Regulation of mammary epithelial cell function: a role for stromal and basement membrane matrices

Summary Interactions between epithelial cells and their environment are critical for normal function. Mammary epithelial cells require hormonal and extracellular matrix (ECM) signalling for the expression of tissue specific characteristics. With regard to ECM, cultured mammary epithelial cells synthesize and secrete milk proteins on stromal collagen I matrices. The onset of function coincides both with morphogenesis of a polarized epithelium and with deposition of basement membrane ECM basal to the cell layer. Mammary specific morphogenesis and biochemical differentiation is induced if mammary cells are cultured directly on exogenous basement membrane (EHS). Thus ECM may effect function by the concerted effect of permissivity for cell shape changes and the direct biochemical signalling of basement membrane molecules.A model is discussed where initial ECM control of mammary epithelial cell function originates in the interstitial matrix of stroma and subsequently transfers to the basement membrane when the epithelial cells have accumulated and deposited an organized basement membrane matrix.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Age-mediated gut microbiota dysbiosis promotes the loss of dendritic cells tolerance

The old age-related loss of immune tolerance inflicts a person with a wide range of autoimmune and inflammatory diseases. Dendritic cells (DCs) are the sentinels of the immune system that maintain immune tolerance through cytokines and regulatory T-cells generation. Aging disturbs the microbial composition of the gut, causing immune system dysregulation. However, the vis-à-vis role of gut dysbiosis on DCs tolerance remains highly elusive. Consequently, we studied the influence of aging on gut dysbiosis and its impact on the loss of DC tolerance. We show that DCs generated from either the aged (DC^Old) or gut-dysbiotic young (DC^Dysbiotic) but not young (DC^Young) mice exhibited loss of tolerance, as evidenced by their failure to optimally induce the generation of Tregs and control the overactivation of CD4⁺ T cells. The mechanism deciphered for the loss of DC^Old and DC^Dysbiotic tolerance was chiefly through the overactivation of NF-κB, impaired frequency of Tregs, upregulation in the level of pro-inflammatory molecules (IL-6, IL-1β, TNF-α, IL-12, IFN-γ), and decline in the anti-inflammatory moieties (IL-10, TGF-β, IL-4, IDO, arginase, NO, IRF-4, IRF-8, PDL1, BTLA4, ALDH2). Importantly, a significant decline in the frequency of the Lactobacillus genus was noticed in the gut. Replenishing the gut of old mice with the Lactobacillus plantarum reinvigorated the tolerogenic function of DCs through the rewiring of inflammatory and metabolic pathways. Thus, for the first time, we demonstrate the impact of age-related gut dysbiosis on the loss of DC tolerance. This finding may open avenues for therapeutic intervention for treating age-associated disorders with the Lactobacillus plantarum.     

Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca²⁺. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca²⁺ is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca²⁺, the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca²⁺ and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca²⁺ protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca²⁺ addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope.     

Bcl-2 protects against oxidative stress while inducing premature senescence

Replicative senescence is a cellular response to stress that has been postulated to serve as a tumor suppression mechanism and a contributor to aging. Numerous experimental studies have demonstrated that an apparently identical senescent state can also be prematurely induced in vitro in different cell types following sublethal oxidative stress stimuli. The former suggests a molecular link between cell cycle regulation and cell survival that could involve regulatory proteins such as Bcl-2. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. The aim of this work was to determine if the protection against oxidative stress mediated by Bcl-2 could prevent or delay oxidative stress-induced senescence. Using a retroviral infection system, Bcl-2 was overexpressed in primary, nonembryonic mice fibroblasts obtained from lungs derived from 2-month-old CD1 mice. Fibroblasts overexpressing Bcl-2 were exposed to 75 microM H2O2 for 2 h to induce SIPS. The rate of proliferation and the increment of senescent cells were then determined. Our results indicate that overexpression of Bcl-2 protected primary fibroblasts against oxidative stress-mediated reduction in cell proliferation, but did not prevent premature senescence.     

