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Idiopathic pulmonary fibrosis is a progressive lung disease that causes scarring and loss of lung function. Macrophages play a key role in fibrosis, but their responses to altered morphological and mechanical properties of the extracellular matrix in fibrosis is relatively unexplored. Our previous work showed functional changes in murine fetal liver-derived alveolar macrophages on fibrous or globular collagen morphologies. In this study, we applied differential proteomics to further investigate molecular mechanisms underlying the observed functional changes. Macrophages cultured on uncoated, fibrous, or globular collagen-coated plastic were analyzed by liquid chromatography-mass spectrometry. The presence of collagen affected expression of 77 proteins, while 142 were differentially expressed between macrophages grown on fibrous or globular collagen. Biological process and pathway enrichment analysis revealed that culturing on any type of collagen induced higher expression of enzymes involved in glycolysis. However, this did not lead to a higher rate of glycolysis, probably because of a concomitant decrease in activity of these enzymes. Our data suggest that macrophages sense collagen morphologies and can respond with changes in expression and activity of metabolism-related proteins. These findings suggest intimate interactions between macrophages and their surroundings that may be important in repair or fibrosis of lung tissue.

Receptor diffusion plays an essential role in cellular signalling via the plasma membrane microenvironment and receptor interactions, but the regulation is not well understood. To aid in understanding of the key determinants of receptor diffusion and signalling, we developed agent-based models (ABMs) to explore the extent of dimerisation of the platelet- and megakaryocyte-specific receptor for collagen glycoprotein VI (GPVI). This approach assessed the importance of glycolipid enriched raft-like domains within the plasma membrane that lower receptor diffusivity. Our model simulations demonstrated that GPVI dimers preferentially concentrate in confined domains and, if diffusivity within domains is decreased relative to outside of domains, dimerisation rates are increased. While an increased amount of confined domains resulted in further dimerisation, merging of domains, which may occur upon membrane rearrangements, was without effect. Modelling of the proportion of the cell membrane which constitutes lipid rafts indicated that dimerisation levels could not be explained by these alone. Crowding of receptors by other membrane proteins was also an important determinant of GPVI dimerisation. Together, these results demonstrate the value of ABM approaches in exploring the interactions on a cell surface, guiding the experimentation for new therapeutic avenues.

Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6tg/tg). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.

Glycoprotein (GP)VI and integrin αIIbβ3 are key signaling receptors in collagen-dependent platelet aggregation and in arterial thrombus formation under shear. The multiple downstream signaling pathways are still poorly understood. Here, we focused on disclosing the integrin-dependent roles of focal adhesion kinase (protein tyrosine kinase 2, PTK2), the shear-dependent collagen receptor GPR56 (ADGRG1 gene), and calcium and integrin-binding protein 1 (CIB1). We designed and synthetized peptides that interfered with integrin αIIb binding (pCIB and pCIBm) or mimicked the activation of GPR56 (pGRP). The results show that the combination of pGRP with PTK2 inhibition or of pGRP with pCIB > pCIBm in additive ways suppressed collagen- and GPVI-dependent platelet activation, thrombus buildup, and contraction. Microscopic thrombus formation was assessed by eight parameters (with script descriptions enclosed). The suppressive rather than activating effects of pGRP were confined to blood flow at a high shear rate. Blockage of PTK2 or interference of CIB1 no more than slightly affected thrombus formation at a low shear rate. Peptides did not influence GPVI-induced aggregation and Ca2+ signaling in the absence of shear. Together, these data reveal a shear-dependent signaling axis of PTK2, integrin αIIbβ3, and CIB1 in collagen- and GPVI-dependent thrombus formation, which is modulated by GPR56 and exclusively at high shear. This work thereby supports the role of PTK2 in integrin αIIbβ3 activation and signaling.

Collagen is the central structural component of extracellular connective tissue, which provides elastic qualities to tissues. For skeletal muscle, extracellular connective tissue transmits contractile force to the tendons and bones. Connective tissue proteins are in a constant state of remodeling and have been shown to express a high level of plasticity. Dietary-protein ingestion increases muscle protein synthesis rates. High-quality, rapidly digestible proteins are generally considered the preferred protein source to maximally stimulate myofibrillar (contractile) protein synthesis rates. In contrast, recent evidence demonstrates that protein ingestion does not increase muscle connective tissue protein synthesis. The absence of an increase in muscle connective tissue protein synthesis after protein ingestion may be explained by insufficient provision of glycine and/or proline. Dietary collagen contains large amounts of glycine and proline and, therefore, has been proposed to provide the precursors required to facilitate connective tissue protein synthesis. This literature review provides a comprehensive evaluation of the current knowledge on the proposed benefits of dietary collagen consumption to stimulate connective tissue remodeling to improve health and functional performance.

Stromal stiffening accompanies malignancy, compromises treatment and promotes tumour aggression. Clarifying the molecular nature and the factors that regulate stromal stiffening in tumours should identify biomarkers to stratify patients for therapy and interventions to improve outcome. We profiled lysyl hydroxylase-mediated and lysyl oxidase-mediated collagen crosslinks and quantified the greatest abundance of total and complex collagen crosslinks in aggressive human breast cancer subtypes with the stiffest stroma. These tissues harbour the highest number of tumour-associated macrophages, whose therapeutic ablation in experimental models reduced metastasis, and decreased collagen crosslinks and stromal stiffening. Epithelial-targeted expression of the crosslinking enzyme, lysyl oxidase, had no impact on collagen crosslinking in PyMT mammary tumours, whereas stromal cell targeting did. Stromal cells in microdissected human tumours expressed the highest level of collagen crosslinking enzymes. Immunohistochemical analysis of biopsies from a cohort of patients with breast cancer revealed that stromal expression of lysyl hydroxylase 2, an enzyme that induces hydroxylysine aldehyde-derived collagen crosslinks and stromal stiffening, correlated significantly with disease specific mortality. The findings link tissue inflammation, stromal cell-mediated collagen crosslinking and stiffening to tumour aggression and identify lysyl hydroxylase 2 as a stromal biomarker.

ObjectivesBone and other human tissues remodel through life, for example, as a response to increasing load, and this prevents permanent destruction of the tissue. Non-traumatic meniscal rupture is a common musculoskeletal disease, but it is unknown if it is caused by inability of the menisci to remodel. The aim of this study was to determine whether meniscal collagen is remodelling throughout life.MethodsThe life-long turnover of the human meniscal collagens was explored by the 14C bomb pulse method. 14C levels were determined in menisci from 18 patients with osteoarthritis and 7 patients with healthy knees.ResultsThere was a negligible turnover of the meniscal collagen in adults. This low turnover was observed in menisci from patients with knee osteoarthritis and in healthy menisci.ConclusionThis study provides evidence that essentially no remodelling occurs in the adult human meniscal collagen structure and explains the clinical degeneration that is often seen in menisci of middle-aged and elderly persons. It suggests that strengthening of the collagen structure of menisci, as response to physical activity, may occur during childhood, while it is not possible in the adult population.

