Merosin Research Articles

Fractone Stem Cell Niche Components Provide Intuitive Clues in the Design of New Therapeutic Procedures/Biomatrices for Neural Repair.

The aim of this study was to illustrate recent developments in neural repair utilizing hyaluronan as a carrier of olfactory bulb stem cells and in new bioscaffolds to promote neural repair. Hyaluronan interacts with brain hyalectan proteoglycans in protective structures around neurons in perineuronal nets, which also have roles in the synaptic plasticity and development of neuronal cognitive properties. Specialist stem cell niches termed fractones located in the sub-ventricular and sub-granular regions of the dentate gyrus of the hippocampus migrate to the olfactory bulb, which acts as a reserve of neuroprogenitor cells in the adult brain. The extracellular matrix associated with the fractone stem cell niche contains hyaluronan, perlecan and laminin α5, which regulate the quiescent recycling of stem cells and also provide a means of escaping to undergo the proliferation and differentiation to a pluripotent migratory progenitor cell type that can participate in repair processes in neural tissues. Significant improvement in the repair of spinal cord injury and brain trauma has been reported using this approach. FGF-2 sequestered by perlecan in the neuroprogenitor niche environment aids in these processes. Therapeutic procedures have been developed using olfactory ensheathing stem cells and hyaluronan as a carrier to promote neural repair processes. Now that recombinant perlecan domain I and domain V are available, strategies may also be expected in the near future using these to further promote neural repair strategies.

Prenatal E‐Cigarette Use Disrupts Blood‐Brain Barrier (BBB) Integrity and Induces Pro‐Inflammatory Cytokines in Postnatal Brain

PurposeElectronic cigarette (e‐Cig) is often considered as a safer alternative to smoking for pregnant women however, its potential toxic effect on neonatal health warrants further investigation. Toxic effects of prenatal tobacco smoking on postnatal health have been reported in several studies however, regarding thorough investigation of e‐Cig exposure during pregnancy is currently unavailable. Due to the staggering growth in e‐Cig usage among pregnant women (10%), it has become critical to explore the consequences of maternal e‐cig use on postnatal health outcomes. Hence, in this study, we have evaluated the consequences of prenatal e‐Cig use during pregnancy on postnatal blood‐brain barrier (BBB) integrity, proinflammatory cytokine levels and behavioral outcomes in offspring.MethodPregnant CD1 mice (E5) were exposed to e‐Cig vapor (2.4% nicotine) till postnatal day (PD) 7. Weight of the offspring was measured up to PD 90. Nicotine and cotinine concentration in plasma and brain were measured in PD 7 offspring by LC‐MS/MS. The expression level of structural elements of the BBB including, tight junction proteins (ZO‐1, claudin‐5, occludin), astrocyte (GFAP), pericyte (PDGFRβ) and basement membrane (Laminin α1, Laminin α4) were analyzed in offspring using western blot and immunohistochemistry at PD 7, PD 23, PD 45 and PD 90. The expression level of pro‐inflammatory cytokines (IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, IL‐12, IL‐17α, KC, LIX, MIP‐2 and TNF‐α) in brain were measured at PD 7 and PD 90. To evaluate behavioral outcomes, open field test, novel object recognition test and morris water maze test were performed on adolescent and adult offspring.ResultsReduced body weight was observed in e‐Cig exposed offspring at all time points till PD 90 compared to control (P <0.05). Plasma concentration of nicotine and cotinine in PD 7 offspring was 3.215 ng/mL and 3.373 ng/mL respectively whereas in brain, these two concentrations were 43.72 ng/mL and 1.123 ng/mL respectively. Significantly reduced expression of brain tight junction proteins and GFAP were observed in male and female offspring at PD 7, PD 23, PD 45 and PD 90 (P <0.05). Moreover, higher level of brain IL‐1α, IL‐6, IL‐7, IL‐10, IL‐17α, KC and TNF‐α was found in prenatally e‐cig exposed offspring at PD 7. However, only IL‐6 and IL‐17α were observed at higher levels in brain for prenatally e‐cig exposed adult offspring at PD 90 (P <0.05). Additionally, disrupted motor, learning and memory function were observed in prenatally e‐Cig exposed adolescent and adult offspring.ConclusionOur findings suggest that prenatal e‐Cig exposure induces potential neurotoxic effects on neonates by disrupting postnatal blood‐brain barrier integrity and inducing proinflammatory brain cytokines which may result in alteration in neurological, cerebrovascular, and behavioral outcomes.

