Alpha and beta adrenergic receptors modulate keratinocyte migration.

Keratinocyte migration into skin wounds is the step of the healing process that correlates with the wound closure rate. Keratinocyte migration, and wound epithelialization are decreased when beta 2-adrenergic receptors (B2AR) are activated by 1 µM epinephrine/adrenaline, resulting in delayed wound healing in human and mouse skin. In the present study, we found paradoxically, that in a subset of keratinocyte strains exposure to low concentrations of epinephrine (0.1 nM) increased, rather than decreased, their migratory rate. We find that both the alpha- and the beta-adrenergic receptors are expressed in human keratinocytes, and expression of alpha-2 AR subtypes demonstrated for the first time. Therefore, we tested if the alpha-AR could be modulating the increased migratory response observed in these cell strains. By using specific inhibitors to alpha-AR, we demonstrated that blocking A2B-AR could reverse the rapid cell migration induced by the 0.1 nM epinephrine. Phosphorylation of ERK was elevated after 1-10 minutes of the low epinephrine treatment and the A2B-AR inhibitor blocked the ERK phosphorylation. The results suggest that both the A2B-AR and B2AR mediate keratinocyte migration, in which with a low level of epinephrine treatment, A2B-AR could alter the B2AR signals and regulate the migration rate.

Bone quality assessment of osteogenic cell cultures by Raman microscopy.

The use of autologous stem/progenitor cells represents a promising approach to the repair of craniofacial bone defects. The calvarium is recognized as a viable source of stem/progenitor cells that can be transplanted in vitro to form bone. However, it is unclear if bone formed in cell culture is similar in quality to that found in native bone. In this study, the quality of bone mineral formed in osteogenic cell cultures were compared against calvarial bone from postnatal mice. Given the spectroscopic resemblance that exists between cell and collagen spectra, the feasibility of extracting information on cell activity and bone matrix quality were also examined. Stem/progenitor cells isolated from fetal mouse calvaria were cultured onto fused-quartz slides under osteogenic differentiation conditions for 28 days. At specific time intervals, slides were removed and analyzed by Raman microscopy and mineral staining techniques. We show that bone formed in culture at Day 28 resembled calvarial bone from 1-day-old postnatal mice with comparable mineralization, mineral crystallinity, and collagen crosslinks ratios. In contrast, bone formed at Day 28 contained a lower degree of ordered collagen fibrils compared with 1-day-old postnatal bone. Taken together, bone formed in osteogenic cell culture exhibited progressive matrix maturation and mineralization but could not fully replicate the high degree of collagen fibril order found in native bone.

Detection and quantification of hypo- and hypergranulated neutrophils on the new Sysmex XN hematology analyzer: establishment of an adapted reference interval for the neutrophil-granularity-intensity compared to XE-technology in adult patients.

BACKGROUND: Since the recent introduction of Sysmex hematology analyzers of the XN-series it can be expected that the values of individual hematological parameters might differ between the new XN and the well-established XE platform. One such parameter is called Neutrophil-Granularity-Intensity or NEUT-GI on the XN-series and NEUT-X on the XE-series. Both parameters are used by clinicians to calculate the Granularity-Index (GI-Index), an important tool to detect hypo- or hypergranulated neutrophils occurring during myelodysplasia or inflammation. The aims of this study were to determine if previously reported reference intervals for NEUT-X can be used for NEUT-GI as well and if the GI-Indices on both analyzer platforms correlate with each other. METHODS: NEUT-GI and NEUT-X were assessed in a set of 789 blood samples (n = 543 samples from adult intensive care units and n = 246 samples from adult "blood-healthy" control patients) and the corresponding Granularity-Indices were calculated for all samples using data obtained from XE-5000 and XN-1000 hematology analyzers. RESULTS: NEUT-GI and NEUT-X correlated significantly with each other (r2: 0.6512; p < 0.0001) with statistically significant higher values for NEUT-GI compared to NEUT-X in the control group (p < 0.0001) as well as in the ICU patients (p < 0.0001). This indicated that previously established reference intervals for NEUT-X cannot be used for NEUT-GI. In contrast, the GI-Indices showed no statistically significant difference between the analyzers in both groups. The GI-Indices were higher in the ICU patients compared to the control group on both analyzer platforms (p < 0.0001), as would be expected. CONCLUSIONS: Our study revealed the emphatic need for a new reference interval for NEUT-GI on the XN platform. The resulting 95% reference intervals were 140.91 - 160.46 channels for NEUT-GI and 129.20 - 142.33 channels for NEUT-X. The GI-Indices showed no significant statistical difference between the XN- and XE-series in both cohorts.

