Antibodies mediate formation of neutrophil extracellular traps in the middle ear and facilitate secondary pneumococcal otitis media

Otitis media (OM) (a middle ear infection) is a common childhood illness that can leave some children with permanent hearing loss. OM can arise following infection with a variety of different pathogens, including a coinfection with influenza A virus (IAV) and Streptococcus pneumoniae (the pneumococcus). We and others have demonstrated that coinfection with IAV facilitates the replication of pneumococci in the middle ear. Specifically, we used a mouse model of OM to show that IAV facilitates the outgrowth of S. pneumoniae in the middle ear by inducing middle ear inflammation. Here, we seek to understand how the host inflammatory response facilitates bacterial outgrowth in the middle ear. Using B cell-deficient infant mice, we show that antibodies play a crucial role in facilitating pneumococcal replication. We subsequently show that this is due to antibody-dependent neutrophil extracellular trap (NET) formation in the middle ear, which, instead of clearing the infection, allows the bacteria to replicate. We further demonstrate the importance of these NETs as a potential therapeutic target through the transtympanic administration of a DNase, which effectively reduces the bacterial load in the middle ear. Taken together, these data provide novel insight into how pneumococci are able to replicate in the middle ear cavity and induce disease.

Activation of matrix metalloproteinase-2 (MMP-2) by membrane type 1 matrix metalloproteinase through an artificial receptor for ProMMP-2 generates active MMP-2

The suggested model for pro-matrix metalloproteinase-2 (proMMP-2) activation by membrane type 1 MMP (MT1-MMP) implicates the complex between MT1-MMP and tissue inhibitor of MMP-2 (TIMP-2) as a receptor for proMMP-2. To dissect this model and assess the pathologic significance of MMP-2 activation, an artificial receptor for proMMP-2 was created by replacing the signal sequence of TIMP-2 with cytoplasmic/transmembrane domain of type II transmembrane mosaic serine protease (MSP-T2). Unlike TIMP-2, MSP-T2 served as a receptor for proMMP-2 without inhibiting MT1-MMP, and generated TIMP-2-free active MMP-2 even at a low level of MT1-MMP. Thus, MSP-T2 did not affect direct cleavage of the substrate testican-1 by MT1-MMP, whereas TIMP-2 inhibited it even at the level that stimulates proMMP-2 processing. Expression of MSP-T2 in HT1080 cells enhanced MMP-2 activation by endogenous MT1-MMP and caused intensive hydrolysis of collagen gel. Expression of MSP-T2 in U87 glioma cells, which express a trace level of endogenous MT1-MMP, induced MMP-2 activation and enhanced cell-associated protease activity, activation of extracellular signal-regulated kinase, and metastatic ability into chick embryonic liver and lung. MT1-MMP can exert both maximum MMP-2 activation and direct cleavage of substrates with MSP-T2, which cannot be achieved with TIMP-2. These results suggest that MMP-2 activation by MT1-MMP potentially amplifies protease activity, and combination with direct cleavage of substrate causes effective tissue degradation and enhances tumor invasion and metastasis, which highlights the complex role of TIMP-2. MSP-T2 is a unique tool to analyze physiologic and pathologic roles of MMP-2 and MT1-MMP in comparison with TIMP-2.

Myogel, a novel, basement membrane-rich, extracellular matrix derived from skeletal muscle, is highly adipogenic in vivo and in vitro

Background/Aims Biological and synthetic scaffolds play important roles in tissue engineering and are being developed towards human clinical applications. Based on previous work from our laboratory, we propose that extracellular matrices from skeletal muscle could be developed for adipose tissue engineering. Methods Extracellular matrices (Myogels) extracted from skeletal muscle of various species were assessed using biochemical assays including ELISA and Western blotting. Biofunctionality was assessed using an in vitro differentiation assay and a tissue engineering construct model in the rat. Results Myogels were successfully extracted from mice, rats, pigs and humans. Myogels contained significant levels of laminin α4- and α2-subunits and collagen I compared to Matrigel™, which contains laminin 1 (α1β1γ1) and collagen IV. Levels of growth factors such as fibroblast growth factor 2 were significantly higher than Matrigel, vascular endothelial growth factor-A levels were significantly lower and all other growth factors were comparable. Myogels reproducibly stimulated adipogenic differentiation of preadipocytes in vitro and the growth of adipose tissue in the rat. Conclusions We found Myogel induces adipocyte differentiation in vitroand shows strong adipogenic potential in vivo, inducing the growth of well-vascularised adipose tissue. Myogel offers an alternative for current support scaffolds in adipose tissue engineering, allowing the scaling up of animal models towards clinical adipose tissue engineering applications.