Defining the cooperation between T cell responses to MHC-I and MHC-II melanoma neoantigens towards developing effective personalized cancer immunotherapies.

Abstract Immune checkpoint therapy (ICT) (e.g. anti-CTLA-4, anti-PD-1) enables durable T-cell dependent anti-tumor immunity in patients with solid tumors. Since not all patients respond to ICT, this work aims at developing a more in-depth understanding of T-cell responses to MHC class I (MHC-I) and MHC class II (MHC-II) tumor antigens that occur as a consequence of aberrant expression of non-mutant antigens or driver and passenger mutations that form tumor neoantigens. We used a poorly immunogenic Brafv600e Pten−/−Cdkn2a−/− YUMM1.7 (Y1.7) murine melanoma line that with a paucity of endogenous neoantigens that is unresponsive to ICT, and introduced model neoantigens previously identified in a murine sarcoma tumor line expressing the same MHC haplotype as the Y1.7 line. The MHC-I neoantigen used was formed by a G1254V point mutation in the Laminin alpha subunit-4 (Lama4) and the MHC-II neoantigen was formed by an A710T point mutation in Integrin beta-1 (Itgb1). Anti-CTLA-4-treated mice bearing the parental Y1.7 melanoma displayed progressive tumor growth, whereas the modified Y1.7 line expressing mLama4 and mItgb1 (Y1.7.mLama4.mItgb1) was rendered sensitive to ICT. Preliminary data showed that anti-CTLA-4 treated Y1.7.mLama4.mItgb1 tumors reject in mice in a CD4+ T cell and CD8+ T cell-dependent manner. Additionally, intratumoral mLama4-specific CD8+ T cells were detected in anti-CTLA-4-treated Y1.7.mLama4.mItgb1 melanoma bearing mice. Overall this model will allow us to better understand T cell mediated anti-tumor responses to both MHC-I and MHC-II neoantigens, as well as shared, non-mutant melanoma antigens (e.g. Pmel/GP100 and Trp2), which may facilitate development of improved neoantigen-based personalized immunotherapies.

Laminin N-terminus α31 is upregulated in invasive ductal breast cancer and changes the mode of tumour invasion.

Laminin N-terminus α31 (LaNt α31) is an alternative splice isoform derived from the laminin α3 gene. The LaNt α31 protein is enriched around the terminal duct lobular units in normal breast tissue. In the skin and cornea the protein influences epithelial cell migration and tissue remodelling. However, LaNt α31 has never been investigated in a tumour environment. Here we analysed LaNt α31 in invasive ductal carcinoma and determined its contribution to breast carcinoma invasion. LaNt α31 expression and distribution were analysed by immunohistochemistry in human breast tissue biopsy sections and tissue microarrays covering 232 breast cancer samples. This analysis revealed LaNt α31 to be upregulated in 56% of invasive ductal carcinoma specimens compared with matched normal tissue, and further increased in nodal metastasis compared with the tumour mass in 45% of samples. 65.8% of triple negative cases displayed medium to high LaNt α31 expression. To study LaNt α31 function, an adenoviral system was used to induce expression in MCF-7 and MDA-MB-231 cells. 2D cell migration and invasion into collagen hydrogels were not significantly different between LaNt α31 overexpressing cells and control treated cells. However, LaNt α31 overexpression reduced the proliferation rate of MCF-7 and MDA-MB-231 cells. Moreover, LaNt α31 overexpressing MDA-MB-231 cells displayed a striking change in their mode of invasion into laminin-containing Matrigel; changing from multicellular streaming to individual cellular-invasion. In agreement with these results, 66.7% of the tumours with the highest LaNt α31 expression were non-cohesive. Together these findings indicate that breast cancer-associated changes in LaNt α31 expression could contribute to the processes involved in tumour invasion and may represent a new therapeutic target.

