Podocyte apoptosis is prevented by blocking the Toll-like receptor pathway.

High serum lipopolysaccharide (LPS) activity in normoalbuminuric patients with type 1 diabetes (T1D) predicts the progression of diabetic nephropathy (DN), but the mechanisms behind this remain unclear. We observed that treatment of cultured human podocytes with sera from normoalbuminuric T1D patients with high LPS activity downregulated 3-phosphoinositide-dependent kinase-1 (PDK1), an activator of the Akt cell survival pathway, and induced apoptosis. Knockdown of PDK1 in cultured human podocytes inhibited antiapoptotic Akt pathway, stimulated proapoptotic p38 MAPK pathway, and increased apoptosis demonstrating an antiapoptotic role for PDK1 in podocytes. Interestingly, PDK1 was downregulated in the glomeruli of diabetic rats and patients with type 2 diabetes before the onset of proteinuria, further suggesting that reduced expression of PDK1 associates with podocyte injury and development of DN. Treatment of podocytes in vitro and mice in vivo with LPS reduced PDK1 expression and induced apoptosis, which were prevented by inhibiting the Toll-like receptor (TLR) signaling pathway with the immunomodulatory agent GIT27. Our data show that LPS downregulates the cell survival factor PDK1 and induces podocyte apoptosis, and that blocking the TLR pathway with GIT27 may provide a non-nephrotoxic means to prevent the progression of DN.

Deciduous tooth crown size and asymmetry in strabismic children.

OBJECTIVES: To explore deciduous tooth crown dimensions in strabismic children and the relationship between the type of strabismus and tooth crown mesio-distal (M-D) and labio-lingual (L-L) size asymmetries. MATERIAL: Dental casts at mixed dentition of 2159 Collaborative Perinatal Study black and white children were measured, 123 of them strabismic at 1 year of age, age ranging from 6 to 12 years. METHODS: Directional and fluctuating asymmetries in antimeric teeth were explored in various types of strabismus having unilateral, bilateral or alternating expression. ANOVA and T-square test were used for size comparisons and calculated asymmetries were explored by comparing the variances and Pearson correlations. RESULTS: Strabismus was associated with significant M-D size increase of deciduous maxillary canines in black boys and white girls, black girls had size reduction in their mandibular canine, but white boys were unaffected. Right side size dominance was found in the strabismic children in the lower second deciduous molar M-D dimensions and in the children with alternating strabismus in their upper deciduous canine M-D dimensions. Children with unilateral strabismus had random fluctuating dental asymmetry in their upper deciduous second molar L-L dimensions when compared with healthy normals. Higher left-right correlations were found in lower second deciduous molar dimensions in strabismic girls when compared with that in controls and in strabismic boys, suggesting better developmental canalization in female. CONCLUSIONS: Asymmetries in the head area, such as promoted here in strabismic children, may have associations with asymmetries in the dentition, focusing the embryonal origins and timing of developmental processes.

Tooth eruption symmetry in functional lateralities.

Dental casts and oral photographs from a cross-sectional sample of 2092 young North Americans with detailed information on functional lateralities (eyedness, handedness and footedness) were examined to compare the proportions of symmetrical and asymmetrical eruption of the antimeric (left-right, contralateral pair) permanent teeth using a four-grade eruption scale. The proportion of symmetrically erupting antimeric teeth was higher for some teeth in those with non-right-sidedness of the feet and eyes, but not significantly so in the case of handedness. Left-footedness was significantly (95% confidence interval) associated with an increased proportion of symmetrical pairs of the maxillary first molar and mandibular lateral incisor, and non-right-eyedness with an increased proportion of symmetrical eruption and left/right non-balanced proportions of asymmetrical eruption in maxillary central incisors. True right-sidedness (hand, foot and eye) was significantly (P< or =0.05) associated with advanced eruption of the left mandibular first molar. It is suggested that while the timing of antimeric tooth emergence and clinical eruption is primarily programmed before crown mineralization, starting approximately at the 30th gestational week in the case of first permanent molars, symmetrical/asymmetrical tooth emergence and eruption may provide information a posteriori on prenatal and early postnatal growth and development.

