Histological and Immunohistochemical Studies to Determine the Mechanism of Cleft Palate Induction after Palatal Fusion in Mice Exposed to TCDD.

Rupture of the basement membrane in fused palate tissue can cause the palate to separate after fusion in mice, leading to the development of cleft palate. Here, we further elucidate the mechanism of palatal separation after palatal fusion in 8-10-week-old ICR female mice. On day 12 of gestation, 40 µg/kg of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), sufficient to cause cleft palate in 100% of mice, was dissolved in 0.4 mL of olive oil containing toluene and administered as a single dose via a gastric tube. Fetal palatine frontal sections were observed by H&E staining, and epithelial cell adhesion factors, apoptosis, and cell proliferation were observed from the anterior to posterior palate. TUNEL-positive cells and Ki67-positive cells were observed around the posterior palatal dissection area of the TCDD-treated group. Moreover, in fetal mice exposed to TCDD, some fetuses exhibited cleft palate dehiscence during fusion. The results suggest that palatal dehiscence may be caused by abnormal cell proliferation in epithelial tissues, decreased intercellular adhesion, and inhibition of mesenchymal cell proliferation. By elucidating the mechanism of cleavage after palatal fusion, this research can contribute to establishing methods for the prevention of cleft palate development.

Chemotherapy-Induced Changes in the Lung Microenvironment: The Role of MMP-2 in Facilitating Intravascular Arrest of Breast Cancer Cells.

Previously, we showed that mice treated with cyclophosphamide (CTX) 4 days before intravenous injection of breast cancer cells had more cancer cells in the lung at 3 h after cancer injection than control counterparts without CTX. At 4 days after its injection, CTX is already excreted from the mice, allowing this pre-treatment design to reveal how CTX may modify the lung environment to indirectly affect cancer cells. In this study, we tested the hypothesis that the increase in cancer cell abundance at 3 h by CTX is due to an increase in the adhesiveness of vascular wall for cancer cells. Our data from protein array analysis and inhibition approach combined with in vitro and in vivo assays support the following two-prong mechanism. (1) CTX increases vascular permeability, resulting in the exposure of the basement membrane (BM). (2) CTX increases the level of matrix metalloproteinase-2 (MMP-2) in mouse serum, which remodels the BM and is functionally important for CTX to increase cancer abundance at this early stage. The combined effect of these two processes is the increased accessibility of critical protein domains in the BM, resulting in higher vascular adhesiveness for cancer cells to adhere. The critical protein domains in the vascular microenvironment are RGD and YISGR domains, whose known binding partners on cancer cells are integrin dimers and laminin receptor, respectively.

Role of albumin on endothelial basement membrane and hemostasis in a rat model of hemorrhagic shock.

BACKGROUND: We sought to determine the extent of loss of endothelial basement membrane (BM), leukocyte recruitment, and changes in coagulation after hemorrhagic shock, followed by limited-volume resuscitation (LVR) with 5% albumin (ALB). METHODS: Anesthetized rats were bled 40% of blood volume and assigned to treatment groups: untreated (n = 6), LVR with normal saline (NS; n = 8), or LVR with ALB (n = 8). Sham rats (n = 6) underwent all procedures except hemorrhage or resuscitation. Blood samples were assayed for active proteases, such as metalloproteinase 9 (MMP-9) and a disintegrin and metalloproteinase 10 (ADAM-10), BM-type heparan sulfate proteoglycan (perlecan), cell count, and coagulation function. Leukocyte transmigration was used to estimate the net efficiency of leukocyte recruitment in cremaster venules. RESULTS: Hemorrhage significantly lowered red cell count, but white cell and platelet counts did not change (vs. sham). Ionized calcium in plasma was significantly reduced in untreated and remained so after NS. In contrast, ionized calcium was normalized after ALB. Plasma expansion after NS and ALB further reduced leukocyte and platelet counts. Metalloproteinase 9, ADAM-10, and perlecan were significantly higher in untreated rats (vs. sham). Albumin normalized MMP-9, ADAM-10, and perlecan levels, while NS further increased MMP-9, ADAM-10, and perlecan (vs. sham). Transmigrated leukocytes doubled in the untreated group and remained elevated after NS (vs. sham) but normalized after ALB. Albumin reduced every stage of the leukocyte recruitment process to sham levels. CONCLUSION: Despite similar plasma expansion, NS weakened platelet function contrary to ALB. Plasma expansion with ALB resulted in restoration of BM integrity and attenuation of leukocyte recruitment to tissues, in contrast to NS. Albumin plays a critical role in restoring BM integrity, attenuating leukocyte recruitment to tissues, and optimizing hemostasis by increasing ionized calcium in plasma.