Fibroblasts are activated to repair the heart following injury. Fibroblast activation in the mammalian heart leads to a permanent fibrotic scar that impairs cardiac function. In other organisms, such as zebrafish, cardiac injury is followed by transient fibrosis and scar-free regeneration. The mechanisms that drive scarring versus scar-free regeneration are not well understood. Here, we show that the homeobox-containing transcription factor Prrx1b is required for scar-free regeneration of the zebrafish heart as the loss of Prrx1b results in excessive fibrosis and impaired cardiomyocyte proliferation. Through lineage tracing and single-cell RNA sequencing, we find that Prrx1b is activated in epicardial-derived cells where it restricts TGFβ ligand expression and collagen production. Furthermore, through combined in vitro experiments in human fetal epicardial-derived cells and in vivo rescue experiments in zebrafish, we conclude that Prrx1 stimulates Nrg1 expression and promotes cardiomyocyte proliferation. Collectively, these results indicate that Prrx1 is a key transcription factor that balances fibrosis and regeneration in the injured zebrafish heart. This article has an associated ‘The people behind the papers’ interview., Summary: Epicardial-expressed Prrx1 ensures a balance between the fibrotic response and myocardial regeneration post-injury in zebrafish.

Ultraviolet radiation (UVR) increases the incidence of cutaneous melanoma1–4. The ageing, sun-exposed dermis accumulates UVR damage5, and older patients develop more melanomas at UVR-exposed sites4,6,7. As fibroblasts are functionally heterogeneous and play key roles in the stromal contribution to cancer8,9, we asked whether UVR modifies dermal fibroblast function. Here we confirmed the expression of collagen-cleaving matrix metalloprotein-1 (MMP1) by UVR-damaged fibroblasts was persistently upregulated to reduce local levels of collagen 1 (COL1A1), and found dermal COL1A1 degradation by MMP1 decreased melanoma invasion. Conversely, we show inhibiting extracellular matrix degradation and MMP1 expression restored melanoma invasion to UVR damaged dermis. We confirmed in vitro findings in a cohort of primary cutaneous melanomas of aged humans, showing more cancer cells invade as single cells at the invasive front of melanomas expressing and depositing more collagen. We found collagen and single melanoma cell invasion are robust predictors of poor melanoma-specific survival. These data indicate melanomas arising over UVR-damaged, collagen-poor skin of the elderly are less invasive, and this reduced invasion improves survival. Consequently, although UVR increases tumour incidence, it delays primary melanoma invasion by degrading collagen. However, we show melanoma-associated fibroblasts can restore invasion in low-collagen primary tumours by increasing collagen synthesis. Finally, we demonstrate high COL1A1 gene expression is a biomarker of poor outcome across a broad range of primary cancers.

Purpose: Keratoconus presents certain specificities in pediatric patients compared with adults. The greatest challenge is because the disease is typically more severe and progresses faster in children. This retrospective study aimed to report crosslinking procedure in patients under 18 years of age and their follow-up for at least 24 months after the procedure. Methods: Overall, 12 eyes from 10 patients were studied and data, such as visual acuity with and without correction, maximum keratometry, corneal thickness, foveal thickness, and endothelial microscopy, were assessed at both preoperative and postoperative visits. Corneal crosslinking was performed in all patients. Results: A tendency toward reduced Kmax and improved Corrected Distance Visual Acuity at all postoperative moments. Only one of the 12 eyes exhibited increased Kmax of more than 1 D during a time frame longer than 12 months. Regarding pachymetry, a tendency for corneal thinning was observed in the first four months after surgery. Conclusion: Encouraging results were obtained regarding the stabilization of the disease, progression, and procedural safety, corroborating to other authors’ findings. The significance of early diagnosis and short-term follow-up were highlighted. RESUMO Objetivo: O ceratocone na população pediátrica apresenta algumas particularidades em relação à população adulta. O maior desafio é devido à doença ser geralmente mais severa e rapidamente progressiva em crianças. Métodos: Este artigo utiliza uma análise retrospectiva para relatar o uso do crosslinking em jovens menores de 18 anos e sua evolução pelo menos 24 meses após o procedimento. Foram estudados 12 olhos de 10 pacientes, e dados como acuidade visual com e sem correção, ceratometria máxima, espessura corneana, espessura foveal e microscopia endotelial avaliados no pré e pós-operatórios. O crosslinking corneano foi realizado em todos os pacientes pelo mesmo cirurgião. Resultados: Observou-se uma tendência de redução do valor do Kmax e melhora da acuidade visual corrigida em todos os momentos de pós operatório. Com relação à paquimetria, observou-se afinamento corneano do ponto mais fino, nos primeiros quatro meses de pós-operatório. Conclusão: Resultados encorajadores foram obtidos com relação à estabilização da doença, progressão e segurança do procedimento, corroborando com as conclusões de outros autores. A importância do diagnóstico precoce e do acompanhamento a curto prazo do paciente deve ser destacada.

Hydrophobic bile salts are considered to promote liver fibrosis in cholestasis. However, evidence for this widely accepted hypothesis remains scarce. In established animal models of cholestasis, e.g., by Mdr2 knockout, cholestasis and fibrosis are both secondary to biliary damage. Therefore, to test the specific contribution of accumulating bile salts to liver fibrosis in cholestatic disease, we applied the unique model of inducible hepatocellular cholestasis in cholate-fed Atp8b1G308V/G308V mice. Glycochenodeoxycholate (GCDCA) was supplemented to humanize the murine bile salt pool, as confirmed by HPLC. Biomarkers of cholestasis and liver fibrosis were quantified. Hepatic stellate cells (HSC) isolated from wild-type mice were stimulated with bile salts. Proliferation, cell accumulation, and collagen deposition of HSC were determined. In cholestatic Atp8b1G308V/G308V mice, increased hepatic expression of &alpha, SMA and collagen1a mRNA and excess hepatic collagen deposition indicated development of liver fibrosis only upon GCDCA supplementation. In vitro, numbers of myofibroblasts and deposition of collagen were increased after incubation with hydrophobic but not hydrophilic bile salts, and associated with EGFR and MEK1/2 activation. We concluded that chronic hepatocellular cholestasis alone, independently of biliary damage, induces liver fibrosis in mice in presence of the human bile salt GCDCA. Bile salts may have direct pro-fibrotic effects on HSC, putatively involving EGFR and MEK1/2 signaling.

