Double-layered collagen gel hemisphere for cell invasion assay: successful visualization and quantification of cell invasion activity

Although various methods for collagen gel-based cell invasion assays have been described, there continues to be a need for a simpler and more objective assay. Here, we describe an easy-to-prepare double-layered collagen gel hemisphere (DL-CGH) system that satisfies these requirements, and we demonstrate the advantages of this new system for visualizing cell movements during invasion. DL-CGH consists of a central core collagen layer surrounded by an outer cover collagen layer. A droplet of collagen I solution (containing cells to be examined) naturally forms a small hemisphere on the bottom of the culture dish. After this central core layer gels, a second droplet is placed atop the first gel, encapsulating it completely. The hemisphere is submerged in the medium and cultured. The invasive activity of cells that infiltrate from the inner to the outer layer can be evaluated optically. Using this in vitro system, we measured the inhibitory effect of E-cadherin expression on cancer cell invasion. DL-CGH also allowed visualization of interactions between invading cancer cells and the stroma. Cancer cells, which lack the proteases required for direct entrance into the three-dimensional collagen matrix, were seen to slip like amoebas through matrix gaps generated by the pericellular proteolytic activity of fibroblasts. [Supplementary materials are available for this article. Go to the publisher's online edition of Cell Communication and Adhesion for the following free supplemental resources: Movies 1-3; 4a and b].     

Lung volume and collapsibility of the passive pharynx in patients with sleep-disordered breathing.

Lung volume dependence of pharyngeal airway patency suggests involvement of lung volume in pathogenesis of obstructive sleep apnea. We examined the structural interaction between passive pharyngeal airway and lung volume independent of neuromuscular factors. Static mechanical properties of the passive pharynx were compared before and during lung inflation in eight anesthetized and paralyzed patients with sleep-disordered breathing. The respiratory system volume was increased by applying negative extrathoracic pressure, thereby leaving the transpharyngeal pressure unchanged. Application of -50-cmH(2)O negative extrathoracic pressure produced an increase in lung volume of 0.72 (0.63-0.91) liter [median (25-75 percentile)], resulting in a significant reduction of velopharyngeal closing pressure of 1.22 (0.14-2.03) cmH(2)O without significantly changing collapsibility of the oropharyngeal airway. Improvement of the velopharyngeal closing pressure was directly associated with body mass index. We conclude that increase in lung volume structurally improves velopharyngeal collapsibility particularly in obese patients with sleep-disordered breathing.     

Myometrial‐derived CXCL12 promotes lipopolysaccharide induced preterm labour by regulating macrophage migration,polarization and function in mice

Preterm birth is a major contributor to neonatal mortality and morbidity. Infection results in elevation of inflammation‐related cytokines followed by infiltration of immune cells into gestational tissue. CXCL12 levels are elevated in preterm birth indicating it may have a role in preterm labour (PTL); however, the pathophysiological correlations between CXCL12/CXCR4 signalling and premature labour are poorly understood. In this study, PTL was induced using lipopolysaccharide (LPS) in a murine model. LPS induced CXCL12 RNA and protein levels significantly and specifically in myometrium compared with controls (3‐fold and 3.5‐fold respectively). Highest levels were found just before the start of labour. LPS also enhanced the infiltration of neutrophils, macrophages and T cells, and induced macrophage M1 polarization. In vitro studies showed that condition medium from LPS‐treated primary smooth muscle cells (SMC) induced macrophage migration, M1 polarization and upregulated inflammation‐related cytokines such as interleukin (IL)‐1, IL‐6 and tumor necrosis factor alpha (TNF‐α). AMD3100 treatment in pregnant mice led to a significant decrease in the rate of PTL (70%), prolonged pregnancy duration and suppressed macrophage infiltration into gestation tissue by 2.5‐fold. Further, in‐vitro treatment of SMC by AMD3100 suppressed the macrophage migration, decreased polarization and downregulated IL‐1, IL‐6 and TNF‐α expression. LPS treatment in pregnant mice induced PTL by increasing myometrial CXCL12, which recruits immune cells that in turn produce inflammation‐related cytokines. These effects stimulated by LPS were completely reversed by AMD3100 through blocking of CXCL12/CXCR4 signalling. Thus, the CXCL12/CXCR4 axis presents an excellent target for preventing infection and inflammation‐related PTL.     