Superoxide Dismutase-3 Downregulates Laminin α5 Expression in Tumor Endothelial Cells via the Inhibition of Nuclear Factor Kappa B Signaling.

Simple SummaryTumor-infiltrating lymphocytes determine cancer prognosis and the response to immunotherapy. The balance of laminin-α4/laminin-α5 isoforms in the endothelial basement membrane plays an instructive function in regulating the diapedesis of immune cells under inflammatory conditions. Previous studies showed that the extracellular superoxide dismutase (SOD)3 induces laminin-α4, which is associated with improved disease-free survival of colorectal cancer patients. The aim of our study was to determine whether SOD3 also affects the expression of laminin-α5 in the tumor vasculature. The results showed that SOD3 differentially regulates laminin-α4 and laminin-α5 in the tumor endothelium. SOD3 promoted notable transcriptomic changes in tumor-stimulated endothelial cells, including the inhibition of the nuclear factor kappa B (NF-κB) pathway, an inductor of laminin α5 transcription. Therefore, high SOD3 levels in the tumor vasculature shifted the laminin α4/α5 balance towards the laminin-α4high/laminin-α5low phenotype, which is permissive for T cell diapedesis into tumors and explains the improved cancer immune surveillance associated to high SOD3 levels.The balance between laminin isoforms containing the α5 or the α4 chain in the endothelial basement membrane determines the site of leukocyte diapedesis under inflammatory conditions. Extracellular superoxide dismutase (SOD3) induces laminin α4 expression in tumor blood vessels, which is associated with enhanced intratumor T cell infiltration in primary human cancers. We show now that SOD3 overexpression in neoplastic and endothelial cells (ECs) reduces laminin α5 in tumor blood vessels. SOD3 represses the laminin α5 gene (LAMA5), but LAMA5 expression is not changed in SOD1-overexpressing cells. Transcriptomic analyses revealed SOD3 overexpression to change the transcription of 1682 genes in ECs, with the canonical and non-canonical NF-κB pathways as the major SOD3 targets. Indeed, SOD3 reduced the transcription of well-known NF-κB target genes as well as NF-κB-driven promoter activity in ECs stimulated with tumor necrosis factor (TNF)-α, an NF-κB signaling inducer. SOD3 inhibited the phosphorylation and degradation of IκBα (nuclear factor of the kappa light polypeptide gene enhancer in B-cells inhibitor alpha), an NF-κB inhibitor. Finally, TNF-α was found to be a transcriptional activator of LAMA5 but not of LAMA4; LAMA5 induction was prevented by SOD3. In conclusion, SOD3 is a major regulator of laminin balance in the basement membrane of tumor ECs, with potential implications for immune cell infiltration into tumors.

Epithelial Basement Membrane Regeneration After PRK-Induced Epithelial-Stromal Injury in Rabbits: Fibrotic Versus Non-fibrotic Corneal Healing.

To study epithelial basement membrane (EBM) regeneration in non-fibrotic and fibrotic corneas after photorefractive keratectomy (PRK). Rabbits (120 total) had either epithelial scrape alone, -4.50 diopters (D) PRK, -9.00 D PRK, or no surgery. Immunohistochemistry was performed on cryofixed corneas at time points from unwounded to 8 weeks (four corneas at each time point in each group). Multiplex immunohistochemistry was performed for EBM components, including collagen type IV, laminin beta-3, laminin alpha-5, perlecan, and nidogen-1. Stromal cellular composition was studied by triplex immunohistochemistry for keratocan, vimentin, and alpha-smooth muscle actin (SMA). PRK-injured EBM significantly regenerated by 4 days after surgery. However, early TGF-beta-regulating perlecan incorporation into the nascent EBM declined 4 to 7 days after surgery in fibrotic corneas. Non-fibrotic corneas that had fully regenerated EBM (with all five components incorporated into the EBM) were transparent and had few SMA-positive myofibroblasts in the stroma. Conversely, corneas with defective nascent EBM that lacked perlecan developed many anterior stromal myofibroblasts and fibrosis at 3 to 4 weeks after surgery and had large amounts of collagen type IV in the nascent EBM and anterior stroma. Myofibroblasts synthesized perlecan but were incompetent to incorporate the heparin sulfate proteoglycan into the nascent EBM. Corneal transparency was restored over several months even in fibrotic corneas, and this was associated with a return of EBM perlecan, myofibroblast disappearance, and reabsorption of disordered extracellular matrix. Defective incorporation of perlecan into the regenerating EBM by subepithelial myofibroblasts, and likely their precursor cells, underlies the development and persistence of stromal fibrosis after PRK corneal injury. [J Refract Surg. 2022;38(1):50-60.].