Collagen damage in articular cartilage is considered nearly irreversible and may be an early indication of cartilage degeneration. Surface fibrillation and internal collagen damage may both develop after overloading. This study hypothesizes that damage develops at these different locations, because the distribution of excessive strains varies with loading rate as a consequence of time-dependent cartilage properties. The objective is to explore whether collagen damage could preferentially occur superficially or internally, depending on the magnitude and rate of overloading. Bovine osteochondral plugs were compressed with a 2 mm diameter indenter to 15, 25, 35 and 45 N, and at 5, 60 and 120 mm/min. Surface fibrillation and internal collagen damage were graded by four observers, based on histology and staining of collagen damage. Results show that loading magnitude affects the degree of collagen damage, while loading rate dominates the location of network damage: low rates predominantly damage superficial collagen, while at high rates, internal collagen damage occurs. The proposed explanation for the rate-dependent location is that internal fluid flows govern the time-dependent internal tissue deformation and therewith the location of overstained and damaged areas. This supports the hypothesis that collagen damage development is influenced by the time-dependent material behaviour of cartilage.

Physiologic turnover of interstitial collagen is mediated by a sequential pathway in which collagen is fragmented by pericellular collagenases, endocytosed by collagen receptors, and routed to lysosomes for degradation by cathepsins. Here, we use intravital microscopy to investigate if malignant tumors, which are characterized by high rates of extracellular matrix turnover, utilize a similar collagen degradation pathway. Tumors of epithelial, mesenchymal, or neural crest origin all display vigorous endocytic collagen degradation. The cells engaged in this process are identified as tumor-associated macrophage (TAM)-like cells that degrade collagen in a mannose receptor-dependent manner. Accordingly, mannose-receptor-deficient mice display increased intratumoral collagen. Whole-transcriptome profiling uncovers a distinct extracellular matrix-catabolic signature of these collagen-degrading TAMs. Lineage-ablation studies reveal that collagen-degrading TAMs originate from circulating CCR2+ monocytes. This study identifies a function of TAMs in altering the tumor microenvironment through endocytic collagen turnover and establishes macrophages as centrally engaged in tumor-associated collagen degradation. Madsen et al. identify a population of tumor-associated macrophages with a distinct matrix catabolic signature as key effectors of collagen turnover during invasive tumor growth. These matrix-degrading macrophages are largely derived from CCR2+ monocytes reprogrammed by the tumor microenvironment and degrade collagen through mannose receptor-dependent cellular uptake.

The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity., Studies examining human podocytopathies have utilised 2D cultured primary or immortalised podocyte cell lines. Here, the authors demonstrate that 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids retain an improved podocyte identity in vitro facilitating disease modelling and toxicity testing.

The hemodynamic functionality of heart valves strongly depends on the distribution of collagen fibers, which are their main load-bearing constituents. It is known that collagen networks remodel in response to mechanical stimuli. Yet, the complex interplay between external load and collagen remodeling is poorly understood. In this study, we adopted a computational approach to simulate collagen remodeling occurring in native fetal and pediatric heart valves. The computational model accounted for several biological phenomena: cellular (re)orientation in response to both mechanical stimuli and topographical cues provided by collagen fibers; collagen deposition and traction forces along the main cellular direction; collagen degradation decreasing with stretch; and cell-mediated collagen prestretch. Importantly, the computational results were well in agreement with previous experimental data for all simulated heart valves. Simulations performed by varying some of the computational parameters suggest that cellular forces and (re)orientation in response to mechanical stimuli may be fundamental mechanisms for the emergence of the circumferential collagen alignment usually observed in native heart valves. On the other hand, the tendency of cells to coalign with collagen fibers is essential to maintain and reinforce that circumferential alignment during development. Statement of Significance: The hemodynamic functionality of heart valves is strongly influenced by the alignment of load-bearing collagen fibers. Currently, the mechanisms that are responsible for the development of the circumferential collagen alignment in native heart valves are not fully understood. In the present study, cell-mediated remodeling of native human heart valves during early development was computationally simulated to understand the impact of individual mechanisms on collagen alignment. Our simulations successfully predicted the degree of collagen alignment observed in native fetal and pediatric semilunar valves. The computational results suggest that the circumferential collagen alignment arises from cell traction and cellular (re)orientation in response to mechanical stimuli, and with increasing age is reinforced by the tendency of cells to co-align with pre-existing collagen fibers.

PURPOSE: Keratoconus (KC) is a multifactorial, ectatic corneal disease. Metabolic changes in the corneal stroma with alterations in collagen fibril stability, oxidative stress, and urea cycle, have previously been reported as key players in KC pathobiology. Recently, corneal collagen cross-linking (CXL) has been introduced as a treatment that can address the progressive nature of KC. While the treatment has been successful in the early days, it is not without clinical ramifications. In this study, we investigated the alterations in KC metabolic profiles due to CXL.METHODS: Primary human corneal fibroblasts (HCFs) from healthy donors and human KC fibroblasts (HKCs) from KC donor patients were plated on transwell polycarbonate membranes and stimulated by a stable vitamin C. At 4 weeks, riboflavin was added to the cultures followed by UVA irradiation (365 nm). Using mass spectrometry, we measured the major differences in metabolites in HKCs compared to HCFs pre- and post CXL.RESULT: The analysis of 276 metabolites in HCFs and HKCs revealed that the most affected metabolites due to CXL were glutathione disulfide, ascorbic acid, proline, and lysine. A significant decrease in the pro-inflammatory biomarkers (myo-inositol and histidine) was also observed. Furthermore, a significant downregulation of many amino acids, lactate levels, and other water-soluble metabolites was noted in HKCs following CXL.CONCLUSION: CXL is a KC treatment available to patients within certain criteria. Surprisingly, the cellular and molecular mechanisms are considerably understudied limiting our ability for more precise and targeted CXL treatments. In this study, for the first time, we report the effects of CXL on KC metabolism.