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 Ω x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

Inhibition of Aquaporin 4 Decreases Amyloid Aß40 Drainage Around Cerebral Vessels.

Aquaporin-4 (AQP4) is located mainly in the astrocytic end-feet around cerebral blood vessels and regulates ion and water homeostasis in the brain. While deletion of AQP4 is shown to reduce amyloid-ß (Aß) clearance and exacerbate Aß peptide accumulation in plaques and vessels of Alzheimer's disease mouse models, the mechanism and clearing pathways involved are debated. Here, we investigated how inhibiting the function of AQP4 in healthy male C57BL/6 J mice impacts clearance of Aß40, the more soluble Aß isoform. Using two-photon in vivo imaging and visualizing vessels with Sulfurodamine 101 (SR101), we first showed that Aß40 injected as a ≤ 0.5-µl volume in the cerebral cortex diffused rapidly in parenchyma and accumulated around blood vessels. In animals treated with the AQP4 inhibitor TGN-020, the perivascular Aß40 accumulation was significantly (P < 0.001) intensified by involving four times more vessels, thus suggesting a generalized clearance defect associated with vessels. Increasing the injecting volume to ≥ 0.5 ≤ 1 µl decreased the difference of Aß40-positive vessels observed in non-treated and AQP4 inhibitor-treated animals, although the difference was still significant (P = 0.001), suggesting that larger injection volumes could overwhelm intramural vascular clearance mechanisms. While both small and large vessels accumulated Aß40, for the ≤ 0.5-µl volume group, the average diameter of the Aß40-positive vessels tended to be larger in control animals compared with TGN-020-treated animals, although the difference was non-significant (P = 0.066). Using histopathology and ultrastructural microscopy, no vascular structural change was observed after a single massive dose of TGN-020. These data suggest that AQP4 deficiency is directly involved in impaired Aß brain clearance via the peri-/para-vascular routes, and AQP4-mediated vascular clearance might counteract blood-brain barrier abnormalities and age-related vascular amyloidopathy.

Ultrastructural Changes in the Air-Blood Barrier of Rats in Acute Intoxication with Furoplast Pyrolysis Products.

Rats were exposed to fluoroplast-4 pyrolysis products (sample weight 2.6 g, pyrolysis temperature 440-750°C, pyrolysis duration 4 min) containing perfluoroisobutylene over 15 min. Lung tissue samples for histological and electron microscopic examination were isolated in 3 and 30 min after intoxication and processed routinely. Histological examination revealed no structural changes in the lungs. In ultrathin sections of rat lungs, some changes in the structure of type I pneumocytes were detected in 3 min after the exposure: detachment of cytoplasmic processes and the appearance of transcytosis pores. These changes attested to impaired cell-cell interactions and their adhesion to the basement membrane, where structural disorganization and edema of the collagen matrix were observed. In 30 min following exposure, the signs of damage to type I pneumocytes became more pronounced. The increase in the equivalents of transcellular and paracellular permeability in the alveolar lining profile was observed. No changes in the pulmonary capillary endotheliocytes were detected, which suggest that type I pneumocytes are the primary target of the toxic effect of perfluoroisobutylene. The vulnerability of a particular cell population, in view of specific metabolism of these cells, can be the key to deciphering of the mechanisms of the toxic effect of pyrolysis products of fluorinated polymer materials.

Low Dose of Genistein Alleviates Mono-(2-Ethylhexyl) Phthalate-Induced Fetal Testis Disorder Based on Organ Culture Model.