Recovery and Cultivation of Keratinocytes From Shipped Mouse Skin.

Murine keratinocyte culture from neonatal skin is an important tool for studying the functional role of specific genes in epithelial biology. However, when the transgenic animal is only available in a geographically distant local, obtaining viable keratinocytes can be problematic. A method for transferring the isolated murine skin from collaborating labs could decrease the cost of shipping live animals, and would allow the efficient use of the tissues from the transgenic animals. Here we optimized shipping conditions and characterized the cells retrieved and cultured from mouse skin shipped for 48 h at 0 °C. The cultured keratinocytes from the control, non-shipped skin and the 2-day shipped skin were 43.6 +/- 7.8% viable, doubled every 2 days, and expressed comparable amounts of heat shock proteins and CD29/integrin beta-1. However, under the same shipping conditions, the 3-day shipped tissue failed to establish colonies in the culture. Therefore, this 2-day shipping technique allows the transfer mouse skin from distant locations with recovery of viable, propagatable keratinocytes, facilitating long-distance collaborations.

Proteolytic cleavage of the disease-related lysosomal membrane glycoprotein CLN7.

CLN7 is a polytopic lysosomal membrane glycoprotein of unknown function and is deficient in variant late infantile neuronal ceroid lipofuscinosis. Here we show that full-length CLN7 is proteolytically cleaved twice, once proximal to the used N-glycosylation sites in lumenal loop L9 and once distal to these sites. Cleavage occurs by cysteine proteases in acidic compartments and disruption of lysosomal targeting of CLN7 results in inhibition of proteolytic cleavage. The apparent molecular masses of the CLN7 fragments suggest that both cleavage sites are located within lumenal loop L9. The known disease-causing mutations, p.T294K and p.P412L, localized in lumenal loops L7 and L9, respectively, did not interfere with correct lysosomal targeting of CLN7 but enhanced its proteolytic cleavage in lysosomes. Incubation of cells with selective cysteine protease inhibitors and expression of CLN7 in gene-targeted mouse embryonic fibroblasts revealed that cathepsin L is required for one of the two proteolytic cleavage events. Our findings suggest that CLN7 is inactivated by proteolytic cleavage and that enhanced CLN7 proteolysis caused by missense mutations in selected luminal loops is associated with disease.

Lysosomal targeting of the CLN7 membrane glycoprotein and transport via the plasma membrane require a dileucine motif.

CLN7 is a polytopic lysosomal membrane protein deficient in variant late infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder. In this study fluorescence protease protection assays and mutational analyses revealed the N- and C-terminal tails of CLN7 in the cytosol and two N-glycosylation sites at N371 and N376. Both partially and non-glycosylated CLN7 were correctly transported to lysosomes. To identify lysosomal targeting motifs, we generated CD4-chimera fused to the N- and C-terminal domains of CLN7. Lysosomal localization of the chimeric proteins requires a consensus acidic dileucine-based motif in the N-terminus and two tandem tyrosine-based signals in the C-terminus. Mutation of these sorting motifs resulted in cell surface redistribution of CD4 chimeras. However, the dileucine-based motif is of critical importance for lysosomal localization of the full-length CLN7 in different cell lines. Cell surface biotinylation revealed that at equilibrium 22% of total CLN7 is localized at the plasma membrane. Mutation of the dileucine motif or the co-expression of dominant-negative mutant dynamin K44A led to a further increase of CLN7 at the plasma membrane. Our data demonstrate that CLN7 contains several cytoplasmic lysosomal targeting signals of which the N-terminal dileucine-based motif is required for the predominant lysosomal targeting along the indirect pathway and clathrin-mediated endocytosis of CLN7.

Osteogenic and adipogenic cell fractions isolated from postnatal mouse calvaria.