Peripheral nerve regeneration is critical and challenging in the adult humans. High level of collagen infiltration (i.e., scar tissue), in the niche of injury, impedes axonal regeneration and path finding. Unfortunately, studies focusing on the modulation of scar tissue in the nerves are scarce. To address part of this problem, we have evaluated the differentiated adipose derived stem cells (dASCs) for their antifibrotic and regenerative effects in a 10 mm nerve gap model in rats. Three different animal groups (N = 5) were treated with fibrin nerve conduits (empty), or seeded with dASCs (F + dASCs) and autograft, respectively. Histological analysis of regenerated nerves, at 12 weeks postoperatively, reveled the high levels of collagen infiltration (i.e., 21.5% ± 6.1% and 24.1% ± 2.9%) in the middle and distal segment of empty conduit groups in comparison with stem cells treated (16.6% ± 2.1% and 12.1% ± 2.9%) and autograft (15.0% ± 1.7% and 12.8% ± 1.0%) animals. Thus, the dASCs treatment resulted in significant reduction of fibrotic tissue formation. Consequently, enhanced axonal regeneration and remyelination was found in the animals treated with dASCs. Interestingly, these effects of dASCs appeared to be equivalent to that of autograft treatment. Thus, the dASCs hold great potential for preventing the scar tissue formation and for promoting nerve regeneration in the adult organisms. Future experiments will focus on the validation of these findings in a critical nerve injury model. Anat Rec, 301:1714–1721, 2018. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists

Nonalcoholic fatty liver disease (NAFLD) is a spectrum comprised of isolated steatosis, nonalcoholic steatohepatitis (NASH), advanced fibrosis, and cirrhosis. The majority of NAFLD subjects do not have NASH and do not carry a significant risk for liver-related adverse outcomes (cirrhosis and mortality). Globally, the prevalence of NAFLD is approximately 25%. In Asia, a gradient of high to low prevalence rates is noted from urban to rural areas. Given the prevalence of NAFLD, the clinical and economic burden of NAFLD and NASH can be substantial. With increasing recognition of NASH as an important liver disease, the diagnosis of NASH still requires a liver biopsy that is suboptimal. Although liver biopsy is the most accurate modality to diagnose and stage the severity of NASH, this method suffers from being invasive, costly, associated with potential complications, and plagued with interobserver variability of individual pathological features. A number of noninvasive modalities to diagnose NASH and stage liver fibrosis are being developed. These modalities include predictive models (NAFLD fibrosis score) and serum biomarkers such as enhanced liver fibrosis (ELF). Other tests are based on radiological techniques, such as transient elastography (TE) or magnetic resonance elastography (MRE), which are used to estimate liver stiffness as a potential surrogate of hepatic fibrosis. Although a dynamic field of research, most of these diagnostic modalities have area under the curve ranging between 0.76 and 0.90%, with MRE having the best predictive performance. In summary, developing safe and easily accessible noninvasive modalities to accurately diagnose and monitor NASH and associated fibrosis is of utmost importance in clinical practice and clinical research. These tests are not only important to risk stratify subjects at the greatest risk for progressive liver disease, but also to serve as appropriate surrogate endpoints for therapeutic clinical trials of NASH. (Hepatology 2018;68:349-360).

Dysregulation of the immune response to microbiota is associated with inflammatory bowel disease (IBD), which can trigger intestinal fibrosis. MyD88 is a key component of microbiota signalling but its influence on intestinal fibrosis has not been clarified. Small bowel resections from donor-mice were transplanted subcutaneously into the neck of recipients C57BL/6 B6-MyD88tm1 Aki (MyD88−/−) and C57BL/6-Tg(UBC-green fluorescence protein (GFP))30Scha/J (GFP-Tg). Grafts were explanted up to 21 days after transplantation. Collagen layer thickness was determined using Sirius Red stained slides. In the mouse model of fibrosis collagen deposition and transforming growth factor-beta 1 (TGF-β1) expression was equal in MyD88+/+ and MyD88−/−, indicating that MyD88 was not essential for fibrogenesis. Matrix metalloproteinase (Mmp)9 expression was significantly decreased in grafts transplanted into MyD88−/− recipients compared to MyD88+/+ recipients (0.2 ± 0.1 vs. 153.0 ± 23.1, respectively, p

The short-lived turquoise killifish Nothobranchius furzeri (Nfu) is a valid model for aging studies. Here, we investigated its age-associated cardiac function. We observed oxidative stress accumulation and an engagement of microRNAs (miRNAs) in the aging heart. MiRNA-sequencing of 5 week (young), 12–21 week (adult) and 28–40 week (old) Nfu hearts revealed 23 up-regulated and 18 down-regulated miRNAs with age. MiR-29 family turned out as one of the most up-regulated miRNAs during aging. MiR-29 family increase induces a decrease of known targets like collagens and DNA methyl transferases (DNMTs) paralleled by 5´methyl-cytosine (5mC) level decrease. To further investigate miR-29 family role in the fish heart we generated a transgenic zebrafish model where miR-29 was knocked-down. In this model we found significant morphological and functional cardiac alterations and an impairment of oxygen dependent pathways by transcriptome analysis leading to hypoxic marker up-regulation. To get insights the possible hypoxic regulation of miR-29 family, we exposed human cardiac fibroblasts to 1% O2 levels. In hypoxic condition we found miR-29 down-modulation responsible for the accumulation of collagens and 5mC. Overall, our data suggest that miR-29 family up-regulation might represent an endogenous mechanism aimed at ameliorating the age-dependent cardiac damage leading to hypertrophy and fibrosis.

Highlights • Pre- and post-somatic segments of type I spiral ganglion neurons (SGNs) are unmyelinated in man. • Following hair cell loss and retrograde nerve degeneration SGNs survive as “mono-polar” cells in human deafness. • Non-myelinated Schwann cells may consolidate the neural cell bodies and protect SGNs from further degeneration. • Human SGNs can persist as electrically excitable mono-polar cells even after long-time deafness. • Robust survival of human SGNs is a prerequisite for cochlear implant function., Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-β2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-β2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-β2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

Purpose To evaluate corneal cross-linking (CXL) in the treatment of keratoconus in pediatric patients. Specifically, this study investigates the impact of CXL on uncorrected distance visual acuity (UDVA), best-corrected distance visual acuity (BDVA), manifest refraction, keratometry (K) measurements, and higher order aberrations. Methods This is a retrospective, observational case series of patients 18 years old or younger with progressive keratoconus who underwent CXL from January 2009 to August 2013. Preoperative and 1-year postoperative data including BDVA, manifest refraction, mean K readings, and corneal aberration measurements were extracted from clinical charts and topographical imaging. Visual acuity was converted to logarithm of the minimum angle of resolution (logMAR) scale, and mean refractive spherical equivalent (MRSE) was calculated from manifest refraction. Results The group consisted of 39 eyes from 28 patients, including 21 males and 7 females (mean age = 16.3 years, range: 11-18, standard deviation [SD] = 1.81). UDVA did not change significantly (preoperative UDVA = 1.20 logMAR, SD = 0.57, and postoperative UDVA = 0.90 logMAR, SD = 0.67, P = 0.19). BDVA did not change significantly (preoperative BDVA = 0.34 logMAR, SD = 0.27, and postoperative BDVA = 0.34 logMAR, SD = 0.23, P = 0.50). There was no significant change in mean K (preoperative K = 48.49, SD = 5.44, and postoperative K = 48.25, SD = 4.74, P = 0.34). Mean MRSE did not change significantly (preoperative MRSE = -3.29 D, SD = 4.04, and postoperative MRSE = -3.53 D, SD = 4.07, P = 0.31). Corneal aberration measurements were available for 10 eyes, and stability of measurements was demonstrated. There were no complications noted. Conclusions This study suggests that CXL is a safe and effective procedure that halts the progression of keratoconus in pediatric patients at 1-year follow-up. To validate these findings, longer follow-up is required.

