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We have studied the reconstitution of basement membrane molecules from extracts prepared from the basement membrane of the EHS tumor. Under physiological conditions and in the presence of added type IV collagen and heparan sulfate proteoglycan, gellike structures form whose ultrastructure appears as interconnected thin sheets resembling the lamina dense zone of basement membrane. The major components of the reconstituted structures include laminin, type IV collagen, heparan sulfate proteoglycan, entactin, and nidogen. These components polymerize in constant proportions on reconstitution, suggesting that they interact in defined proportions. Molecular sieve studies on the soluble extract demonstrate that laminin, entactin, and nidogen are associated in large but dissociable complexes which may be a necessary intermediate in the deposition of basement membrane. The reconstituted matrix was biologically active and stimulated the growth and differentiation of certain cells.

The members of the Tear Film Subcommittee reviewed the role of the tear film in dry eye disease (DED). The Subcommittee reviewed biophysical and biochemical aspects of tears and how these change in DED. Clinically, DED is characterized by loss of tear volume, more rapid breakup of the tear film and increased evaporation of tears from the ocular surface. The tear film is composed of many substances including lipids, proteins, mucins and electrolytes. All of these contribute to the integrity of the tear film but exactly how they interact is still an area of active research. Tear film osmolarity increases in DED. Changes to other components such as proteins and mucins can be used as biomarkers for DED. The Subcommittee recommended areas for future research to advance our understanding of the tear film and how this changes with DED. The final report was written after review by all Subcommittee members and the entire TFOS DEWS II membership.

Electron microscopic immunostaining of rat duodenum and incisor tooth was used to examine the location of four known components of the basement-membrane region: type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin. Antibodies or antisera against these substances were localized by direct or indirect peroxidase methods on 60-/~m thick slices of formaldehyde-fixed tissues.In the basement-membrane region of the duodenal epithelium, enamel-organ epithelium, and blood-vessel endothelium, immunostaining for all four components was observed in the basal lamina (also called lamina densa). The bulk of the lamina lucida (rara) was unstained, but it was traversed by narrow projections of the basal lamina that were immunostained for all four components. In the subbasement-membrane fibrous elements or reticular lamina, immunostaining was confined to occasional "bridges" extending from the epithelial basal-lamina to that of adjacent capillaries.The joint presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basal lamina indicates that these substances do not occur in separate layers but are integrated into a common structure.

Laminin is a multifunctional protein with diverse biological activities. Like fibronectin, it can influence cell adhesion, growth, morphology, differentiation, migration, and agglutination as well as the assembly of the extracellular matrix. Laminin primarily affects cells of epithelial origin, and the response varies depending on the cell. Because most differentiated cells are difficult to maintain in culture, laminin may be an important supplement in studies on cell differentiation in vitro.

Renewal of nongermative epithelia is poorly understood. The novel mitogen “lacritin” is apically secreted by several nongermative epithelia. We tested 17 different cell types and discovered that lacritin is preferentially mitogenic or prosecretory for those types that normally contact lacritin during its glandular outward flow. Mitogenesis is dependent on lacritin's C-terminal domain, which can form an α-helix with a hydrophobic face, as per VEGF's and PTHLP's respective dimerization or receptor-binding domain. Lacritin targets downstream NFATC1 and mTOR. The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Gαi or Gαo–PKCα-PLC–Ca2+–calcineurin–NFATC1 and Gαi or Gαo–PKCα-PLC–phospholipase D (PLD)–mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1. This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi–translocated PKCα upstream of both Ca2+ mobilization and PLD activation in a complex with PLCγ2. Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.

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