Mono-(2-ethylhexyl) phthalate (MEHP) and genistein have been classified as endocrine-disrupting chemicals (EDCs) which interfere with the differentiation and development of the male reproductive system. However, how these two EDCs would affect fetal rat testis development at a low dose was rarely studied. In this study, we established the organ culture system and applied it to evaluate testicular effects following multiple EDC exposure at a low dose. 15.5 days postcoitum fetal rat testes were dissected, cultured, and exposed to vehicle (control), GEN (1 µmol/L, G), MEHP (1 µmol/L, M), or GEN (1 µmol/L)+MEHP (1 µmol/L, G+M). Testicular cell markers, testosterone concentration, redox state, testicular histology, and testicular ultrastructure were evaluated. Our results showed that a low dose of MEHP suppressed the development of Sertoli cells, Leydig cells, and gonocytes by triggering oxidative injuries, which was consistent with the ultrastructural findings. However, coadministration of genistein at a low dose could partially attenuate MEHP-induced fetal testis damage through antioxidative action. Cotreatment of genistein at a low dose may have a promising future on its protecting role for attenuating other EDC-induced reproductive disorders during early life. Based on the results, it can be speculated that dietary intake of isoflavones may make the fetal testis less susceptible to phthalate-induced injury.

Peroxidasin is essential for endothelial cell survival and growth signaling by sulfilimine crosslink-dependent matrix assembly.

Peroxidasin (PXDN) has been reported to crosslink the C-terminal non-collagenous domains of collagen IV (Col IV) by forming covalent sulfilimine bond. Here, we explored the physiological role of PXDN and its mechanism of action in endothelial cell survival and growth. Silencing of PXDN using siRNAs decreased cell proliferation without increase of the number of detached cells and decreased cell viability under serum-starved condition with increased fragmented nuclei and caspase 3/7 activity. Conditioned medium (CM) containing wild-type PXDN restored the proliferation of PXDN-depleted cells, but CM containing mutant PXDN with deletion of either N-terminal extracellular matrix (ECM) motifs or peroxidase domain failed to restore PXDN function. Accordingly, anti-PXDN antibody [raised against IgC2 (3-4) subdomain within ECM motifs] and peroxidase inhibitor phloroglucinol prevented the rescue of the PXDN-depleted cells by PXDN-containing CM. PXDN depletion resulted in loss of sulfilimine crosslinks, and decreased dense fibrillar network assembly of not only Col IV, but also fibronectin and laminin like in Col IV knockdown. Exogenous PXDN-containing CM restored ECM assembly as well as proliferation of PXDN-depleted cells. Accordingly, purified recombinant PXDN protein restored the proliferation and ECM assembly, and prevented cell death of the PXDN-depleted cells. PXDN depletion also showed reduced growth factors-induced phosphorylation of FAK and ERK1/2. In addition, siPXDN-transfected cell-derived matrix failed to provide full ECM-mediated activation of FAK and ERK1/2. These results indicate that both the ECM motifs and peroxidase activity are essential for the cellular function of PXDN and that PXDN is crucial for ECM assembly for survival and growth signaling.

The endothelial basement membrane acts as a checkpoint for entry of pathogenic T cells into the brain.

The endothelial cell basement membrane (BM) is a barrier to migrating leukocytes and a rich source of signaling molecules that can influence extravasating cells. Using mice lacking the major endothelial BM components, laminin 411 or 511, in murine experimental autoimmune encephalomyelitis (EAE), we show here that loss of endothelial laminin 511 results in enhanced disease severity due to increased T cell infiltration and altered polarization and pathogenicity of infiltrating T cells. In vitro adhesion and migration assays reveal higher binding to laminin 511 than laminin 411 but faster migration across laminin 411. In vivo and in vitro analyses suggest that integrin α6ß1- and αvß1-mediated binding to laminin 511-high sites not only holds T cells at such sites but also limits their differentiation to pathogenic Th17 cells. This highlights the importance of the interface between the endothelial monolayer and the underlying BM for modulation of immune cell phenotype.

Human embryoid bodies as a 3D tissue model of the extracellular matrix and α-dystroglycanopathies.