A G protein-coupled receptor responsive to bile acids

So far some nuclear receptors for bile acids have been identified. However, no cell surface receptor for bile acids has yet been reported. We found that a novel G protein-coupled receptor, TGR5, is responsive to bile acids as a cell-surface receptor. Bile acids specifically induced receptor internalization, the activation of extracellular signal-regulated kinase mitogen-activated protein kinase, the increase of guanosine 5'-O-3-thio-triphosphate binding in membrane fractions, and intracellular cAMP production in Chinese hamster ovary cells expressing TGR5. Our quantitative analyses for TGR5 mRNA showed that it was abundantly expressed in monocytes/macrophages in human and rabbit. Treatment with bile acids was found to suppress the functions of rabbit alveolar macrophages including phagocytosis and lipopolysaccharide-stimulated cytokine productions. We prepared a monocytic cell line expressing TGR5 by transfecting a TGR5 cDNA into THP-1 cells that did not express TGR5 originally. Treatment with bile acids suppressed the cytokine productions in the THP-1 cells expressing TGR5, whereas it did not influence those in the original THP-1 cells, suggesting that TGR5 is implicated in the suppression of macrophage functions by bile acids.     

Histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow (Capricornis crispus, Artiodactyla, Mammalia) with special reference to glandular structures

The histochemistry of glycoconjugates in the nasolabial skin of the Japanese serow ( Capricornis crispus ) was studied by light microscopic histochemical methods, particularly lectin histochemistry. The eccrine glands present exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine, alpha-D-galactose and N-acetyl-neuraminic acid) especially in the cells of the secretory acini, the free surface of the collecting duct cells also showed distinct positive reactions with most of the histochemical methods. The thick epidermis of the nasolabial skin contained smaller amounts of glycoproteins. The results obtained are discussed with regard to possible functions of the glandular secretions. This substance mixture may particularly improve water retention on the skin surface, and protect against physical damage as well as microbial contamination. There seem to be no basic differences of muzzle functions between wild and domesticated bovine species.     

Distinct Roles of Urinary Liver-Type Fatty Acid-Binding Protein in Non-Diabetic Patients with Anemia

BackgroundVarious stresses including ischemia are known to up-regulate renal L-FABP gene expression and increase the urinary excretion of L-FABP. In diabetic patients with anemia, the urinary excretion of L-FABP is significantly increased. We studied the clinical significance of urinary L-FABP and its relationship with anemia in non-diabetic patients.ResultsUrinary L-FABP levels were significantly higher in patients with anemia compared to those in patients without anemia. Similarly, the urinary L-FABP levels were significantly higher in patients with albuminuria compared to those in patients without albuminuria. Urinary L-FABP levels correlated with urinary albumin-to-creatinine ratios, estimated glomerular filtration rates, body mass index, and hemoglobin levels. Multivariate linear regression analysis determined that hemoglobin levels (β = -0.249, P = 0.001) and urinary albumin-to-creatinine ratios (β = 0.349, P < 0.001) were significant predictors of urinary L-FABP levels.ConclusionsUrinary L-FABP is strongly associated with anemia in non-diabetic patients.     

Heart rate variability responses to hypoxic and hypercapnic exposures in different mouse strains.