Increased protection from vaccinia virus infection in mice genetically prone to lymphoproliferative disorders

Mutations in the genes that encode Fas or Fas ligand (FasL) can result in poor restraints on lymphocyte activation and in increased susceptibility to autoimmune disorders. Because these mutations portend a continuously activated immune state, we hypothesized that they might in some cases confer resistance to infection. To examine this possibility, the immune response to, morbidity caused by, and clearance of vaccinia virus (VACV) Western Reserve was examined in 5- to 7-week-old Fas mutant (lpr) mice, before an overt lymphoproliferative disorder was observable. On day 6 after VACV infection, C57BL/6-lpr (B6-lpr) mice had decreased morbidity, decreased viral titers, and an increased percentage and number of CD4(+) and CD8(+) T cells. As early as day 2 after infection, B6-lpr mice had decreased liver and spleen viral titers and increased numbers of and increased gamma interferon (IFN-γ) production by several different effector cell populations. Depletion of individual effector cell subsets did not inhibit the resistance of B6-lpr mice. Uninfected B6-lpr mice also had increased numbers of NK cells, γδ(+) T cells, and CD44(+) CD4(+) and CD44(+) CD8(+) T cells compared to uninfected B6 mice. Antibody to IFN-γ resulted in increased virus load in both B6 and B6-lpr mice and eliminated the differences in viral titers between them. These results suggest that IFN-γ produced by multiple activated leukocyte populations in Fas-deficient hosts enhances resistance to some viral infections.     

Physical mapping of the human carbonic anhydrase gene cluster on chromosome 8

A cluster of genes encoding the three cytoplasmic carbonic anhydrase isozymes CAI, CAII, and CAIII lie on the long arm of chromosome 8 (8q22) in humans. These genes have been mapped using pulsed-field gel electrophoresis. The genes lie in the order CA2, CA3, CA1. CA2 and CA3 are separated by 20 kb and are transcribed in the same direction, away from CA1. CA1 is separated from CA3 by over 80 kb and is transcribed in the direction opposite to CA2 and CA3. The arrangement of the genes is consistent with proposals that the duplication event which gave rise to CA1 predated the duplication which gave rise to CA2 and CA3. The order of these three genes differs from that suggested for the mouse based on recombination frequency.     

Analysis of the odontogenic and osteogenic potentials of dental pulp in vivo using a Col1a1-2.3-GFP transgene

Recently, transgenic mice that carry a Green Fluorescent Protein (GFP) reporter gene fused to 2.3 kb fragment of rat Col1a1 regulatory sequences (pOBCol2.3GFPemd) were generated. In the present study, we have examined the patterns of expression of Col1a1-2.3-GFP during odontoblast differentiation in this transgenic line. We report that Col1a1-2.3-GFP is expressed in newly differentiated odontoblasts secreting predentin and fully differentiated odontoblasts. The pattern of expression of Col1a1-2.3-GFP in odontoblasts is correlated with that of dentin sialophosphoprotein (DSPP). Col1a1-2.3-GFP is also expressed in the osteoblasts and osteocytes of alveolar bone. The pattern of expression of Col1a1-2.3-GFP in osteocytes is correlated with the expression of Dmp1. These observations indicate the 2.3 kb rat Col1a1 promoter fragment has sufficient strength and specificity to monitor the stage-specific changes during both odontoblast and osteoblast differentiation. We also used coronal pulp tissues isolated from postnatal pOBCol2.3GFPemd transgenic animals to follow their differentiation after transplantation under the kidney capsule. Our observations provide direct evidence that the dental pulp contains competent progenitor cells capable of differentiating into new generations of odontoblast-like cells which express high levels of Col1a1-2.3-GFP and DSPP and secrete tubular containing reparative dentin. We also report that the dental pulp is capable of giving rise to atubular bone-like tissue containing osteocytes expressing high levels of Col1a1-2.3-GFP and Dmp1. Our studies indicate that pOBCol2.3GFPemd transgenic animals provide a powerful tool for direct examination of the underlying mechanisms and the signaling pathways involved in dentin regeneration and repair, stem cell properties and heterogeneity of the dental pulp.     

On the Importance of Hysteresis in Numerical Modeling of Surfactant-Induced Unsaturated Flow

It is known that surfactants can induce flow in unsaturated porous media due to the dependence of capillary pressure on surface tension. A commonly observed feature in systems with surfactant-induced flow is a transient wetting/drying/wetting sequence associated with the propagation of a surfactant solute front under monotonic flow conditions. Previous efforts to model surfactant-induced flow in relatively complex (e.g., two-dimensional systems) have not successfully incorporated hysteretic moisture retention properties. In this research, hysteretic, two-dimensional simulations of surfactant-induced flow were performed to assess the potential importance of considering hysteresis in such simulations. Hysteretic simulation results were compared to experimental data and to non-hysteretic simulations. The results suggest that the inclusion of hysteresis in numerical simulations can improve the match between simulated and experimental results in systems with surfactant-induced unsaturated flow. Furthermore, the inclusion of hysteresis in numerical simulations played a significant role in predicting the distribution of the contaminant and correct pressure head/moisture condition at the end of the experiment.