Kidney allograft failure due to chronic injury/rejection remains the main cause of graft loss in renal transplant recipients (RTR). Here, we investigated whether specific biomarkers of extracellular matrix (ECM) turnover are associated with allograft function and chronic kidney disease (CKD) stage in RTR. Seventy-eight patients who attended the University Medical Center Groningen for a routine check-up after kidney transplantation were enrolled in the study. Plasma and/or 24h-urine samples were collected and specific matrix-metalloproteinase- generated neo-epitope fragments of collagens were measured by enzyme-linked immunosorbent assay. Our results demonstrated that urinary levels of C3M, a marker for collagen type III degradation, correlated with estimated glomerular filtration rate (eGFR; r = 0.58, p

Lymph node stromal cells (LNSCs) have newly been promoted to the rank of new modulators of T cell responses. The different non-hematopoietic cell subsets in lymph node (LN) were considered for years as a simple scaffold, forming routes and proper environment for antigen (Ag)-lymphocyte encountering. Deeper characterization of those cells has recently clearly shown their impact on both dendritic cell and T cell functions. In particular, lymphatic endothelial cells (LECs) control lymphocyte trafficking and homeostasis in LNs and limit adaptive immune responses. Therefore, the new role of LECs in shaping immune responses has drawn the attention of immunologists. Striking is the discovery that LECs, among other LNSCs, ectopically express a large range of peripheral tissue-restricted Ags (PTAs), and further present PTA-derived peptides through major histocompatibility class I molecules to induce self-reactive CD8(+) T cell deletional tolerance. In addition, both steady-state and tumor-associated LECs were described to be capable of exogenous Ag cross-presentation. Whether LECs can similarly impact CD4(+) T cell responses through major histocompatibility class II restricted Ag presentation is still a matter of debate. Here, we review and discuss our current knowledge on the contribution of Ag-presenting LECs as regulators of peripheral T cell responses in different immunological contexts, including autoimmunity and cancer.

SummaryPharmacological treatments targeting CXC chemokines and the associated neutrophil activation and recruitment into atherosclerotic plaques hold promise for treating cardiovascular disorders. Therefore, we investigated whether FK866, a nicotinamide phosphoribosyltransferase (NAMPT) inhibitor with anti-inflammatory properties that we recently found to reduce neutrophil recruitment into the ischaemic myocardium, would exert beneficial effects in a mouse atherosclerosis model. Atherosclerotic plaque formation was induced by carotid cast implantation in ApoE-/- mice that were fed with a Western-type diet. FK866 or vehicle were administrated intraperitoneally from week 8 until week 11 of the diet. Treatment with FK866 reduced neutrophil infiltration and MMP-9 content and increased collagen levels in atherosclerotic plaques compared to vehicle. No effect on other histological parameters, including intraplaque lipids or macrophages, was observed. These findings were associated with a reduction in both systemic and intraplaque CXCL1 levels in FK866-treated mice. In vitro, FK866 did not affect MMP-9 release by neutrophils, but it strongly reduced CXCL1 production by endothelial cells which, in the in vivo model, were identified as a main CXCL1 source at the plaque level. CXCL1 synthesis inhibition by FK866 appears to reflect interference with nuclear factor-κB signalling as shown by reduced p65 nuclear levels in endothelial cells pre-treated with FK866. In conclusion, pharmacological inhibition of NAMPT activity mitigates inflammation in atherosclerotic plaques by reducing CXCL1-mediated activities on neutrophils. These results support further assessments of NAMPT inhibitors for the potential prevention of plaque vulnerability.

Introduction The renin angiotensin system plays a crucial role in erectile function. It has been shown that elevated angiotensin‐II levels contribute to the development of erectile dysfunction (ED). Oppositely, angiotensin‐(1‐7) (Ang‐[1‐7]) mediates penile erection by activation of receptor Mas. Recently, we have developed a formulation based on Ang‐(1‐7) inclusion in cyclodextrin (CyD) [Ang‐(1‐7)‐CyD], which allows for the oral administration of Ang‐(1‐7). Aim In the present study, we evaluated the effects of chronic treatment with Ang‐(1‐7)‐CyD on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemic mice. Methods Apolipoprotein(Apo)E −/− mice fed a Western‐type diet for 11 weeks received Ang‐(1‐7)‐CyD or vehicle during the final 3 weeks. Collagen content and reactive oxygen species (ROS) production within the corpus cavernosum were evaluated by Sirius red and dihydroethidium staining, respectively. Protein expression of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH) subunits (p67‐phox and p22‐phox), and AT1 and Mas receptors in the penis was assessed by Western blotting. Nitric oxide (NO) production was measured by Griess assay in the mice serum. Cavernosal strips were mounted in an isometric organ bath to evaluate the endothelial function. Main Outcome Measures The effect of Ang‐(1‐7)‐CyD treatment on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemia‐induced ED. Results Ang‐(1‐7)‐CyD treatment reduced collagen content in the corpus cavernosum of ApoE −/− mice. This effect was associated with an attenuation of ROS production and a diminished expression of NADPH. Furthermore, Ang‐(1‐7)‐CyD treatment augmented the expression of nNOS and eNOS in the penis and elevated vascular NO production. Importantly, these effects were accompanied by an improvement in cavernosal endothelial function. Conclusion Long‐term treatment with Ang‐(1‐7)‐CyD reduces penile fibrosis associated with attenuation of oxidative stress. Additionally, cavernosal endothelial function in hypercholesterolemic mice was markedly improved. These results suggest that Ang‐(1‐7)‐CyD might have significant therapeutic benefits for the treatment of erectile dysfunction. Fraga‐Silva RA, Costa‐Fraga FP, Savergnini SQ, De Sousa FB, Montecucco F, da Silva D, Sinisterra RD, Mach F, Stergiopulos N, da Silva RF, and Santos RAS. An oral formulation of angiotensin‐(1–7) reverses corpus cavernosum damages induced by hypercholesterolemia. J Sex Med 2013;10:2430–2442.