Bilateral sphenoid wing metastases of prostate cancer presenting with extensive brain edema.

A 76-year-old man insidiously developed diffuse neurological symptoms: cognitive decline, dysphagia, dysphasia and mental disturbance. Computed tomography of the cranium revealed widespread bilateral brain edema and symmetrical bilateral sphenoid wing hyperostosis. Adjacent to the hyperostosis that resembled skull base meningiomas, two separate parenchymatous temporal lobe lesions enhancing with contrast medium were observed. The patient had earlier been diagnosed to have prostatic carcinoma. Dexamethasone therapy resulted in discontinuation of the neurological symptoms. The diagnosis of metastasized adenocarcinoma of the prostate was confirmed histologically on autopsy after a sudden death from pneumonia. Intracranial metastases of prostate cancer may have a predilection site at the sphenoid wing, and can mimic a skull base meningioma. Intracranial spread of prostatic adenocarcinoma should be considered in elderly men as a treatable cause of gradual neurological deterioration, especially if cranial malignancy or hyperostosis is found.

A cloned human germ cell tumor-derived cell line differentiating in culture.

We have derived a clonal cell line (HGCT-1) from a lymph node metastasis of a primary testicular germ cell tumor (GCT). The tumor was negative for the embryonal carcinoma (EC) cell marker BerH2 but positive for vimentin, cytokeratin (CK) and desmin. Comparative genomic hybridization (CGH) revealed a high-level amplification at 12p that was observed in both the metastatic tumor and in the cultured HGCT-1 cells. In vitro, the phenotype of HGCT-1 cells was modulated by the culture conditions. In the presence of 10% fetal calf serum (FCS), the majority of HGCT-1 cells lacked CK and desmin. If cultured in 0.5% FCS, HGCT-1 cells acquired a uniform co-expression of vimentin, CK and desmin. Upon treatment with retinoic acid (RA), HGCT-1 cells lost the expression of desmin, but exhibited abundant CK filaments. Simultaneously, they started to express desmoplakin, form desmosomes and flatten on the culture substratum. The RA-induced changes were irreversible, whereas those following the culture in 0.5% FCS were at least partially reversible. When xenografted into an immunosuppressed rat, HGCT-1 cells formed a tumor consisting of epithelial- and mesenchymal-like structures. HGCT-1 cells thus represent a pluripotential cell system with a capacity for reversible phenotypic modulation and for irreversible differentiation into epithelial-type cells. The behavior of this novel cell line, distinct from established EC cell models, suggests a complex regulation of GCT cell differentiation.

Maternal smoking and tooth formation in the foetus. III. Thin mandibular incisors and delayed motor development at 1 year of age.

Dental casts from 2159 black and white Americans with detailed neurological data available from the Collaborative Perinatal Study were examined to investigate the relationship of maternal smoking during pregnancy and delayed motor development at 1 year of age to morphological traits in the dentition. Earlier results have indicated that maternal smoking during pregnancy may cause selected tooth size metric reductions in the deciduous dentition and at least in some of the permanent teeth with prenatal crown formation, these features being influenced by sex and race differences. The present results suggest that a thinning of the incisal parts of the permanent mandibular incisors is associated with heavy maternal smoking during pregnancy, and those white girls, in whom this dental variant is found, have probably experienced more severe central damage during the smoking sensitive gestational months, as is also seen in a delayed motor development at the age of 1 year.

FGF-2 inhibits apoptosis in human teratocarcinoma cells during differentiation on collagen substratum.