The basal lamina is a specialized sheet of dense extracellular matrix (ECM) linked to the plasma membrane of specific cell types in their tissue context, which serves as a structural scaffold for organ genesis and maintenance. Disruption of the basal lamina and its functions is central to many disease processes, including cancer metastasis, kidney disease, eye disease, muscular dystrophies and specific types of brain malformation. The latter three pathologies occur in the α-dystroglycanopathies, which are caused by dysfunction of the ECM receptor α-dystroglycan. However, opportunities to study the basal lamina in various human disease tissues are restricted owing to its limited accessibility. Here, we report the generation of embryoid bodies from human induced pluripotent stem cells that model the basal lamina. Embryoid bodies cultured via this protocol mimic pre-gastrulation embryonic development, consisting of an epithelial core surrounded by a basal lamina and a peripheral layer of ECM-secreting endoderm. In α-dystroglycanopathy patient embryoid bodies, electron and fluorescence microscopy reveal ultrastructural basal lamina defects and reduced ECM accumulation. By starting from patient-derived cells, these results establish a method for the in vitro synthesis of patient-specific basal lamina and recapitulate disease-relevant ECM defects seen in the α-dystroglycanopathies. Finally, we apply this system to evaluate an experimental ribitol supplement therapy on genetically diverse α-dystroglycanopathy patient samples.This article has an associated First Person interview with the first author of the paper.

Laminin-111 protein therapy after disease onset slows muscle disease in a mouse model of laminin-α2 related congenital muscular dystrophy.

Laminin-α2 related congenital muscular dystrophy (LAMA2-CMD) is a fatal muscle disease caused by mutations in the LAMA2 gene. Laminin-α2 is critical for the formation of laminin-211 and -221 heterotrimers in the muscle basal lamina. LAMA2-CMD patients exhibit hypotonia from birth and progressive muscle loss that results in developmental delay, confinement to a wheelchair, respiratory insufficiency and premature death. There is currently no cure or effective treatment for LAMA2-CMD. Several studies have shown laminin-111 can serve as an effective protein-replacement therapy for LAMA2-CMD. Studies have demonstrated early treatment with laminin-111 protein results in an increase in life expectancy and improvements in muscle pathology and function. Since LAMA2-CMD patients are often diagnosed after advanced disease, it is unclear if laminin-111 protein therapy at an advanced stage of the disease can have beneficial outcomes. In this study, we tested the efficacy of laminin-111 protein therapy after disease onset in a mouse model of LAMA2-CMD. Our results showed laminin-111 treatment after muscle disease onset increased life expectancy, promoted muscle growth and increased muscle stiffness. Together these studies indicate laminin-111 protein therapy either early or late in the disease process could serve as an effective protein replacement therapy for LAMA2-CMD.

Targeting the OXE receptor as a potential novel therapy for asthma.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is an arachidonic acid metabolite formed by oxidation of the 5-lipoxygenase (5-LO) product 5S-hydroxy-6,8,11,14-eicosatetraenoic acid (5S-HETE) by the NADP+-dependent enzyme 5-hydroxyeicosanoid dehydrogenase. It is the only 5-LO product with appreciable chemoattractant activity for human eosinophils. Its actions are mediated by the selective OXE receptor, which is highly expressed on eosinophils, basophils, neutrophils and monocytes. Orthologs of the OXER1 gene, which encodes this receptor, are found in many species except for rodents. Intradermal injection of 5-oxo-ETE into humans and monkeys elicits eosinophil infiltration into the skin, raising the possibility that it may play a pathophysiological role in eosinophilic diseases. To investigate this and possibly identify a novel therapy we sought to prepare synthetic antagonists that could selectively block the OXE receptor. We synthesized a series of indole-based compounds bearing substituents that mimic the regions of 5-oxo-ETE that are required for biological activity, which we modified to reduce metabolism. The most potent of these OXE receptor antagonists is S-Y048, which is a potent inhibitor of 5-oxo-ETE-induced calcium mobilization (IC50, 20 pM) and has a long half-life following oral administration. S-Y048 inhibited allergen-induced eosinophil infiltration into the skin of rhesus monkeys that had been experimentally sensitized to house dust mite and inhibited pulmonary inflammation resulting from challenge with aerosolized allergen. These data provide the first evidence for a pathophysiological role for 5-oxo-ETE in mammals and suggest that potent and selective OXE receptor antagonists such as S-Y048 may be useful therapeutic agents in asthma and other eosinophilic diseases.