BACKGROUND: Platelets play a pivotal role in coagulation, inflammation and wound healing. Suitable animal models that have the potential to mimic human platelet function are limited. The objective of the current study was to compare platelet aggregation response in the whole blood of baboons and humans using impedance aggregometry.METHODS: Blood was drawn from 24 anesthetised male baboons and 25 healthy volunteers. The platelet aggregation response was determined by impedance aggregometry (Multiplate®). Platelets in the hirudinised whole blood samples were stimulated with four different activators: adenosine diphosphate (ADP), collagen (COL), thrombin receptor activating peptide-6 (TR1AP), and activation of PAR-4 thrombin receptor subtype (TR4AP) at standard concentrations. Higher than standard concentrations were tested in a subgroup of the animals.RESULTS: The cell counts showed no differences between baboons and humans. The platelet aggregation response was significantly lower in baboons compared to humans when stimulated with the platelet agonists ADP (pCONCLUSION: The current study revealed that testing the platelet function in baboon blood by impedance aggregometry is feasible with ADP, COL and TR4AP, but not with TR1AP. Compared to humans, the aggregation response is lower in baboons. Considering the limitations in accordance to these results, baboons might represent a potential species for further platelet research.

INTRODUCTION AND HYPOTHESIS: Steroid soaking may decrease mesh-triggered inflammatory reaction in tissue. We aimed to investigate the tissue reaction to a steroid-soaked mesh material and an unsoaked mesh material in the rat model. METHODS: Neutral and steroid-soaked type I macroporous polypropylene (PP) monofilament and polyvinylidene fluoride (PVF) mesh materials were implanted on the rectus abdominis muscle of 20 mature Wistar albino rats. Animals were divided into four groups: PP mesh with steroid (PP-S), PP mesh without steroid, PVF mesh with steroid (PVF-S), and PVF mesh without steroid. The rats were killed after 12 weeks, and histologic, immunohistochemical and electron microscopic examinations were performed. For immunohistochemical analysis, polyclonal rabbit anti-mouse CD3, rabbit anti-mouse CD68, rabbit anti-mouse CD15, and rabbit anti-mouse CD34 antibodies were used for the detection of lymphocytes, macrophages, polymorphonuclear leukocyte foreign body giant cells, and fibromyocyte stem cells, respectively. Samples were stained with hematoxylin and eosin for the histologic evaluation of inflammation and with Masson's trichrome stain for the evaluation of collagen deposition. Pore size and mesh ultrastructure were evaluated by electron microscopy. RESULTS: Expression of CD3 was lower in the PVF, PVF-S and PP-S groups, and expression of CD34 was higher in the PVF-S and PP-S groups than in the PP groups (p < 0.05). Collagen deposition was lower in the PVF, PVF-S and PP-S groups (p < 0.05). Histologically, the intensity of inflammation was lower in the PVF-S and PP-S groups than in the PP mesh group (p < 0.05). There were no significant differences among the groups in terms of pore size and mesh ultrastructure on electron microscopic examination (p > 0.05). CONCLUSIONS: PVF mesh induces less inflammation than PP mesh, and in both mesh types steroid soaking further decreases inflammation without changing the pore size.

The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD). In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT) in the decorin (DCN) gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS), to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal.

Aims Anti-Apolipoprotein A-1 auto-antibodies (anti-ApoA-1 IgG) represent an emerging prognostic cardiovascular marker in patients with myocardial infarction or autoimmune diseases associated with high cardiovascular risk. The potential relationship between anti-ApoA-1 IgG and plaque vulnerability remains elusive. Thus, we aimed to investigate the role of anti-ApoA-1 IgG in plaque vulnerability. Methods and results Potential relationship between anti-ApoA-1 IgG and features of cardiovascular vulnerability was explored both in vivo and in vitro. In vivo , we investigated anti-ApoA-1 IgG in patients with severe carotid stenosis ( n = 102) and in ApoE−/− mice infused with polyclonal anti-ApoA-1 IgG. In vitro , anti-ApoA-1 IgG effects were assessed on human primary macrophages, monocytes, and neutrophils. Intraplaque collagen was decreased, while neutrophil and matrix metalloprotease (MMP)-9 content were increased in anti-ApoA-1 IgG-positive patients and anti-ApoA-1 IgG-treated mice when compared with corresponding controls. In mouse aortic roots (but not in abdominal aortas), treatment with anti-ApoA-1 IgG was associated with increased lesion size when compared with controls. In humans, serum anti-ApoA-1 IgG levels positively correlated with intraplaque macrophage, neutrophil, and MMP-9 content, and inversely with collagen. In vitro , anti-ApoA-1 IgG increased macrophage release of CCL2, CXCL8, and MMP-9, as well as neutrophil migration towards TNF-α or CXCL8. Conclusion These results suggest that anti-ApoA-1 IgG might be associated with increased atherosclerotic plaque vulnerability in humans and mice.

Corneal collagen crosslinking (CXL) with riboflavin and ultraviolet-A light is a method for treating progressive keratectasia. The currently accepted treatment parameters induce collagen crosslinking in the anterior 250 to 350 microm of corneal stroma. To protect the endothelium, CXL inclusion criteria require a minimum corneal thickness of 400 microm after removal of the epithelium. In advanced keratoconus, however, progressive corneal thinning often leads to a remaining stromal thickness of less than 400 microm. We have therefore modified the current treatment protocol by preoperatively swelling thin corneas to a stromal thickness of at least 400 microm using hypoosmolar riboflavin solution. This treatment protocol was performed in a case series of 20 patients, and no complications were observed. Preoperative swelling of the cornea safely broadens the spectrum of CXL indications to thin corneas that would otherwise not be eligible for treatment.

http://hdl.handle.net/20.500.11768/96914 A 32-year-old man with keratoconus developed corneal melting 5 days after riboflavin/ultraviolet-A corneal collagen crosslinking (CXL). Corneal scraping was positive for Acanthamoeba. The patient was unaware that he was wearing a bandage contact lens and repeatedly rinsed his face and eyelids with tap water. Because of corneal perforation, a large therapeutic keratoplasty a chaud was performed. Although CXL is considered a safe procedure, this case emphasizes the potential risks. We discuss the potential effects of deepithelialization, contact lens placement, instillation of topical nonsteroidal antiinflammatory drugs and anesthetic agents, and the possible role of apoptosis when performing CXL treatment for keratoconus.

Background— Atherosclerosis is a chronic inflammatory disease that represents the primary cause of death through coronary disease and stroke. Chemokines are known to play a crucial role in this disease by recruiting inflammatory leukocytes to the endothelium. Recently, the chemokine variant [ 44 AANA 47 ]-RANTES was shown to impair inflammatory cell recruitment in vivo by interfering with heparin binding and oligomerization. Methods and Results— In this study we report that curative treatment with [ 44 AANA 47 ]-RANTES limits atherosclerotic plaque formation in LDLr −/− mice. This was associated with reduced infiltration of T cells and macrophages and reduced production of matrix metalloproteinase (MMP)-9. By contrast, the relative smooth muscle cell and collagen content was increased, indicating a more stable plaque phenotype. In addition, we provide evidence for direct inhibition of leukocyte recruitment into aortic root lesions, attenuated leukocyte rolling and arrest in mesenteric vessels, as well as a reduced proinflammatory response following Con A stimulation in vitro. Conclusions— Interference with chemokine oligomerization and chemokine/heparin interactions is a powerful novel approach that inhibits progression of established atherosclerosis in mice. By inhibiting leukocyte recruitment into plaques, [ 44 AANA 47 ]-RANTES mediates a less inflammatory plaque phenotype and thus reduced systemic inflammatory state.

