens were detected in conditioned media and in basal keratinocytes. Despite production of these BM components, however, BM was rarely observed at the dermal-epidermal junction. One possible explanation for the absence of BM in SEs is that matrix metalloproteinases (MMPs) degrade newly synthesized extracellular matrices. In fact, several MMPs, such as MMPs-1, 2, 3, and 9, were observed to be present in conditioned media and some of them were in active forms. Tissue inhibitor of metalloproteinase (TIMP)-2 was not detected, although TIMP-1 was present. BM degradation activity presumably exceeds BM formation activity in the SE, resulting in the absence of lamina densa at the dermal-epidermal junction. Synthetic MMP inhibitors CGS27023A and MMP inhibitor I, which inhibit MMPs 1, 2, 3, and 9, markedly augmented deposition of laminin 5 and type IV and VII collagens at the dermal-epidermal junction, resulting in formation of continuous epidermal BM. These results suggest that the balance between synthesis and degradation of BM components is important for BM formation.     

Building collagen IV smart scaffolds on the outside of cells

Collagen IV scaffolds assemble through an intricate pathway that begins intracellularly and is completed extracellularly. Multiple intracellular enzymes act in concert to assemble collagen IV protomers, the building blocks of collagen IV scaffolds. After being secreted from cells, protomers are activated to initiate oligomerization, forming insoluble networks that are structurally reinforced with covalent crosslinks. Within these networks, embedded binding sites along the length of the protomer lead to the “decoration” of collagen IV triple helix with numerous functional molecules. We refer to these networks as “smart” scaffolds, which as a component of the basement membrane enable the development and function of multicellular tissues in all animal phyla. In this review, we present key molecular mechanisms that drive the assembly of collagen IV smart scaffolds.     

Noncontiguous T cell epitopes in autoimmune diabetes: From mice to men and back again

Type 1 diabetes (T1D) is a T cell–mediated autoimmune disease that affects the insulin-producing beta cells of the pancreatic islets. The nonobese diabetic mouse is a widely studied spontaneous model of the disease that has contributed greatly to our understanding of T1D pathogenesis. This is especially true in the case of antigen discovery. Upon review of existing knowledge concerning the antigens and peptide epitopes that are recognized by T cells in this model, good concordance is observed between mouse and human antigens. A fascinating recent illustration of the contribution of the nonobese diabetic mouse in the area of epitope identification is the discovery of noncontiguous CD4⁺ T cell epitopes. This novel epitope class is characterized by the linkage of an insulin-derived peptide to, most commonly, a fragment of a natural cleavage product of another beta cell secretory granule constituent. These so-called hybrid insulin peptides are also recognized by T cells in patients with T1D, although the precise mechanism for their generation has yet to be defined and is the subject of active investigation. Although evidence from the tumor immunology arena documented the existence of noncontiguous CD8⁺ T cell epitopes, generated by proteasome-mediated peptide splicing involving transpeptidation, such CD8⁺ T cell epitopes were thought to be a rare immunological curiosity. However, recent advances in bioinformatics and mass spectrometry have challenged this view. These developments, coupled with the discovery of hybrid insulin peptides, have spurred a search for noncontiguous CD8⁺ T cell epitopes in T1D, an exciting frontier area still in its infancy.     

Differential expression of type IV collagen isoforms in rat glomerular endothelial and mesangial cells

Type IV collagen, which is encoded by six genetically distinct alpha-chains (alpha 1-alpha 6), is a major component of the kidney glomerulus. The alpha 1(IV) and alpha 2(IV) chains are present predominantly in the mesangial matrix, whereas the alpha 3(IV), alpha 4(IV), and alpha 5(IV) chains are localized almost exclusively to the glomerular basement membrane (GBM). Thickening of the GBM and expansion of the mesangial matrix are believed to contribute to the pathogenesis of diabetic nephropathy. In the present study, we evaluated the expression of alpha 1(IV), alpha 3(IV), and alpha 5(IV) chains in rat glomerular endothelial (GEndC) and mesangial cells (GMC). Under physiological concentrations of glucose (5 mM), alpha 1(IV) and alpha 5(IV) chains were detectable in GMCs, with an obvious absence of alpha 3(IV) chain. All three isoforms tested were present in GEndCs. At diabetic concentrations of glucose (25 mM), alpha 1(IV) was up-regulated in GMCs, whereas expression level of alpha 1(IV) remained unaltered in GEndCs. The alpha 3(IV) and alpha 5(IV) chains were up-regulated in GEndCs, but remained unchanged in GMCs under diabetic glucose concentrations (25 mM). Collectively, our results demonstrate that GMC might contribute to mesangial matrix expansion, mediated by alpha 1(IV) collagen, while GEndC might contribute to thickening of GBM, mediated by alpha 3(IV) collagen, in patients with diabetic nephropathy.     