Pericyte abnormalities precede strial capillary basement membrane thickening in Alport mice.

In 129 Sv autosomal Alport mice, the strial capillary basement membranes (SCBMs) progressively thicken between 5 and 9 weeks of age resulting in a hypoxic microenvironment with metabolic stress and induction of pro-inflammatory cytokines and chemokines. These events occur concomitant with a drop in endocochlear potential and a susceptibility to noise-induced hearing loss under conditions that do not permanently affect age/strain-matched littermates. Here we aimed to gain an understanding of events that occur before the onset of SCBM thickening. Alport stria has normal thickness and shows levels of extracellular matrix (ECM) molecules in the SCBMs commensurate with wild-type mice. Hearing thresholds in the 3-week Alport mice do not differ from those of wild-type mice. We performed RNAseq analysis using RNA from stria vascularis isolated from 3-week Alport mice and wild type littermates. Data was processed using Ingenuity Pathway Analysis software and further distilled using manual procedures. RNAseq analysis revealed significant dysregulation of genes involved in cell adhesion, cell migration, formation of protrusions, and both actin and tubulin cytoskeletal dynamics. Overall, the data suggested changes in the cellular architecture of the stria might be apparent. To test this notion, we performed dual immunofluorescence analysis on whole mounts of the stria vascularis from these same animals stained with anti-isolectin gs-ib4 (endothelial cell marker) and anti-desmin (pericyte marker) antibodies. The results showed evidence of pericyte detachment and migration as well as the formation of membrane ruffling on pericytes in z-stacked confocal images from Alport mice compared to wild type littermates. This was confirmed by TEM analysis. Earlier work from our lab showed that endothelin A receptor blockade prevents SCBM thickening and ECM accumulation in the SCBMs. Treating cultured pericytes with endothelin-1 induced actin cytoskeletal rearrangement, increasing the ratio of filamentous to globular actin. Collectively, these findings suggest that the change in type IV collagen composition in the Alport SCBMs results in cellular insult to the pericyte compartment, activating detachment and altered cytoskeletal dynamics. These events precede SCBM thickening and hearing loss in Alport mice, and thus constitute the earliest event so far recognized in Alport strial pathology.

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells.

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

Curcumin Attenuates Both Acute and Chronic Immune Nephritis.

Curcumin is known to have immunomodulatory potential in addition to anti-oxidant, anti-inflammatory and anti-carcinogenic effects. The aim of the present study is to investigate the therapeutic effects of curcumin on immune-mediated renal disease in an anti-glomerular basement membrane (GBM) model (representing acute kidney Injury, AKI) and murine lupus model (representing chronic kidney disease, CKD). In the AKI model, female anti-GBM 129/svj mice were administered with curcumin right before disease induction. In the CKD model, female MRL.lpr mice at the age of 8-10 weeks old were treated with curcumin or placebo via oral gavage daily for two months. After treatment, serum autoantibody levels, splenomegaly and spleen cellularity were reduced in murine lupus. Collectively, curcumin ameliorated kidney disease in the two mouse models with either acute or chronic nephritis, as marked by reduced proteinuria, blood urea nitrogen, glomerulonephritis, crescent formation, tubule-interstitial disease, and renal infiltration by lymphocytes. In addition, curcumin treatment reduced activation of the NFkB, MAPK, AKT and pBAD pathways either systemically, or within the inflamed kidneys. These findings suggest that natural food supplements could become an alternative approach to ameliorating immune-mediated kidney diseases.

Corneal Endothelial Cell Toxicity Determines Long-Term Outcome After Ocular Exposure to Sulfur Mustard Vapor.