Understanding vascular pathologies requires insight in the structure and function, and, hence, an imaging technique combining subcellular resolution, large penetration depth, and optical sectioning. We evaluated the applicability of two-photon laser-scanning microscopy (TPLSM) in large elastic and small muscular arteries under physiological conditions. Elastic (carotid) and muscular (uterine, mesenteric) arteries of C57BL/6 mice were mounted in a perfusion chamber. TPLSM was used to assess the viability of arteries and to visualize the structural components elastin, collagen, nuclei, and endothelial glycocalyx (EG). Functionality was determined using diameter changes in response to noradrenaline and acetylcholine. Viability and functionality were maintained up to 4 h, enabling the assessment of structure-function relationships. Structural vessel wall components differed between elastic and muscular arteries: size (1.3 vs. 2.1 microm) and density (0.045 vs. 0.57 microm(-2)) of internal elastic lamina fenestrae, smooth muscle cell density (3.50 vs. 1.53 microm(-3)), number of elastic laminae (3 vs. 2), and adventitial collagen structure (tortuous vs. straight). EG in elastic arteries was 4.5 microm thick, covering 66% of the endothelial surface. TPLSM enables visualization and quantification of subcellular structures in vital and functional elastic and muscular murine arteries, allowing unraveling of structure-function relationships in healthy and diseased arteries.

The tumor suppressor protein BARD1, originally discovered as BRCA1-binding protein, acts in conjunction with BRCA1 as ubiquitin ligase. BARD1 and BRCA1 form a stable heterodimer and dimerization, which is required for most tumor suppressor functions attributed to BRCA1. In addition, BARD1 has BRCA1-independent functions in apoptosis, and a role in control of tissue homeostasis was suggested. However, cancer-associated mutations of BARD1 are rare; on the contrary, overexpression of truncated BARD1 was found in breast and ovarian cancer and correlated with poor prognosis. Here we report that human cytotrophoblasts, which show a strong similarity with cancer cells in respect of their invasive behavior and capacity of matrix metalloprotease production, overexpress isoforms of BARD1 derived from differential splicing. We demonstrate that expression of BARD1 and its isoforms is temporally and spatially regulated by human chorionic gonadotropin and by hypoxia, both factors known to regulate the invasive phase and proliferation of cytotrophoblasts. Interestingly, we found a subset of BARD1 isoforms secreted by cytotrophoblasts. BARD1 repression by siRNAs, mitigates the interference of cytotrophoblasts with cell adhesion of collagen matrix-dependent epithelial cells, suggesting a role of BARD1 isoforms in extracellular matrix remodelling and in cytotrophoblasts invasion.

The use of nonablative thermal energy to shrink soft-tissue collagen results in ultrastructural and mechanical changes at temperatures 60 °C or above. Due to this effect, the fibrils undergo shortening and shrinkage. Arthroscopic thermal capsulorrhaphy has been used in the treatment of shoulder instabilities and posterior impingement syndrome; in particular, the presence of a Bankart lesion or a superior labral anterior posterior lesion requires a labral or capsulolabral repair. Despite ease of application, thermal techniques have higher complication rates, with no proven superiority over traditional suture techniques. Further studies are required to develop the most appropriate technique for tissue shrinkage without any associated tissue destruction. The mechanical properties and long-term durability of the newly produced collagen need to be analyzed, as well., Yumuşak doku kollajenini büzüştürmek için kullanılan termal enerji, 60 °C ve üzerinde dokuda yapısal ve mekanik değişiklikler meydana getirir. Bu etkiyle fibriller kısalmakta ve büzüşmektedir. Artroskopik termal kapsülorafi, tekyönlü ve çokyönlü omuz instabiliteleri ile posterior internal sıkışma sendromunun tedavisinde kullanılmaktadır. Bankart veya süperiorlabrum anterior posterior lezyonunun varlığı labral veya kapsülolabral tamiri gerektirir. Termal tekniğin kolay olmasına karşın komplikasyon oranı yüksektir ve geleneksel dikiş tekniklerine üstünlüğü gösterilmemiştir. Doku zararı oluşturmaksızın, dokuda büzüştürme oluşturabilecek en uygun yöntemi geliştirmek için ileri çalışmalara ihtiyaç vardır. Ayrıca, yeni oluşan kollajenin mekanik özellikleri ve uzun dönem dayanıklılığı da araştırılmalıdır.

PURPOSE: To investigate the effect of fluorescein on the antimicrobial efficacy of photoactivated riboflavin in corneal collagen cross-linking (CXL) in vitro. METHODS: The ultraviolet light-A (UVA) absorption by fluorescein and riboflavin in different concentrations was analyzed with a spectrophotometer. The killing rate of Staphylococcus aureus strains after CXL with UV-A irradiation using different solutions containing riboflavin and/or fluorescein, was evaluated in vitro. RESULTS: Fluorescein absorbed UV-A to a significant extent, which augmented with increasing concentration. Moreover, addition of fluorescein to the riboflavin solution contributed to a significant reduction of the killing rate of S. aureus strains after 30 minutes of UV-A irradiation in vitro with a killing rate of 85%, whereas it was 47% in the presence of 2% fluorescein ( P = .0247). CONCLUSIONS: The results indicate that fluorescein competes with riboflavin for the absorption of UV-A during CXL and reduces the antimicrobial effect of the procedure. The authors recommend that physicians treating infectious ulcerative keratitis by CXL should not stain the cornea with fluorescein for visualization of the corneal ulceration prior to CXL. [ J Refract Surg. 2013:29(12):842–845.]