The use of stem/progenitor cells represents a promising approach to treat craniofacial bone defects, but successful treatments will rely on the availability of cells that can be expanded in vitro and which will differentiate appropriately in vivo. The calvaria may represent a source of autologous cells for such purposes. We demonstrate expression of stem cell antigen-1 (Sca-1) in mouse calvaria. We isolated Sca-1(+) and Sca-1(-) cells at high purity and tested the ability of these cells to differentiate into adipose and bone. We show that the Sca-1(+) cell fraction has adipogenic differentiation potential and that the cell Sca-1(-) fraction has osteogenic differentiation potential. The Sca-1(+) cell fraction partially retains its adipogenic differentiation potential and the Sca-1(-) cell fraction partially retains its osteogenic differentiation potential after in vitro expansion. These data suggest that the calvaria may be used as a source of stem/progenitor cells that can be expanded in vitro and transplanted in vivo for craniofacial tissue regeneration.

Cell surface expression of stem cell antigen-1 (Sca-1) distinguishes osteo-, chondro-, and adipoprogenitors in fetal mouse calvaria.

The flat bones of the skull (calvaria) develop by balanced cell proliferation and differentiation in the calvarial sutures and the bone tips. As the brain grows and the calvaria expand, cells within the sutures must remain undifferentiated to maintain suture patency, but osteoprogenitors also need to be recruited into the osteogenic fronts. The exact identity of calvarial osteoprogenitors is currently not known. We used immunomagnetic cell sorting to isolate Sca-1+ and Sca-1(-) cells from fetal mouse calvaria and determined their differentiation potential in in vitro differentiation asssays and in vivo subcutaneous transplantations. Cells within the Sca-1+ cell fraction have a higher adipogenic potential, whereas cells within the Sca-1(-) cell fraction have a higher osteogenic and chondrogenic potential. The Sca-1(-) fraction retains its chondrogenic potential after in vitro expansion but not its osteogenic potential. The Sca-1+ fraction does not retain its adipogenic potential after in vitro expansion. Subcutaneous transplantation resulted in islands of bone and cartilage in implants that had been seeded with Sca-1(-) cells. In conclusion, immunomagnetic cell sorting with Sca-1 antibodies can be used to separate a Sca-1+ cell fraction with adipogenic potential from a Sca-1(-) cell fraction with osteogenic and chondrogenic potential. Isolation of pure populations of calvarial adipoprogenitors, osteoprogenitors, and chondroprogenitors will be beneficial for cellular studies of calvarial development, adipogenesis, osteogenesis, and chondrogenesis. Calvaria-derived osteogenic cell populations may be useful in craniofacial tissue regeneration and repair.

Raman spectroscopic evidence for octacalcium phosphate and other transient mineral species deposited during intramembranous mineralization.

UNLABELLED: To understand early mineralization events, we studied living murine calvarial tissue by Raman spectroscopy using fibroblast growth factor 2 (FGF2)-soaked porous beads. We detected increased levels of a transient phase resembling octacalcium phosphate in sutures undergoing premature suture closure. INTRODUCTION: Several calcium phosphates have been postulated as the earliest inorganic precursors to bone mineral. They are unstable and have not been previously detected in tissue specimens. Whether the same intermediates are formed in sutures undergoing premature closure is also unknown. METHODS: Six coronal suture tissue specimens from fetal day 18.5 B6CBA F1/J wild-type mice were studied. Three sutures specimens were treated with FGF2-soaked heparin acrylic beads to induce accelerated mineralization and premature suture closure. Three control specimens were treated with empty heparin acrylic beads. All sutures were maintained as organ cultures to permit repeated spectral analyses at 12-24 h intervals over a 72-h period. RESULTS: During the first 24 h, the spectra contained bands of octacalcium phosphate (OCP) or an OCP-like mineral. The main phosphorus-oxygen stretch was at 955 cm(-1), instead of the 957-959 cm(-1) seen in bone mineral, and there was an additional band at 1010-1014 cm(-1), as expected for OCP. A broad band was found at 945 cm(-1), characteristic of a highly disordered or amorphous calcium phosphate. An increased amount of mineral was observed in FGF2-treated sutures, but no qualitative differences in Raman spectra were observed between experimental and control specimens. CONCLUSIONS: Inorganic mineral deposition proceeds through transient intermediates, including an OCP-like phase. Although this transient phase has been observed in purely inorganic model systems, this study is the first to report OCP or an OCP-like intermediate in living tissue. Raman microspectroscopy allows observation of this transient mineral and may allow observation of other precursors as well.