PURPOSE: Ocular exposure to sulfur mustard (SM) vapor causes acute loss of corneal endothelial cells (CECs). Persistent corneal endothelial pathologies are observed in eyes that do not recover from SM exposure, suggesting that endothelial toxicity contributes to mustard gas keratopathy (MGK). Here, we evaluated the contributions of endothelial loss to acute and chronic corneal injuries in SM-exposed eyes. METHODS: Rabbit eyes were exposed in vivo to equivalent doses of SM using 9-, 11-, or 14-mm vapor caps. The effects of exposure area on corneal injury progression were longitudinally evaluated over 12 weeks using clinical evaluations. The effects of exposure area on CEC morphology, endothelial and epithelial ultrastructure, and endothelial barrier function were determined from 1 day to 12 weeks. RESULTS: SM exposure caused loss of CECs and failure of endothelial barrier integrity at 1 day, independent of exposure cap size. By 3 weeks, eyes exposed with the 14-mm vapor cap exhibited increased corneal permeability, repopulation of the endothelium by cells with fibroblastic morphology, and abnormal deposition of extracellular matrix. Eyes exposed with 9- or 11-mm vapor caps exhibited transient symptoms of injury that fully resolved, with the rate of recovery correlated with cap size. CONCLUSIONS: The nonlinear correlation between endothelial lesion size and probability of developing MGK suggests that the CEC loss is a determinative factor for emergence of MGK. These studies illustrate the importance of endothelial repair in preventing MGK. Furthermore, they exclude chemical modification of basement membrane as a mechanistic cause of recurrent epithelial erosions in MGK eyes.

Omalizumab may decrease the thickness of the reticular basement membrane and fibronectin deposit in the bronchial mucosa of severe allergic asthmatics.

Introduction: Immunoglobulin E is an important modulator of the inflammatory reaction in allergic asthma. It also contributes to airway remodeling in the course of the disease. The authors evaluated airway structural changes in severe allergic asthma during the omalizumab therapy. Patients and methods: The study included 13 patients with severe allergic asthma treated with omalizumab for at least one year. In each patient clinical, laboratory, and spirometry parameters were evaluated before and after the treatment. In addition, bronchoscopy with bronchial mucosa biopsy and bronchoalveolar lavage was performed. The basal lamina thickness, inflammatory cell infiltration, fibronectin, as well as type I and III collagen accumulation were assessed in bronchial mucosa specimens, together with the assessment of bronchoalveolar lavage cellularity. Results: The omalizumab therapy led to a decrease in the basal lamina thickness (p = 0.002), and to a reduction in fibronectin (p = 0.02), but not collagen deposits in the bronchial mucosa. The decrease in fibronectin accumulation was associated with an improvement in asthma control and quality of life (p = 0.01, both), and a diminished dose of systemic corticosteroids (p = 0.001). It was also associated with a tendency towards reduction of the eosinophil count in the peripheral blood, bronchoalveolar lavage fluid, and bronchial mucosa specimens. Conclusion: Our study has shown that omalizumab, effective in the treatment of severe allergic asthma, may also decrease unfavorable structural airway changes in allergic asthmatics, at least with respect to the fibronectin deposit and an increased thickness of the basal lamina. However, more extensive observational studies are needed to verify the above hypothesis.

Anti-Wrinkle Benefits of Peptides Complex Stimulating Skin Basement Membrane Proteins Expression.

The dermal-epidermal junction (DEJ) provides a physical and biological interface between the epidermis and the dermis. In addition to providing a structural integrity, the DEJ also acts as a passageway for molecular transport. Based on the recently reported importance of the DEJ in skin aging, novel peptide derivatives have been tested for their effects on basement membrane (BM) protein expressions in cultured human epidermal keratinocytes. As a result, protein expressions of collagen XVII, laminin and nidogen were stimulated by the test peptide and peptides complex. Further ex vivo evaluation using excised human skin, confirmed that the topical application of the peptides complex significantly increased dermal collagen expression, as well as expressions of collagen XVII and laminin. Interestingly, while the origin of the laminin protein is epidermal keratinocytes, the immunohistochemical staining of skin showed that laminin was only detected in the uppermost layer of the dermis, which suggests a tight assembly of laminin protein onto the dermal side of the DEJ. These results suggest that a peptide complex could improve the structural properties of the DEJ through its ability to stimulate BM proteins. In order to evaluate the anti-wrinkle benefits of the peptide complex in vivo, a clinical study was performed on 22 healthy Asian female volunteers older than 40 years. As a result, significant improvements in skin wrinkles for all of the five sites were observed after two weeks, as assessed by skin topographic measurements. Collectively, these results demonstrate the anti-aging efficacy of the peptides complex.

