A novel isolator-based system promotes viability of human embryos during laboratory processing., On the fate of primordial germ cells injected into early mouse embryos.

Polyclonal and monoclonal antibodies against chicken gizzard 5‘-nucleotidase inhibit the spreading process of chicken embryonic fibroblasts on laminin substratum.

by Yasmin
Polyclonal and monoclonal antibodies raised against chicken gizzard 5′-nucleotidase were tested in adhesion assays of embryonic chicken fibroblasts (CEF) for their ability to interfere with the adhesion process of these cells on either laminin or fibronectin substrata. The initial attachment process of CEF on fibronectin and laminin substrata was not influenced by preincubating these cells with antibodies against chicken gizzard 5′-nucleotidase.
However, the subsequent spreading process of these cells was found to be inhibited for at least 2 h on a laminin substratum. This effect was obtained with a polyclonal antibody as well as with one from 12 monoclonal antibodies raised against the native enzyme purified from chicken gizzard.
In vitro assays demonstrated a competition of laminin and this monoclonal antibody for the binding site on purified 5′-nucleotidase. Spreading-arrested and rounded CEF do not develop prominent intracellular stress-fibers like control cells, instead they seem to concentrate their available actin in areas of presumptive initial contact with the laminin substratum.

Clinical and immunological heterogeneity of canine subepidermal blistering dermatoses with anti-<em>laminin</em>-332 (<em>laminin</em>-<em>5</em>) auto-<em>antibodies</em>.

Laminin-332 (laminin-5) is a basement membrane heterotrimeric protein composed of alpha-3, beta-3 and gamma-2 laminin chains. Laminin-332 polypeptides are targeted by auto-antibodies in human patients with mucous membrane (cicatricial) pemphigoid or, more rarely, subepidermal vesicular diseases that resemble epidermolysis bullosa acquisita (EBA) or bullous pemphigoid (BP).
The objectives of this report were to characterize the clinical, histopathological and immunological characteristics of nine dogs with auto-antibodies targeting laminin-332. Immunological investigations consisted of direct immunofluorescence (IF), indirect IF with intact and salt-split canine gingival, and salt-split normal or laminin-332-deficient human skin, immunoblotting with purified human laminin-332 and immunoblotting with recombinant NC1 domain of human collagen VII.
All dogs exhibited varying degrees of skin blistering and ulceration associated with microscopic subepidermal vesiculation with or without inflammatory cells. Indirect IF established that circulating IgG auto-antibodies bound the dermal side of salt-split canine lip and human skin. In five dogs, IgG variably recognized the basement membrane of laminin-332-deficient human skin (three dogs negative, two dogs positive).
In all nine dogs, IgG auto-antibodies detected purified human laminin-332 by immunoblotting. In two dogs, additional targeting of collagen VII-NC1 was present.
These observations establish laminin-332 as a novel basement membrane antigen in dogs with autoimmune blistering diseases with variable clinical phenotypes. The names ‘acquired junctional epidermolysis bullosa’, ‘anti-laminin-332 mucous membrane pemphigoid (MMP)’ and ‘mixed auto-immune subepidermal blistering dermatosis’ are proposed for dogs with clinical signs reminiscent of EBA, MMP or BP respectively.

Bullous pemphigoid positive for anti-BP180 and anti-<em>laminin</em> <em>5</em> <em>antibodies</em> in a patient with graft-vs-host disease.

We report the case of a 55-year-old female with bullous pemphigoid (BP) who was positive for anti-BP180 and anti-laminin 5 antibodies after development of graft-vs-host disease (GVHD) caused by a bone marrow transplant. She had tense blisters on her trunk and extremities.
Histologic examination showed a subepidermal blister and marked lymphocytic infiltration, especially eosinophils. Direct immunofluorescence revealed a linear deposition of IgG on the base membrane zone. Indirect immunofluorescence on 1M NaCl split skin revealed a linear IgG deposition to both sides of the epidermal and the dermal layers.
Immunoblot assays using human epidermal extracts and BP180 NC16a domain recombinant protein confirmed the presence of IgG antibodies against BP180 and recombinant BP180 NC16a domain protein. Furthermore, immunoblotting using laminin 5 purified from human keratinocyte extract as the substrate demonstrated reactivity against the gamma2 and beta3 subunits but not the alpha3 subunit of laminin 5.
We diagnosed BP and treated her with prednisolone (40 mg/day). Both skin and oral lesions resolved without leaving scars on the bulla. Immune disturbance as well as destruction of basal epidermal cells and base membrane by GVHD may result in the induction of autoimmune blistering diseases with unusual clinical and laboratory manifestations.