Characterization of continuous B‐cell epitopes in the N‐terminus of glutamate decarboxylase67 using monoclonal antibodies

Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type‐1 diabetes (T1D) and stiff‐person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ‐aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme‐linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N‐terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14–23 and 91–99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side‐chain functionality, rather than amino acid side‐chain specificity. Additionally, results suggested that non‐contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide‐based therapeutics against SPS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Epitope mapping of the N‐terminal portion of tissue transglutaminase protein antigen to identify linear epitopes in celiac disease

Celiac disease (CD) is an autoimmune mediated disease with complex and multifactorial etiology. Gluten intake triggers a composite immune response involving T‐cells and B‐cells and leading to the secretion of autoantibodies if a genetic predisposition is present. Untreated CD patients show high levels of circulating autoantibodies directed to different auto‐antigens present in the intestinal mucosa. The most important auto‐antigen is the endomysial enzyme tissue transglutaminase (tTG). Both IgA and IgG antibody isotypes to tTG are known, but only the IgA antibodies demonstrate the highest disease specificity and thus are considered disease biomarkers. Because the pathogenicity and exact tTG binding properties of these autoantibodies are still unclear, the characterization of tTG antigenic domains is a crucial step in understanding CD onset and the autoimmune pathogenesis. Overlapping peptide libraries can be used for epitope mapping of selected protein portions to determine antigenic fragments contributing to the immunological activity and possibly develop innovative peptide‐based tools with high specificity and sensitivity for CD. We performed an epitope mapping study to characterize putative linear auto‐antigenic epitopes present in the tTG N‐terminal portion (1–230). A library of 23 overlapping peptides spanning tTG(1–230) was generated by Fmoc/tBu solid‐phase peptide synthesis and screened by immunoenzymatic assays employing patients' sera. The results indicate that four synthetic peptides, that is, Ac‐tTG(1–15)‐NH₂, Ac‐tTG(41–55)‐NH₂, Ac‐tTG(51–65)‐NH₂, and Ac‐tTG(151–165)‐NH₂, are recognized by IgA autoantibodies circulating in CD patients' sera. These results offer important insight on the nature of the antigen‐antibody interaction. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Rabbit anti-HMG-17 antibodies recognize similar epitopes on the HMG-17 molecule as lupus autoantibodies. Relation with histone H1 defined epitopes.

HMG-17 is a nucleosomal protein which is an immune target of autoantibodies in systemic lupus erythematosus (SLE) and other autoimmune diseases. Autoantibody production in SLE is believed to result from autoantigen specific immune stimulation and subsequently, it is expected that antigenic determinants recognized by SLE autoantibodies and induced antibodies by immunization are quite similar. To examine this issue, rabbits were immunized with purified HMG-17. The produced antiserum showed cross reactivity on blots and in inhibition ELISA with histone H1, even after its affinity purification with immobilized HMG-17. Finally, purification of the antiserum over H1 absorbed on nitrocellulose membrane produced specific anti-HMG-17 antibodies in the supernatant and anti-HMG-17/H1 antibodies that were bound to H1. SLE sera positive for HMG-17 had also cross reactivity with H1, and following the same procedure as before we received HMG-17 specific SLE autoantibodies and anti-HMG-17/H1 autoantibodies. Using the multipin epitope mapping technology, 19 overlapping 15-mer HMG-17 peptides and six 15-peptides, corresponding to known epitopes of histone H1, were synthesized. Four major epitopes were identified on the HMG-17 molecule, reactive with induced anti-HMG-17 antibodies, and these were the same as major autoepitopes In SLE. The sequence 25-51 of HMG-17, part of its DNA-binding domain, was recognized by the anti-HMG-17/H1 antibodies that were bound to H1. These antibodies recognized also defined epitopes of H1. Our results show that SLE autoantibodies can be directed against the same or similar epitopes as do IgGs evoked during the active immunization of animals, and provide additional evidence that autosensitization with an autoantigen might be operative. The possibility that the same or similar epitopes are found on different molecules (in this study HMG-17 and H1) supports the fact that there are rules by which nature selects the most dominant immunodeterminant to a given protein, which often represents functional or structural sites in the autoantigen.     