PURPOSE: To evaluate the need for and quantify the extent of nomogram adjustments to compensate for potential changes in the amount of effective corneal stroma ablated in previously cross-linked corneas. METHODS: Ex vivo porcine corneas were divided into two groups (the corneal cross-linking [CXL] group, n = 30; and the control group, n = 3): these experimental corneas underwent CXL including deepithelialization, instillation of riboflavin solution for 25 minutes, and ultraviolet-A irradiation at 9 mW/cm 2 for 10 minutes. The control group was deepithelialized only. Four consecutive excimer laser ablations of 50 µ m each were performed (AMARIS 750S; SCHWIND eye-tech-solutions, Kleinostheim Germany), and stromal bed thickness was measured with a built-in optical coherence pachymeter. To determine the potential influence of riboflavin, a third group (the riboflavin group, n = 12) underwent deepithelialization and instillation of riboflavin, but no ultraviolet-A irradiation. RESULTS: The mean individual ablation depth across the four ablations was significantly smaller in cross-linked corneas (−17%) when compared to untreated control corneas ( P < .001). A consistent reduction of 12% was observed via a cumulative analysis when assessing the relative isolated effect of CXL on the ablation rate. There was no significant effect from riboflavin in the deeper ablations, except for the first ablation (68.6 ± 1.1 mm [range: 1 to 50 µ m]). This may be due to a measurement error in pachymetric readings due to the thin film of riboflavin on the surface that resists even extensive rinsing. CONCLUSIONS: CXL reduces the corneal ablation depth of excimer lasers in the anterior 200 µ m of the porcine cornea by approximately 12%. Further clinical studies are needed to validate these findings in human corneas. [ J Refract Surg . 2014;30(9):628–632.]

PURPOSE: To report the use of accelerated photoactivated chromophore for keratitis–corneal collagen cross-linking (PACK-CXL) as a first-line treatment in a patient with an atypical fungal keratitis. METHODS: Case report and literature review. RESULTS: A patient who presented with a painful peripheral corneal infiltrate underwent PACK-CXL with a local limited abrasion and accelerated ultraviolet-A irradiation at 365 μm and 9 mW/cm 2 for 10 minutes. Cultures grew Aureobasidium pullulans . The corneal epithelium closed completely within 3 days and the infiltrate was completely eradicated without administration of antibiotics. CONCLUSIONS: Accelerated PACK-CXL was successfully used as a first-line and sole treatment in a case of early fungal keratitis caused by Aureobasidium pullulans . Further characterization of the antifungal effect of PACK-CXL is needed in prospective studies. [ J Refract Surg . 2014;30(12):855–857.]

Here we integrated multiphoton laser scanning microscopy and the registration of second harmonic generation images of collagen fibers to overcome difficulties in tracking stromal cell-matrix interactions for several days in live mice. We show that the matrix-modifying hormone relaxin increased tumor-associated fibroblast (TAF) interaction with collagen fibers by stimulating beta1-integrin activity, which is necessary for fiber remodeling by matrix metalloproteinases.

BACKGROUND: To ascertain whether changes in proteoglycans are involved in the pathogenesis of the nephrotic syndrome in Denys-Drash syndrome (DDS), we analysed the glycosaminoglycan (GAG) content and composition of the glomerular basement membrane (GBM) in one child with this disorder and in children of comparable age who had died from unrelated disorders.METHODS: The diagnosis of DDS was confirmed by the presence of a previously described mutation in the WT1 gene (a tumour suppressor gene). The GAG content and composition of the GBM and tubular basement membrane (TBM), both in the Denys-Drash patient as well as age-matched control infants, was analysed by biochemical studies and indirect immunofluorescence studies. Finally we investigated the urinary GAG excretion of the Drash patient.RESULTS: The biochemical studies revealed that the total GAG content in the GBM as well as TBM was comparable in the Drash patient and the control group. However, the GAG composition of the GBM of the patient was clearly different, with relatively more chondroitin sulphate. The urinary GAG content (expressed as mg GAG/mmol creatinine) was elevated in the Denys-Drash patient due to an increased heparan sulphate (HS(GAG)) excretion. Indirect immunofluorescence (IF) studies for the core protein of human GBM heparan sulphate proteoglycan (HSPG) showed a similar linear staining of all renal basement membranes in the patient and the controls. A monoclonal antibody directed against the HS chain of HSPG (MoAb 403) displayed a strong GBM and a weak TBM staining of normal kidneys. Kidney tissue from the Drash patient displayed a reduced staining of the GBM with MoAb 403. IF studies for chondroitin sulphate proteoglycan (CSPG) showed increased staining of the mesangium and glomerular capillary loops in the Denys-Drash patient which is in agreement with the biochemical studies. No discernible differences in distribution or quality of staining with antibodies against collagen type IV and laminin were observed.CONCLUSIONS: These biochemical and immunohistochemical results indicate that in our patient the proteoglycan composition of the GBM is altered. This alteration may play a role in the pathogenesis of proteinuria in this syndrome.

PURPOSE: To report a patient who developed a left paracentral stromal scar due to infectious keratitis that occurred after corneal collagen cross-linking (CXL) for progressive keratoconus. The flattening effect of the scar led to an increase in visual acuity. METHODS: The corneal scar and flattening effect on the anterior corneal curvature were assessed by slit-lamp photography, high-resolution Scheimpflug imaging, and corneal confocal microscopy. RESULTS: Three days after CXL, a corneal bacterial infection occurred in the left cornea and was treated with local antibiotics that led to a paracentral scar. Twenty-one days after CXL, a flattening of the anterior curvature of >11.00 diopters was observed. As a consequence, corrected distance visual acuity improved by five lines. CONCLUSIONS: Corneal remodeling may lead to a homogenization of the anterior corneal surface and an increase in visual acuity. Remodeling may not only occur spontaneously following CXL, but also following an event that results in focal corneal scarring, such as corneal infection. In a highly irregular keratoconic cornea, the benefit of the flattening effect of a scar may outweigh the increase in aberrations and light scatter.

Diabetic nephropathy is characterized by albuminuria which proceeds to overt proteinuria. The highly negatively stained HS side chain of heparan sulphate proteoglycan (HSPG) is a major determinant of the charge-dependent permeability of the GBM. We set out to study the presence of HS and HSPG in the GBM of patients with diabetic nephropathy using newly developed monoclonal antibodies, and to compare HSPG expression to the expression of other previously investigated glomerular extracellular matrix compounds. Immunohistochemically, glomerular extracellular matrix components were analysed in 14 renal biopsies of patients with diabetic nephropathy and compared with those of normal control subjects. Monoclonal antibodies used were: JM403 against the HS side chain of GBM HSPG and JM72 against the HSPG-core protein. Also, a polyclonal antiserum (B31) against human GBM-HSPG-core protein was used. Additionally, antibodies were used against collagen types I, III, IV and against alpha 1 (IV)NC, alpha 3(IV)NC and fibronectin. Staining was scored for intensity and for staining pattern by four independent observers who had no previous knowledge of the sample origin. No glomerular staining was seen for collagen type I. Collagen type III was present in some diabetic nodules. Anti-collagen type IV showed a decreased GBM staining in patients with diabetic nephropathy (p = 0.04). With anti-alpha 1 (IV)NC no changes in GBM staining intensity were observed; with anti-alpha 3 (IV)NC brilliant GBM staining was seen in both groups. Increased mesangial staining (p = 0.003) was seen with anti-collagen type IV in biopsies with nodular lesions.(ABSTRACT TRUNCATED AT 250 WORDS)