Royal Jelly Extract Accelerates Keratinocyte Proliferation, and Upregulates Laminin <i>α</i>3 and Integrin <i>β</i>1 mRNA Expression, via Akt/mTOR/HIF-1<i>α</i> Pathway

Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract (RJ) on keratinocytes have not been fully elucidated. Objective: The primary objectives of this study were to reveal the effects of RJ on keratinocytes and explore the underlying mechanism. Methods: HaCaT cells, an immortal human epidermis-derived keratinocyte cell line, were used in this study. Laminin α3 (LAMA3), integrin β1 (ITGB1), and hypoxia-inducible factor-1α (HIF-1α) mRNA expression levels were determined using real-time PCR. Cell proliferation rate was measured using a bromodeoxyuridine uptake assay. Results: RJ treatment upregulated LAMA3, ITGB1 and HIF-1α mRNA expression, and accelerated HaCaT cell proliferation. Akt and mTOR inhibitors suppressed the RJ-induced HIF-1α expression and cell proliferation. HIF-1α silencing abrogated RJ-induced LAMA3 and ITGB1 mRNA expression and cell proliferation, whereas LAMA3 silencing and antibody-mediated ITGB1 blockade did not affect the effects of RJ. Conclusion: RJ upregulates LAMA3 and ITGB1 mRNA expression levels by HIF-1α expression enhancement. In addition, RJ accelerates keratinocyte proliferation via Akt/mTOR/HIF-1α/NF-κB signaling pathway. These suggest that RJ is beneficial for anti-aging, as a skin care product ingredient.

Critical role of Lama4 for hematopoiesis regeneration and acute myeloid leukemia progression

AbstractImpairment of normal hematopoiesis and leukemia progression are 2 well-linked processes during leukemia development and are controlled by the bone marrow (BM) niche. Extracellular matrix proteins, including laminin, are important BM niche components. However, their role in hematopoiesis regeneration and leukemia is unknown. Laminin α4 (Lama4), a major receptor-binding chain of several laminins, is altered in BM niches in mice with acute myeloid leukemia (AML). So far, the impact of Lama4 on leukemia progression remains unknown. We here report that Lama4 deletion in mice resulted in impaired hematopoiesis regeneration following irradiation-induced stress, which is accompanied by altered BM niche composition and inflammation. Importantly, in a transplantation-induced MLL-AF9 AML mouse model, we demonstrate accelerated AML progression and relapse in Lama4−/− mice. Upon AML exposure, Lama4−/− mesenchymal stem cells (MSCs) exhibited dramatic molecular alterations, including upregulation of inflammatory cytokines that favor AML growth. Lama4−/− MSCs displayed increased antioxidant activities and promoted AML stem cell proliferation and chemoresistance to cytarabine, which was accompanied by increased mitochondrial transfer from the MSCs to AML cells and reduced reactive oxygen species in AML cells in vitro. Similarly, we detected lower levels of reactive oxygen species in AML cells from Lama4−/− mice post–cytarabine treatment. Notably, LAMA4 inhibition or knockdown in human MSCs promoted human AML cell proliferation and chemoprotection. Together, our study for the first time demonstrates the critical role of Lama4 in impeding AML progression and chemoresistance. Targeting Lama4 signaling pathways may offer potential new therapeutic options for AML.

Regulation and Function of Laminin A5 during Mouse and Human Decidualization.