Scleral matrix metalloproteinases, serine proteinase activity and hydrational capacity are increased in myopia induced by retinal image degradation

In the avian model of myopia, retinal image degradation quickly leads to ocular enlargement. We now give evidence that regionally specific changes in ocular size are correlated with both biomechanical indices of scleral remodeling, e.g. hydration capacity and with biochemical changes in proteinase activities. The latter include a 72 kDa matrix metalloproteinase (putatively MMP-2), other gelatin-binding MMPs, an acid pH MMP and a serine protease. Specifically, we have found that increases in scleral hydrational capacity parallel increases in collagen degrading activities. Gelatin zymography reveals that eyes with 7 days of retinal image degradation have elevated levels (1.4-fold) of gelatinolytic activities at 72 and 67 kDa M(r) in equatorial and posterior pole regions of the sclera while, after 14 days of treatment, increases are no longer apparent. Lower M(r) zymographic activities at 50, 46 and 37 kDa M(r) are collectively increased in eyes treated for both 7 and 14 days (1.4- and 2.4-fold respectively) in the equator and posterior pole areas of enlarging eyes. Western blot analyses of scleral extracts with an antibody to human MMP-2 reveals immunoreactive bands at 65, 30 and 25 kDa. Zymograms incubated under slightly acidic conditions reveal that, in enlarging eyes, MMP activities at 25 and 28 kDa M(r) are increased in scleral equator and posterior pole (1.6- and 4.5-fold respectively). A TIMP-like protein is also identified in sclera and cornea by Western blot analysis. Finally, retinal-image degradation also increases (~2.6-fold) the activity of a 23.5 kDa serine proteinase in limbus, equator and posterior pole sclera that is inhibited by aprotinin and soybean trypsin inhibitor. Taken together, these results indicate that eye growth induced by retinal-image degradation involves increases in the activities of multiple scleral proteinases that could modify the biomechanical properties of scleral structural components and contribute to tissue remodeling and growth.

Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins α-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, α-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

The role of biological extracellular matrix scaffolds in vascularized three-dimensional tissue growth in vivo

An in vivo murine vascularized chamber model has been shown to generate spontaneous angiogenesis and new tissue formation. This experiment aimed to assess the effects of common biological scaffolds on tissue growth in this model. Either laminin-1, type I collagen, fibrin glue, hyaluronan, or sea sponge was inserted into silicone chambers containing the epigastric artery and vein, one end was sealed with adipose tissue and the other with bone wax, then incubated subcutaneously. After 2, 4, or 6 weeks, tissue from chambers containing collagen I, fibrin glue, hyaluronan, or no added scaffold (control) had small amounts of vascularized connective tissue. Chambers containing sea sponge had moderate connective tissue growth together with a mild "foreign body" inflammatory response. Chambers containing laminin-1, at a concentration 10-fold lower than its concentration in Matrigel™, resulted in a moderate adipogenic response. In summary, (1) biological hydrogels are resorbed and gradually replaced by vascularized connective tissue; (2) sponge-like matrices with large pores support connective tissue growth within the pores and become encapsulated with granulation tissue; (3) laminin-containing scaffolds facilitate adipogenesis. It is concluded that the nature and chemical composition of the scaffold exerts a significant influence on the amount and type of tissue generated in this in vivo chamber model.

ST7-mediated suppression of tumorigenicity of prostate cancer cells is characterized by remodeling of the extracellular matrix

Multiple lines of evidence have provided compelling evidence for the existence of a tumor suppressor gene (TSG) on chromosome 7q31.1. ST7 may be the target of this genetic instability but its designation as a TSG is controversial. In this study, we show that, functionally, ST7 behaves as a tumor suppressor in human cancer. ST7 suppressed growth of PC-3 prostate cancer cells inoculated subcutaneously into severe combined immunodeficient mice, and increased the latency of tumor detection from 13 days in control tumors to 23 days. Re-expression of ST7 was also associated with suppression of colony formation under anchorage-independent conditions in MDA-MB-231 breast cancer cells and ST7 mRNA expression was downregulated in 44% of primary breast cancers. Expression profiling of PC-3 cells revealed that ST7 predominantly induces changes in genes involved in re-modeling the extracellular matrix such as SPARC, IGFBP5 and several matrix metalloproteinases. These data indicate that ST7 may mediate tumor suppression through modification of the tumor microenvironment.