Ocular ‘non-scarring’ mucous membrane pemphigoid associated with anti-<em>laminin</em>-<em>5</em> <em>antibodies</em>.

Mucous membrane pemphigoid is a rare, chronic autoimmune disease characterized by subepidermal blistering and scarring, predominantly affecting mucous membranes. Ocular involvement frequently occurs and often represents the only manifestation of the disease.
We describe a 62-year-old woman with a bilateral 18-month duration of conjunctival hyperaemia, associated with erythema and oedema of the eyelids, lacking any typical ocular signs of mucous membrane pemphigoid such as sub-conjuctival fibrosis and scarring. Histology was not significant.
Direct immunofluorescence of the conjunctiva showed IgG, IgA and complement deposition along the basement membrane zone. Immunoprecipitation analysis of affinity purified laminin-5 revealed a band consistent with the beta3 chain of laminin-5. This represents the first case of pure ocular mucous membrane pemphigoid associated with anti-laminin-5 antibodies.

Detection of <em>laminin</em> <em>5</em>-specific auto-<em>antibodies</em> in mucous membrane and bullous pemphigoid sera by ELISA.

Mucous membrane pemphigoid (MMP) is an autoimmune bullous disease that primarily affects mucous membranes leading to a scarring phenotype. MMP patients produce auto-antibodies (auto-ab) that preferentially recognize two components of the dermoepidermal basement membrane zone (BMZ): bullous pemphigoid (BP)180 and laminin 5 (LN5). Since detection of disease-specific auto-ab may be critical for the diagnosis of MMP, we developed an ELISA with affinity-purified native human LN5. A total of 24 MMP, 72 BP, and 51 control sera were analyzed for LN5-specific auto-ab: 18/24 (75.0%) MMP and 29/72 (40.3%) BP sera were LN5 reactive.
Sensitivity and specificity of the LN5 ELISA for MMP were 75% and 84.3%, respectively, and 40.3% and 88.2% for BP, respectively.
The LN5 ELISA was more sensitive than a dot blot assay with native LN5, which detected LN5-reactive IgG in 14/24 (58.3%) MMP and 16/72 (22.2%) BP sera. In MMP, but not BP, levels of LN5-reactive IgG correlated with disease severity. Furthermore, IgG reactivity to LN5 of the MMP and BP sera was not significantly associated with IgG reactivity against other autoantigens of the BMZ, such as BP180 or BP230. Thus, the established LN5 ELISA holds great promise as a novel diagnostic and prognostic parameter for MMP.

<em>Antibody</em>-induced activation of beta1 integrin receptors stimulates cAMP-dependent migration of breast cells on <em>laminin</em>-<em>5</em>.

The beta1 integrin-stimulating antibody TS2/16 induces cAMP-dependent migration of MCF-10A breast cells on the extracellular matrix protein laminin-5. TS2/16 stimulates a rise in intracellular cAMP within 20 min after plating. Pertussis toxin, which inhibits both antibody-induced migration and cAMP accumulation, targets the Galphai3 subunit of heterotrimeric G proteins in these cells, suggesting that Galphai3 may link integrin activation and migration via a cAMP signaling pathway.

Anti-epiligrin cicatricial pemphigoid with <em>antibodies</em> against the gamma2 subunit of <em>laminin</em> <em>5</em>.

BACKGROUND
Cicatricial pemphigoid (CP) is a scarring subepithelial mucocutaneous blistering disease characterized by anti-basement membrane zone autoantibodies. Anti-epiligrin CP is an uncommon variant that has been recently characterized. Severe laryngeal involvement is infrequently observed in all forms of CP and has been documented in only 2 patients with anti-epiligrin CP.
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METHODS
We report a case of CP exhibiting extensive laryngeal and ocular involvement. Histological, immunofluorescence, and immunoprecipitation studies confirmed the diagnosis of anti-epiligrin CP. Immunoblotting studies demonstrated the presence of antibodies against the alpha3 and the gamma2 subunit of laminin 5.
CONCLUSIONS
This article expands the diversity of the clinical and immunopathologic features of this newly characterized variant of CP.