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1.  Murine membranous nephropathy: Immunization with α3(IV) collagen fragment induces subepithelial immune complexes and FcγR-independent nephrotic syndrome 
Membranous nephropathy (MN) is a leading cause of nephrotic syndrome in adults and a significant cause of end-stage renal disease, yet current therapies are non-specific, toxic, and often ineffective. The development of novel targeted therapies requires a detailed understanding of the pathogenic mechanisms, but progress is hampered by the lack of a robust mouse model of disease. We report that DBA/1 mice as well as congenic FcγRIII−/− and FcRγ−/− mice immunized with a fragment of α3(IV) collagen developed massive albuminuria and nephrotic syndrome, due to subepithelial deposits of mouse IgG and C3 with corresponding basement membrane reaction and podocyte foot process effacement. The clinical presentation and histopathologic findings were characteristic of MN. Even though immunized mice produced genuine anti-α3NC1 autoAbs which bound to kidney and lung basement membranes, neither crescentic glomerulonephritis nor alveolitis ensued, likely due to the predominance of mIgG1 over mIgG2a and mIgG2b autoAbs. The ablation of activating IgG Fc receptors did not ameliorate injury, implicating subepithelial deposition of immune complexes and consequent complement activation as a major effector pathway. We have thus established an active model of murine MN. This model, leveraged by the availability of genetically engineered mice and mouse-specific reagents, will be instrumental for studying the pathogenesis of MN and for evaluating the efficacy of novel experimental therapies.
PMCID: PMC3311769  PMID: 22371398
autoimmune; autoantibody; mouse model; glomerulonephritis; subepithelial immune complexes; podocytes
2.  A New Model of Development of the Mammalian Ovary and Follicles 
PLoS ONE  2013;8(2):e55578.
Ovarian follicular granulosa cells surround and nurture oocytes, and produce sex steroid hormones. It is believed that during development the ovarian surface epithelial cells penetrate into the ovary and develop into granulosa cells when associating with oogonia to form follicles. Using bovine fetal ovaries (n = 80) we identified a novel cell type, termed GREL for Gonadal Ridge Epithelial-Like. Using 26 markers for GREL and other cells and extracellular matrix we conducted immunohistochemistry and electron microscopy and chronologically tracked all somatic cell types during development. Before 70 days of gestation the gonadal ridge/ovarian primordium is formed by proliferation of GREL cells at the surface epithelium of the mesonephros. Primordial germ cells (PGCs) migrate into the ovarian primordium. After 70 days, stroma from the underlying mesonephros begins to penetrate the primordium, partitioning the developing ovary into irregularly-shaped ovigerous cords composed of GREL cells and PGCs/oogonia. Importantly we identified that the cords are always separated from the stroma by a basal lamina. Around 130 days of gestation the stroma expands laterally below the outermost layers of GREL cells forming a sub-epithelial basal lamina and establishing an epithelial-stromal interface. It is at this stage that a mature surface epithelium develops from the GREL cells on the surface of the ovary primordium. Expansion of the stroma continues to partition the ovigerous cords into smaller groups of cells eventually forming follicles containing an oogonium/oocyte surrounded by GREL cells, which become granulosa cells, all enclosed by a basal lamina. Thus in contrast to the prevailing theory, the ovarian surface epithelial cells do not penetrate into the ovary to form the granulosa cells of follicles, instead ovarian surface epithelial cells and granulosa cells have a common precursor, the GREL cell.
PMCID: PMC3567121  PMID: 23409002
3.  Dynamics of extracellular matrix in ovarian follicles and corpora lutea of mice 
Cell and Tissue Research  2009;339(3):613-624.
Despite the mouse being an important laboratory species, little is known about changes in its extracellular matrix (ECM) during follicle and corpora lutea formation and regression. Follicle development was induced in mice (29 days of age/experimental day 0) by injections of pregnant mare’s serum gonadotrophin on days 0 and 1 and ovulation was induced by injection of human chorionic gonadotrophin on day 2. Ovaries were collected for immunohistochemistry (n=10 per group) on days 0, 2 and 5. Another group was mated and ovaries were examined on day 11 (n=7). Collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2 and perlecan were present in the follicular basal lamina of all developmental stages. Collagen type XVIII was only found in basal lamina of primordial, primary and some preantral follicles, whereas laminin α2 was only detected in some preantral and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. In the corpora lutea, staining was restricted to capillary sub-endothelial basal laminas containing collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. Laminins α4 and α5 were not immunolocalised to any structure in the mouse ovary. The ECM composition of the mouse ovary has similarities to, but also major differences from, other species with respect to nidogens 1 and 2 and perlecan.