Reversibility of established diabetic glomerulopathy by anti-TGF-beta antibodies in db/db mice

Treatment with a neutralizing anti-transforming growth factor-beta (TGF-beta) antibody can prevent the development of diabetic nephropathy in the db/db mouse, a model of type 2 diabetes. However, it is unknown whether anti-TGF-beta therapy can reverse the histological lesions of diabetic glomerulopathy once they are established. Diabetic db/db mice and their non-diabetic db/m littermates were allowed to grow until 16 weeks of age, by which time the db/db mice had developed glomerular basement membrane (GBM) thickening and mesangial matrix expansion. The mice were then treated with an irrelevant control IgG or a panselective, neutralizing anti-TGF-beta antibody for eight more weeks. Compared with control db/m mice, the db/db mice treated with IgG had developed increased GBM width (16.64+/-0.80 nm vs. 21.55+/-0.78 nm, P<0.05) and increased mesangial matrix fraction (4.01+/-0.81% of total glomerular area vs. 9.55+/-1.04%, P<0.05). However, the db/db mice treated with anti-TGF-beta antibody showed amelioration of GBM thickening (18.40+/-0.72 nm, P<0.05 vs. db/db-IgG) and mesangial matrix accumulation (6.32+/-1.79%, P<0.05 vs. db/db-IgG). Our results demonstrate that inhibiting renal TGF-beta activity can partially reverse the GBM thickening and mesangial matrix expansion in this mouse model of type 2 diabetes. Anti-TGF-beta regimens would be useful in the treatment of diabetic nephropathy.     

Structural analysis of nested neutralizing and non‐neutralizing B cell epitopes on ricin toxin's enzymatic subunit

In this report, we describe the X‐ray crystal structures of two single domain camelid antibodies (V_HH), F5 and F8, each in complex with ricin toxin's enzymatic subunit (RTA). F5 has potent toxin‐neutralizing activity, while F8 has weak neutralizing activity. F5 buried a total of 1760 Ų in complex with RTA and made contact with three prominent secondary structural elements: α‐helix B (Residues 98–106), β‐strand h (Residues 113–117), and the C‐terminus of α‐helix D (Residues 154–156). F8 buried 1103 Ų in complex with RTA that was centered primarily on β‐strand h. As such, the structural epitope of F8 is essentially nested within that of F5. All three of the F5 complementarity determining regions CDRs were involved in RTA contact, whereas F8 interactions were almost entirely mediated by CDR3, which essentially formed a seventh β‐strand within RTA's centrally located β‐sheet. A comparison of the two structures reported here to several previously reported (RTA‐V_HH) structures identifies putative contact sites on RTA, particularly α‐helix B, associated with potent toxin‐neutralizing activity. This information has implications for rational design of RTA‐based subunit vaccines for biodefense. Proteins 2016; 84:1162–1172. © 2016 Wiley Periodicals, Inc.     

Epitope mapping of anti‐myelin oligodendrocyte glycoprotein (MOG) antibodies in a mouse model of multiple sclerosis: microwave‐assisted synthesis of the peptide antigens and ELISA screening

The role of pathologic auto‐antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35–55). In this scenario, we analyzed the anti‐MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1–117). To assess the presence of a B‐cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1–117 sequence of MOG, including MOG(35–55). For this purpose, we cloned, expressed in Escherichia coli and on‐column refolded MOG(1–117), and we applied an optimized microwave‐assisted solid‐phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid‐phase immunoenzymatic assay testing both naïve and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35–55), we hypothesize the presence of both linear and conformational epitopes on MOG(35–55) sequence. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Functional differentiation of mouse uterine epithelial cells grown on collagen gels or reconstituted basement membranes

Summary Epithelial cells were isolated from mouse endometrium and cultured on two types of extracellular matrix, namely, rat-tail collagen (type I) gels and basement membrane extract (BME) derived from the Engelbreth-Holm-Swarm murine sarcoma. Cell attachment in serum-free medium during the initial 24 h after seeding was approximately twofold higher on BME compared with collagen type I. Addition of serum to the medium enhanced cell attachment on both matrices. On both collagen and BME, uterine cells grew as smooth-bordered colonies, and within a week of culture the cells became cuboidal to columnar in shape. Electron microscopy revealed the presence of apical microvilli associated with a glycocalyx, junctional complexes, tonofilaments, short strands of undilated endoplasmic reticulum, Golgi complex, and lipid droplets. However, cells on BME showed a higher degree of differentiation as assessed by occasional formation of small patches of basement membranelike structure subjacent to the flattened basal surface and formation of glandlike structures within the matrix. Proliferation of these cells as measured by radioactive thymidine incorporation into DNA was increased threefold by addition of epidermal growth factor (EGF) and insulin to the medium, but was not changed by 17β-estradiol. The expression of progesterone receptors by uterine epithelial cells grown on both matrices was doubled by addition of EGF and estradiol to the medium. This work was supported in part by a Rockefeller Foundation postdoctoral fellowship (D.G.), and NIh grant 23511.     