Molecular, histological and ultrastructural characterization of cytotoxic effects of amitraz on the ovaries of engorged females of Rhipicephalus (Boophilus) annulatus.

In the present study, the cytotoxic effects of amitraz, an octopamine receptor agonist on the reproductive system of engorged adult females of Rhipicephalus (Boophilus) annulatus were assessed using histology, electron microscopy and octopamine beta (OCTß) receptor transcriptional expression analysis. Adult immersion test (AIT) was performed by immersing the fully engorged female ticks for 2 min in different concentrations of amitraz (200, 250, 300, 350 ppm). Amitraz at the dose of 300 ppm, caused an adult tick mortality of 16.66 ±â€¯6.80 per cent, inhibition of fecundity of 75.80 per cent and hatching of 50 per cent of ova laid by treated ticks. Histological changes in the ovaries of ticks collected after 24 h of treatment with amitraz (300 ppm), in comparison with controls (distilled water/methanol) were identified by microscopical examination of sections (4  µm) stained using haematoxylin and eosin. These changes included reduction in size and basophilia of stage I oocytes, presence of cytoplasmic vacuoles of various sizes around germinal vesicle of stage II oocytes, wavy basement membrane of stage III oocytes and reduction in size and number of mature stage IV and V oocytes. Electron microscopy was employed for understanding the structural changes in the ultrathin sections (60 nm) of ovaries. Ticks treated with amitraz showed major ultrastructural changes such as irregular nuclear membrane, crystolysis of mitochondria and detachment of external and internal layers of basal lamina of oocytes. The cDNA synthesized from the total RNA of whole ticks and ovaries of ticks treated with amitraz along with controls were used for relative quantification of Octopamine ß receptor (OCTß-R) expression based on the 2-ΔΔCT method by quantitative real time PCR (qRT PCR). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as endogenous control. Down regulation of expression of OCTß-R mRNA in the ovaries of amitraz treated ticks was observed compared to controls. Thus, the inhibition of fecundity observed in the ticks treated with amitraz can be attributed to the major structural changes and decreased expression of OCT ß receptor mRNA induced by it in the ovary.

Functional regeneration of tissue engineered skeletal muscle in vitro is dependent on the inclusion of basement membrane proteins.

Skeletal muscle has a high regenerative capacity, injuries trigger a regenerative program which restores tissue function to a level indistinguishable to the pre-injury state. However, in some cases where significant trauma occurs, such as injuries seen in military populations, the regenerative process is overwhelmed and cannot restore full function. Limited clinical interventions exist which can be used to promote regeneration and prevent the formation of non-regenerative defects following severe skeletal muscle trauma. Robust and reproducible techniques for modelling complex tissue responses are essential to promote the discovery of effective clinical interventions. Tissue engineering has been highlighted as an alternative method, allowing the generation of three-dimensional in vivo like tissues without laboratory animals. Reducing the requirement for animal models promotes rapid screening of potential clinical interventions, as these models are more easily manipulated, genetically and pharmacologically, and reduce the associated cost and complexity, whilst increasing access to models for laboratories without animal facilities. In this study, an in vitro chemical injury using barium chloride is validated using the C2C12 myoblast cell line, and is shown to selectively remove multinucleated myotubes, whilst retaining a regenerative mononuclear cell population. Monolayer cultures showed limited regenerative capacity, with basement membrane supplementation or extended regenerative time incapable of improving the regenerative response. Conversely tissue engineered skeletal muscles, supplemented with basement membrane proteins, showed full functional regeneration, and a broader in vivo like inflammatory response. This work outlines a freely available and open access methodology to produce a cell line-based tissue engineered model of skeletal muscle regeneration.