Decidualization is essential to the establishment of pregnancy in rodents and primates. Laminin A5 (encoding by Laminin α5) is a member of the laminin family, which is mainly expressed in the basement membranes. Although laminins regulate cellular phenotype maintenance, adhesion, migration, growth, and differentiation, the expression, function, and regulation of laminin A5 during early pregnancy are still unknown. Therefore, we investigated the expression and role of laminin A5 during mouse and human decidualization. Laminin A5 is highly expressed in mouse decidua and artificially induced deciduoma. Laminin A5 is significantly increased under in vitro decidualization. Laminin A5 knockdown significantly inhibits the expression of Prl8a2, a marker for mouse decidualization. Progesterone stimulates the expression of laminin A5 in ovariectomized mouse uterus and cultured mouse stromal cells. We also show that progesterone regulates laminin A5 through the PKA-CREB-C/EBPβ pathway. Laminin A5 is also highly expressed in human pregnant decidua and cultured human endometrial stromal cells during in vitro decidualization. Laminin A5 knockdown by siRNA inhibits human in vitro decidualization. Collectively, our study reveals that laminin A5 may play a pivotal role during mouse and human decidualization via the PKA-CREB-C/EBPβ pathway.

Laminin Isoforms in Human Dental Pulp: Lymphatic Vessels Express Laminin-332, and Schwann Cell-Associated Laminin-211 Modulates CD163 Expression of M2-like Macrophages.

Laminin, a basement membrane heterotrimeric glycoprotein composed of α/β/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3β3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2-coated plates upregulated CD163 expression, compared with laminin-511/E8-α5-coated plates. Integrin α3- and integrin α6-neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin-laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

Clear Evidence of LAMA5 Gene Biallelic Truncating Variants Causing Infantile Nephrotic Syndrome.

Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid-resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only seven patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome, and one patient with SRNS with biallelic LAMA5 missense variants. We conducted comprehensive gene screening of Japanese patients with severe proteinuria. With the use of targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Biallelic truncating variants in the LAMA5 gene (NM_005560) were detected in three patients from two families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.[Arg3078*]) and a splice site variant (c.1282 + 1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.[Arg2720*]) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin β1 and γ1 chains. Patient 4 showed SRNS at the age of 8 years, and carried compound heterozygous missense variants (c.1493C>T, p.[Ala498Val] and c.8399G>A, p.[Arg2800His]). Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathologic characteristics observed in LAMA5-related nephropathy. LAMA5 variant screening should be performed in patients with congenital/infantile nephrotic syndrome.

Regeneration in the phylogenetic empire: an interview with Peter Currie.

Regeneration in the phylogenetic empire: an interview with Peter Currie.

Gene expression profiling of laminin α3-blocked keratinocytes reveals an immune-independent mechanism of blistering.

Laminin-332 pemphigoid is a rare and chronic autoimmune blistering disease which results in subepidermal blisters and erosive lesions predominantly localized to mucous membranes. As histologic inflammation is variable and non-complement-fixing IgG antibodies against laminin-332 are the predominant class of autoantibodies deposited at the epidermal basement membrane zone, we hypothesized that complement-independent pro-inflammatory and blistering pathways existed similarly to that previously shown in bullous pemphigoid. As autoantibodies to laminin α3 are most prevalent, we studied the major cellular response to blockade of laminin α3 using a well-characterized monoclonal antibody (P3H9-2). RNA-seq revealed upregulation of numerous desmosomal genes (DSG1, DSG3, DSC1, DSC3 and DSP) as well as KRT1 and KRT10. Additionally, P3H9-2-treated cells demonstrated downregulation of most hemidesmosomal genes. A pro-inflammatory response was not appreciated. Using pharmacological inhibitors, we identified both protein kinase C and NOTCH as key regulators of P3H9-2 induced differentiation. We lastly utilized 3D human skin equivalents to determine whether blockade of laminin α3 would lead to delayed blistering, consistent with keratinocyte differentiation. Significant blistering was noted after 72h of treatment, with only minimal separation at 24h. In summary, blockade of laminin α3 alters keratinocyte differentiation, representing a potential complement-independent mechanism of blistering.