A surface plasmon resonance assay for characterisation and epitope mapping of anti‐GLP‐1 antibodies

The incretin hormone glucagon‐like peptide‐1 (GLP‐1) has been subject to substantial pharmaceutical research regarding the treatment of type 2 diabetes mellitus. However, quantification of GLP‐1 levels remains complicated due to the low circulation concentration and concurrent existence of numerous metabolites, homologous peptides, and potentially introduced GLP‐1 receptor agonists. Surface plasmon resonance (SPR) facilitates real‐time monitoring allowing a more detailed characterisation of the interaction compared with conventional enzyme‐linked immunosorbent assays (ELISA). In this paper, we describe the development of the first SPR assays for characterisation of anti‐GLP‐1 antibodies for ELISA purposes. Binding responses were obtained on covalently immobilised anti‐GLP‐1 antibodies at 12°C, 25°C, and 40°C and fitted to a biomolecular (1:1) interaction model showing association rates of 1.01 × 10³ to 4.54 × 10³ M^?1 s^?1 and dissociation rates of 3.56 × 10^?5 to 1.56 × 10^?3 s^?1 leading to affinities of 35.2 to 344 nM, depending on the temperature. Determination of thermodynamic properties revealed an enthalpy driven interaction (ΔH < ΔS < 0) with higher affinities at lower temperatures due to the formation and stabilisation of hydrogen bonds within the binding site primarily composed of polar amino acids (ΔC_p < 0). Pair‐wise epitope mapping was performed on captured anti‐GLP‐1 antibodies followed by subsequent interaction with GLP‐1 (7‐36) and other anti‐GLP‐1 antibodies. A global evaluation of every binding response led to an epitope map elucidating the potential of various anti‐GLP‐1 antibody pairs for sandwich ELISA and hence pinpointing the optimal antibody combinations. The SPR assays proved capable of providing vital information for ELISA development endorsing it as a useful optimisation tool.     

Eye‐mediated immune tolerance to Type II collagen in arthritis‐prone strains of mice

Type II collagen (CII) is a cartilage structural protein that plays important roles in joint function, arthritis and ageing. In studying the ability of CII to induce eye‐mediated specific immune tolerance, we have recently proven that CII is capable of inducing anterior chamber‐associated immune deviation (ACAID) in Balb/c mice. Here, we study the ability of CII to induce eye‐mediated immune tolerance in strains of mice that are prone to the induction of rheumatoid arthritis. Thus, we hypothesized that CII induces ACAID in DBA/1 mice and in C57BL/6 mice through the AC route (direct injection) or the intravenous route (adoptive transfer of in vitro‐generated CII‐specific ACAID macrophages or of CII‐specific in vitro‐generated T regulatory cells). Specific immune tolerance induction was assessed using both delayed‐type hypersensitivity (DTH) and local adoptive transfer (LAT) assays. Results indicated the ability of CII to generate CII‐specific ACAID‐mediated immune tolerance in vivo and in vitro in both DBA/1 mice and C57BL/6 mice. These findings could be beneficial in studies of immune tolerance induction using CII.     

Silencing of NONO inhibits abdominal aortic aneurysm in apolipoprotein E‐knockout mice via collagen deposition and inflammatory inhibition

The role of Non‐POU‐domain‐containing octamer‐binding protein (NONO) in the formation and development of angiotensin II (Ang II)‐induced abdominal aortic aneurysm (AAA) in apolipoprotein E‐knockout (ApoE^?/?) mice is still unknown. In Part I, the protein level of NONO was suggestively greater in the AAA tissues compare to that in the normal abdominal aortas. In Part II, 20 ApoE^?/? male mice were used to examine the transfection efficiency of lentivirus by detecting GFP fluorescence. In Part III, mice were arbitrarily separated into two groups: one was the control group without Ang II infusion, and another was the Ang II group. Mice treated with Ang II were further randomly divided into three groups to receive the same volume of physiological saline (NT group), sh‐negative control lentivirus (sh‐NC group) and si‐NONO lentivirus (sh‐NONO group). NONO silencing suggestively reduced the occurrence of AAA and abdominal aortic diameter. Compare to the NT group, NONO silencing markedly augmented the content of collagen and vascular smooth muscle cells but reduced macrophage infiltration in AAA. In addition, knockdown of NONO also increased the expression of prolyl‐4‐hydroxylase α1, whereas also decreased the levels of collagen degradation and pro‐inflammatory cytokines in AAA. We detected the interface of NONO and NF‐κB p65, and found that NONO silencing inhibited both the nuclear translocation and the phosphorylation levels of NF‐κB p65. Silencing of NONO prevented Ang II‐influenced AAA in ApoE^?/? mice through increasing collagen deposition and inhibiting inflammation. The mechanism may be that silencing of NONO decreases the nuclear translocation and phosphorylation of NF‐κB.     