The influence of extracellular matrix on the generation of vascularized, engineered, transplantable tissue

In a recently described model for tissue engineering, an arteriovenous loop comprising the femoral artery and vein with interposed vein graft is fabricated in the groin of an adult male rat, placed inside a polycarbonate chamber, and incubated subcutaneously. New vascularized granulation tissue will generate on this loop for up to 12 weeks. In the study described in this paper three different extracellular matrices were investigated for their ability to accelerate the amount of tissue generated compared with a no-matrix control. Poly-D,L-lactic-co-glycolic acid (PLGA) produced the maximal weight of new tissue and vascularization and this peaked at two weeks, but regressed by four weeks. Matrigel was next best. It peaked at four weeks but by eight weeks it also had regressed. Fibrin (20 and 80 mg/ml), by contrast, did not integrate with the generating vascularized tissue and produced less weight and volume of tissue than controls without matrix. The limiting factors to growth appear to be the chamber size and the capacity of the neotissue to integrate with the matrix. Once the sides of the chamber are reached or tissue fails to integrate, encapsulation and regression follow. The intrinsic position of the blood supply within the neotissue has many advantages for tissue and organ engineering, such as ability to seed the construct with stem cells and microsurgically transfer new tissue to another site within the individual. In conclusion, this study has found that PLGA and Matrigel are the best matrices for the rapid growth of new vascularized tissue suitable for replantation or transplantation.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

A computational model for BMP movement in sea urchin embryos

Bone morphogen proteins (BMPs) are distributed along a dorsal-ventral (DV) gradient in many developing embryos. The spatial distribution of this signaling ligand is critical for correct DV axis specification. In various species, BMP expression is spatially localized, and BMP gradient formation relies on BMP transport, which in turn requires interactions with the extracellular proteins Short gastrulation/Chordin (Chd) and Twisted gastrulation (Tsg). These binding interactions promote BMP movement and concomitantly inhibit BMP signaling. The protease Tolloid (Tld) cleaves Chd, which releases BMP from the complex and permits it to bind the BMP receptor and signal. In sea urchin embryos, BMP is produced in the ventral ectoderm, but signals in the dorsal ectoderm. The transport of BMP from the ventral ectoderm to the dorsal ectoderm in sea urchin embryos is not understood. Therefore, using information from a series of experiments, we adapt the mathematical model of Mizutani et al. (2005) and embed it as the reaction part of a one-dimensional reaction–diffusion model. We use it to study aspects of this transport process in sea urchin embryos. We demonstrate that the receptor-bound BMP concentration exhibits dorsally centered peaks of the same type as those observed experimentally when the ternary transport complex (Chd-Tsg-BMP) forms relatively quickly and BMP receptor binding is relatively slow. Similarly, dorsally centered peaks are created when the diffusivities of BMP, Chd, and Chd-Tsg are relatively low and that of Chd-Tsg-BMP is relatively high, and the model dynamics also suggest that Tld is a principal regulator of the system. At the end of this paper, we briefly compare the observed dynamics in the sea urchin model to a version that applies to the fly embryo, and we find that the same conditions can account for BMP transport in the two types of embryos only if Tld levels are reduced in sea urchin compared to fly.

Characterization of the tail-specific protease (Tsp) from Legionella

Bacterial tail-specific proteases (Tsps) have been attributed a wide variety of functions including intracellular virulence, cell wall morphology, proteolytic signal cascades and stress response. This study tested the hypothesis that Tsp has a key function for the transmissive form of Legionella pneumophila. A tsp mutant was generated in Legionella pneumophila 130b and the characteristics of this strain and the isogenic wild-type were examined using a range of growth and proteomic analyses. Recombinant Tsp protein was also produced and analyzed. The L. pneumophila tsp mutant showed no defect in growth on rich media or during thermo-osmotic stress conditions. In addition, no defects in cellular morphology were observed when the cells were examined using transmission electron microscopy. Purified recombinant Tsp was found to be an active protease with a narrow substrate range. Proteome analysis using iTRAQ (5% coverage of the proteome) found that, of those proteins detected, only 5 had different levels in the tsp mutant compared to the wild type. ACP (Acyl Carrier Protein), which has a key role for Legionella differentiation to the infectious form, was reduced in the tsp mutant; however, tsp(-) was able to infect and replicate inside macrophages to the same extent as the wild type. Combined, these data demonstrate that Tsp is a protease but is not essential for Legionella growth or cell infection. Thus, Tsp may have functional redundancy in Legionella.