A little bit of LOGIC goes a long way: Multidisciplinary management of LOGIC Syndrome‐associated oral manifestations

AbstractLOGIC syndrome (laryngeal and ocular granulation tissues in children from the Indian subcontinent), also known as Laryngo‐onycho‐cutaneous (LOC) syndrome, is a rare, autosomal recessive condition that is a subtype of junctional epidermolysis bullosa (JEB), primarily occurring in individuals of Punjabi ancestry. First observed by Shabbir et al in 1986, it has since been discovered that the syndrome is a result of autosomal recessive mutations in the LAMA3 gene. The clinical manifestations of the syndrome can be life threatening and are characterised by excessive granulation tissue deposition on the skin and mucous membranes including laryngeal, ocular and oral sites. Oral manifestations can drastically affect the quality of life. We present a case of LOGIC syndrome to highlight the importance of clinicians understanding the oral manifestations of the syndrome and implementing a multidisciplinary approach with good communication across specialities, including pathologists.

A novel model of nephrotic syndrome results from a point mutation in Lama5 and is modified by genetic background

Nephrotic syndrome is characterized by severe proteinuria, hypoalbuminaemia, edema and hyperlipidaemia. Genetic studies of nephrotic syndrome have led to the identification of proteins playing a crucial role in slit diaphragm signaling, regulation of actin cytoskeleton dynamics and cell-matrix interactions. The laminin α5 chain is essential for embryonic development and, in association with laminin β2 and laminin γ1, is a major component of the glomerular basement membrane, a critical component of the glomerular filtration barrier. Mutations in LAMA5 were recently identified in children with nephrotic syndrome. Here, we have identified a novel missense mutation (E884G) in the uncharacterized L4a domain of LAMA5 where homozygous mice develop nephrotic syndrome with severe proteinuria with histological and ultrastructural changes in the glomerulus mimicking the progression seen in most patients. The levels of LAMA5 are reduced in vivo and the assembly of the laminin 521 heterotrimer significantly reduced in vitro. Proteomic analysis of the glomerular extracellular fraction revealed changes in the matrix composition. Importantly, the genetic background of the mice had a significant effect on aspects of disease progression from proteinuria to changes in podocyte morphology. Thus, our novel model will provide insights into pathologic mechanisms of nephrotic syndrome and pathways that influence the response to a dysfunctional glomerular basement membrane that may be important in a range of kidney diseases.

The inclusion of leukocytes into platelet rich plasma reduces scaffold stability and hinders extracellular matrix remodelling.

BackgroundScaffolds should have controllable degradation rate and allow cells to produce their own extracellular matrix. Platelet rich plasma (PRP) is a source of autologous growth factors and proteins embedded in a 3D fibrin scaffold. There is no consensus regarding the obtaining conditions and composition of PRPs. The aim of this study was to evaluate how the inclusion of leukocytes (L-PRP) in plasma rich in growth factors (PRGF) may alter the process of fibrinolysis. The effect of different combinations of cellular phenotypes with PRGF and L-PRP clots on both the fibrinolysis and matrix deposition process was also determined. MethodsPRGF and L-PRP clots were incubated for 14 days and D-dimer and type I collagen were determined in their conditioned media to evaluate clots’ stability. For remodelling assays, gingival fibroblasts, alveolar osteoblasts and human umbilical vein endothelial cells (HUVEC) were seeded onto the two types of clots for 14 days. D-dimer, type I collagen, and laminin α4 were measured by ELISA kits in their conditioned media. Morphological and histological analysis were also performed. Cell proliferation was additionally determined ResultsPRGF clots preserved their stability as shown by the low levels of both D-dimer and collagen type I compared to those obtained for L-PRP clots. The inclusion of both gingival fibroblasts and alveolar osteoblasts stimulated a higher fibrinolysis in the PRGF clots. In contrast to this, the degradation rates of both PRGF and L-PRP clots remained unchanged after culturing with the endothelial cells. In all cases, type I collagen and laminin α4 levels were in line with the degree of clots’ degradation. In all phenotypes, cell proliferation was significantly higher in PRGF than in L-PRP clots. ConclusionThe inclusion of leukocytes in PRGF scaffolds reduced their stability, decreased cell number and slowed down cell remodelling.