Heart rate variability (HRV) is a well-characterized, noninvasive means of assessing cardiac autonomic nervous system activity. This study examines the basic cardiac responses to hypoxic and hypercapnic challenges in seven strains of commonly used inbred mice (A/J, BALB/cJ, C3H/HeJ, C57BL/6J, CBA/J, DBA/2J, and FVB/J). Adult male mice, 8-12 wk of age, were chronically instrumented to a femoral artery catheter for the continuous measurement of systemic arterial blood pressure and heart rate. Mice were exposed to multiple 4-min periods of hypoxia (10% O2), hypercapnia (5% CO2), and combined hypoxia/hypercapnia (10% O2 + 5% CO2). HRV was derived from pulse intervals of the blood pressure tracings. Hypoxia induced increases in high-frequency HRV power and decreased low-frequency (LF) HRV power in most strains. Hypercapnia led to decreased high-frequency HRV power and increased LF HRV power in most strains. Strain differences were most notable in regard to the concomitant exposures of hypoxia and hypercapnia, with FVB/J mice mirroring their own response to hypercapnia alone, whereas CBA/J mice mirrored their own responses to hypoxia. As blood pressure is most likely the driving factor for heart rate changes via the baroreflex pathway, it is interesting that LF, considered to reflect cardiac sympathetic activity, was negatively correlated with heart rate, suggesting that LF changes are driven by baroreflex oscillation and not necessarily by absolute sympathetic or parasympathetic activity to the heart. These findings suggest that genetic background can influence the centrally mediated cardiovascular responses to basic hypoxic and hypercapnic challenges.     

Spectrocolorimetric assessment of cartilage plugs after autologous osteochondral grafting: correlations between color indices and histological findings in a rabbit model

We investigated the use of a commercial spectrocolorimeter and the application of two color models (L* a* b* colorimetric system and spectral reflectance distribution) to describe and quantify cartilage plugs in a rabbit model of osteochondral autografting. Osteochondral plugs were removed and then replaced in their original positions in Japanese white rabbits. The rabbits were sacrificed at 4 or 12 weeks after the operation and cartilage samples were assessed using a spectrocolorimeter. The samples were retrospectively divided into two groups on the basis of the histological findings (group H: hyaline cartilage, successful; group F: fibrous tissue or fibrocartilage, failure) and investigated for possible significant differences in the spectrocolorimetric analyses between the two groups. Moreover, the relationships between the spectrocolorimetric indices and the Mankin histological score were examined. In the L* a* b* colorimetric system, the L* values were significantly lower in group H than in group F (P = 0.02), whereas the a* values were significantly higher in group H than in group F (P = 0.006). Regarding the spectral reflectance distribution, the spectral reflectance percentage 470 (SRP470) values, as a coincidence index for the spectral reflectance distribution (400 to 470 nm in wavelength) of the cartilage plugs with respect to intact cartilage, were 99.8 +/- 6.7% in group H and 119.8 +/- 10.6% in group F, and the difference between these values was significant (P = 0.005). Furthermore, the a* values were significantly correlated with the histological score (P = 0.004, r = -0.76). The SRP470 values were also significantly correlated with the histological score (P = 0.01, r = 0.67). Our findings demonstrate the ability of spectrocolorimetric measurements to predict the histological findings of cartilage plugs after autologous osteochondral grafting. In particular, the a* values and SRP470 values can be used to judge the surface condition of an osteochondral plug on the basis of objective data. Therefore, spectrocolorimetry may contribute to orthopedics, rheumatology and related research in arthritis, and arthroscopic use of this method may potentially be preferable for in vivo assessment.     

Synthesis and in vitro insulin-mimetic activities of zinc(ii) complexes of ethyl 2,5-dihydro-4-hydroxy-5-oxo-1H-pyrrole-3-carboxylates

Several Zn(II) complexes (2a-e) of 1-arylmethyl-2,5-dihydro-4-hydroxy-5-oxo-1H-pyrrole-3-carboxylates (1a-e), known drug candidates for diabetic complications, were synthesized and proved to have in vitro insulin-mimetic activities, suggesting that these complexes are potential chemotherapeutics that are effective against both diabetes and diabetic complications.