Morphofunctional studies of the glomerular wall in mice lacking entactin-1.

The architecture of the basement membranes is essential for proper function. This architecture is based on interactions among its components, which assemble in a complex network. Entactin-1 appears to be the mastermind of this assembling. In entactin-1-null transgenic mice, immunocytochemistry established the absence of entactin-1 in the glomerular basement membrane, and morphological thickening of this membrane was demonstrated. This prompted us to investigate the organization of other components of the glomerular basement membrane in the transgenic animals. The distribution of type IV collagen and laminin remained unchanged, whereas that of anionic charges was significantly altered. We also evaluated the impact of the absence of entactin-1 on cell relays by studying the alpha(3)- and the alpha(v)-integrins along the endothelial and epithelial glomerular cell plasma membranes. Only the density of alpha(v) was found to be increased. Finally, the filtration properties of the glomerular wall were evaluated by revealing endogenous albumin distribution across the basement membrane. This was altered in transgenic animals, suggesting changes in permselectivity properties. Entactin-1 appears to be an essential component in basement membranes because its absence appears to modify the molecular organization leading to alterations in functional properties.     

Age‐related ultrastructural changes of the basement membrane in the mouse blood‐brain barrier

The blood‐brain barrier (BBB) is essential for a functional neurovascular unit. Most studies focused on the cells forming the BBB, but very few studied the basement membrane (BM) of brain capillaries in ageing. We used transmission electron microscopy and electron tomography to investigate the BM of the BBB in ageing C57BL/6J mice. The thickness of the BM of the BBB from 24‐month‐old mice was double as compared with that of 6‐month‐old mice (107 nm vs 56 nm). The aged BBB showed lipid droplets gathering within the BM which further increased its thickness (up to 572 nm) and altered its structure. The lipids appeared to accumulate toward the glial side of the BM. Electron tomography showed that the lipid‐rich BM regions are located in small pockets formed by the end‐feet of astrocytes. These findings suggest an imbalance of the lipid metabolism and that may precede the structural alteration of the BM. These alterations may favour the accretion of abnormal proteins that lead to neurodegeneration in ageing. These findings warrant further investigation of the BM of brain capillaries and of adjoining cells as potential targets for future therapies.     

Immunoprofiling reveals unique cell‐specific patterns of wall epitopes in the expanding Arabidopsis stem

The Arabidopsis inflorescence stem undergoes rapid directional growth, requiring massive axial cell‐wall extension in all its tissues, but, at maturity, these tissues are composed of cell types that exhibit markedly different cell‐wall structures. It is not clear whether the cell‐wall compositions of these cell types diverge rapidly following axial growth cessation, or whether compositional divergence occurs at earlier stages in differentiation, despite the common requirement for cell‐wall extensibility. To examine this question, seven cell types were assayed for the abundance and distribution of 18 major cell‐wall glycan classes at three developmental stages along the developing inflorescence stem, using a high‐throughput immunolabelling strategy. These stages represent a phase of juvenile growth, a phase displaying the maximum rate of stem extension, and a phase in which extension growth is ceasing. The immunolabelling patterns detected demonstrate that the cell‐wall composition of most stem tissues undergoes pronounced changes both during and after rapid extension growth. Hierarchical clustering of the immunolabelling signals identified cell‐specific binding patterns for some antibodies, including a sub‐group of arabinogalactan side chain‐directed antibodies whose epitope targets are specifically associated with the inter‐fascicular fibre region during the rapid cell expansion phase. The data reveal dynamic, cell type‐specific changes in cell‐wall chemistry across diverse cell types during cell‐wall expansion and maturation in the Arabidopsis inflorescence stem, and highlight the paradox between this structural diversity and the uniform anisotropic cell expansion taking place across all tissues during stem growth.