Descemet's membrane injury and regeneration, and posterior corneal fibrosis, in rabbits

The purpose of this investigation was to study Descemet's membrane and corneal endothelial regeneration, myofibroblast generation and disappearance, and TGF beta-1 localization after Descemet's membrane-endothelial excision (Descemetorhexis) in rabbits. Thirty-six rabbits had 8 mm Descemetorhexis and standardized slit lamp photos at 1, 2 and 4 days, 1, 2 and 4 weeks, and 2, 4 and 6 months, as well as multiplex IHC for stromal cell markers keratocan, vimentin, and alpha-smooth muscle actin (SMA); basement membrane (BM) components perlecan, nidogen-1, laminin alpha-5, and collagen type IV; and corneal endothelial marker Na,K-ATPase β1, and TGF beta-1, with ImageJ quantitation. Stromal transparency increased from the periphery beginning at two months after injury and progressed into the central cornea by six months. At six months, central transparency was primarily limited by persistent mid-stromal neovascularization. Stromal myofibroblast zone thickness in the posterior stroma peaked at one month after injury, and then progressively decreased until to six months when few myofibroblasts remained. The regeneration of a laminin alpha-5 and nidogen-1 Descemet's membrane “railroad track” structure was accompanied by corneal endothelial closure and stromal cell production of BM components in corneas from four to six months after injury. TGF beta-1 deposition at the posterior corneal surface from the aqueous humor peaked at one day after Descemetorhexis and diminished even before regeneration of the endothelium and Descemet's membrane. This decrease was associated with collagen type IV protein production by corneal fibroblasts, and possibly myofibroblasts, in the posterior stroma. Descemet's membrane and the corneal endothelium regenerated in the rabbit cornea by six months after eight mm Descemetorhexis. Real-time quantitative RT-PCR experiments in vitro with marker-verified rabbit corneal cells found that 5 ng/ml or 10 ng/ml TGF beta-1 upregulated col4a1 or col4a2 mRNA expression after 6 h or 12 h of exposure in corneal fibroblasts, but not in myofibroblasts. Stromal cells produced large amounts of collagen type IV that likely decreased TGF beta-1 penetration into the stroma and facilitated the resolution of myofibroblast-generated fibrosis.

A Mosaic Mutation in the LAMA2 Gene in a Case of Merosin-deficient Congenital Muscular Dystrophy

Merosine deficient congenital muscular dystrophy is one of the most common forms of congenital muscular dystrophy. This disease is caused by a primary deficiency or a functionally inactive form of the protein merosin in muscle tissue. The type of inheritance of this disease is autosomal recessive. De novo variants with this type of inheritance are rare, and it is quite possible that the de novo variant may hide a mosaic form in the parent of an affected child. We present a birth family with two affected children who inherited a previously undescribed pathogenic variant c.1755del from their mother and a previously described pathogenic variant c.9253C > T in the LAMA2 gene from their mosaic father. LAMA2 gene mutation analysis was performed by mass parallel sequencing and direct sequencing of genomic DNAs.

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Lysosomes and the pathogenesis of merosin-deficient congenital muscular dystrophy.

Congenital muscular dystrophy type 1A (MDC1A), the most common congenital muscular dystrophy in Western countries, is caused by recessive mutations in LAMA2, the gene encoding laminin alpha 2. Currently, no cure or disease modifying therapy has been successfully developed for MDC1A. Examination of patient muscle biopsies revealed altered distribution of lysosomes. We hypothesized that this redistribution was a novel and potentially druggable aspect of disease pathogenesis. We explored this hypothesis using candyfloss (caf), a zebrafish model of MDC1A. We found that lysosome distribution in caf zebrafish was also abnormal. This altered localization was significantly associated with fiber detachment and could be prevented by blocking myofiber detachment. Overexpression of transcription factor EB, a transcription factor that promotes lysosomal biogenesis, led to increased lysosome content and decreased fiber detachment. We conclude that genetic manipulation of the lysosomal compartment is able to alter the caf zebrafish disease process, suggesting that lysosome function may be a target for disease modification.