PMCID: PMC2831189  PMID: 20033213
Follicle; Corpus luteum; Extracellular matrix; Collagen; Laminin; Perlecan; Nidogen; Mouse (CBAxC57BL/6F1)
4.  Loss of Alpha3(IV) Collagen Expression Associated with Corneal Keratocyte Activation 
To determine whether changes in the expression of type IV α1, α2, or α3 collagen isoforms are stringently associated with corneal stromal cell activation.
Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-β1-, or fetal bovine serum (FBS)-supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents.
Immunohistochemical analysis indicated that type IV α1, α2, and α3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, α3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-β1. α3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-β1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels.
The loss in the expression of α3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-β1, and it accompanies the downregulation of keratocan expression.
PMCID: PMC2714546  PMID: 17251459
5.  The extracellular matrix of hydra is a porous sheet and contains type IV collagen 
Zoology (Jena, Germany)  2008;111(5):410-418.
Hydra, as an early diploblastic metazoan, has a well defined extracellular matrix (ECM) called-mesoglea. It is organized in a tri-laminar pattern with one centrally located interstitial matrix that contains type I collagen and two sub-epithelial zones that resemble a basal lamina containing laminin and possibly type IV collagen. This study used monoclonal antibodies to the three hydra mesoglea components (type I, type IV collagens and laminin) and immunofluorescent staining to visualize hydra mesoglea structure and the relationship between these mesoglea components. In addition, hydra mesoglea was isolated free of cells and studied with immunofluorescence and SEM. Our results show that type IV collagen co-localizes with laminin in the basal lamina whereas type I collagen forms a grid pattern of fibers in the interstitial matrix. The isolated-mesoglea can maintain its structural stability without epithelial cell attachment. Hydra mesogleais porous with multiple trans-mesoglea pores ranging from 0.5 to 1 µm in diameter and about 6 pores per 100 µm2 in density. We think these trans-mesoglea pores provide a structural base for epithelial cells on both sides to form multiple trans-mesoglea cell-cell contacts. Based on these findings, we propose a new model of hydra mesoglea structure.
PMCID: PMC2560992  PMID: 18602803
Hydra; Extracellular matrix; Mesoglea; Basement membrane; Cell-ECM interaction
6.  Compositional Differences between Infant and Adult Human Corneal Basement Membranes 
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. In the infant DM, type IV collagen α1-α6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
The distribution of laminin γ3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
PMCID: PMC2151758  PMID: 17962449
7.  Lymphocytes from Enlarged Iliac Lymph Nodes as Fusion Partners for the Production of Monoclonal Antibodies after a Single Tail Base Immunization Attempt 
A novel method of preparing hybridomas producing mouse monoclonal antibodies was ­established, called “the mouse iliac lymph node method”. Lymphocytes from enlarged iliac lymph nodes from mice injected intramuscularly at the tail base with an emulsion of antigen and Freund’s adjuvant were used for cell fusion. For the most part, lymph node lymphocytes from two mice were used for a single cell fusion attempt. Ovalbumin was used as the antigen and the results of fusion were compared with the results of a previous report (Cell Struct. Funct. 20; 151–156, 1995). Approximately 100 positive wells producing antibody of interest were identified using this method. By comparison, approximately 10 positive wells were identified using the more conventional mouse spleen method after three immunization injections. The relative proportions of hybridomas producing IgM, IgG1, IgG2a, IgG2b, and IgG3, following fusion using iliac lymph node lymphocytes obtained 14 days after injection were 14.0%, 78.7%, 3.2%, 3.5% and 0.5%, respectively. This method demonstrated the following advantages: (1) a single injection of the antigen emulsion was sufficient, (2) the lymph nodes were ready for use 14 days after injection, and (3) a high yield of positive hybridomas was obtained.
PMCID: PMC1790974  PMID: 17327928
mouse monoclonal antibody; iliac lymph node; cell fusion; tail base immunization

Results 1-7 (7)