Tera-2 is a human teratocarcinoma cell line, which is induced to differentiate into neuronal direction by retinoic acid. Once differentiated, the cells form an almost nondividing population that can be maintained for weeks under conventional culture conditions. If differentiation by retinoic acid is induced while the cells are growing on type I collagen or if the already-differentiated cells are transferred onto collagen, they survive only a few days unless the cultures are repeatedly supplied with FGF-2. Lack of this growth factor induces programmed cell death (apoptosis) detectable after 24-48 h, as marked by DNA cleavage and nuclear fragmentation. The undifferentiated stem cells survive and proliferate readily on collagen without addition of FGF-2. Tera-2 cells express two members of the FGF family, FGF-2 and FGF-4. The expression of both FGFs is turned off during differentiation on collagen substratum, whereas when cultivated on plain tissue culture dish, the expression of only FGF-4 becomes undetectable. The results indicate that signaling through cell surface FGF receptors is vital for the cells, and differentiation on collagen substratum results in complete extinction of the autocrine stimulatory loop. In vivo, such induction of growth factor dependency upon differentiation would result in apoptotic death of those cells which fail to find adequate conditions for continuing FGF stimulation.

Fibroblast growth factor-mediated stimulation of differentiating teratocarcinoma cells: evidence for paracrine growth regulation.

Tera 2 human embryonal carcinoma cells proliferate rapidly in culture but are capable of differentiating into quiescent cells with neuronal features. We have characterized the effects of exogenous and endogenous fibroblast growth factors on the proliferation of differentiating Tera 2 cells. Exogenous basic fibroblast growth factor (bFGF) stimulated DNA synthesis and induced the proliferation-associated antigen Ki 67 in differentiated Tera 2 cells. Heparin-binding growth factors isolated from the undifferentiated cells excerted a similar stimulatory effect on their differentiated derivatives. The functional potential of these endogenous growth factors was further demonstrated by their ability to stimulate plasminogen activator production by capillary endothelial cells. A major part of the growth promoting activity was removed by absorption with immobilized bFGF antibodies. bFGF was also detected in Tera 2 cells by immunoblotting. The production of heparin-binding growth-promoting activity decreased during differentiation. The results demonstrate a potential role for heparin-binding growth factors in the autocrine or paracrine growth regulation of teratocarcinoma cells.

A simplified representation of biochemical principles based on the dynamics of chemical flow systems.

This work suggests that the amount of information included in biochemistry texts is artificially increased, because the knowledge presented contains tautologies that are obscured by the use of inappropriate methods of representation. The work then proposes alternative methods of representation for describing biochemical systems that are based on the dynamics of an idealized chemical open-flow system. They would clarify the fact, in an open system, the concentrations of the reactants and reaction products depend not only on the equilibrium constants but on the absolute velocities of the reactions as well. Similar rules apply to phenomena involving other processes, such as diffusion of ions. Biochemical systems are considered as a set of chemical flow systems in which individual processes have the potential for interactions if their end products influence the rate of other processes. These interactions are used to explain how biochemical systems maintain themselves in states of high order. By use of these formulations, some of the logical sequences by which biochemical principles can be deduced from the principles of chemistry can be given simple and illustrative expression.

Maternal smoking and tooth formation in the foetus. II. Tooth crown size in the permanent dentition.

Altogether 2159 pregnancies among black and white Americans in the Collaborative Perinatal Study and dental casts from their children at the age of 6-12 years were studied to determine the effect of maternal smoking on permanent tooth crown dimensions. A trend of reduction, similar to that observed in the deciduous second molars, was found in the permanent first molars and also in the mesio-distal dimension of permanent incisors in relation to sex and race of the children and smoking habits of the mother. In terms of peak in their mitotic growth, the results can be interpreted to indicate a sensitive period of intra uterine development from the 24th to 28th gestational weeks. Comparisons of postnatal body size and differential correlation patterns in affected tooth dimensions with early postnatal body and head size between smokers and non-smokers, suggests that maternal smoking during pregnancy may have an effect on basic growth of the head and body and/or the developmental process that impacts tooth development at some specific sensitive period also during the postnatal formation of these tooth crowns.

Implanted solid human tumours grow under the renal capsule of cyclosporin-immunosuppressed rats.

Cyclosporin (CsA) is a potent immunosuppressive drug widely used in organ transplantation. We transplanted fresh surgical samples from human solid malignant tumours into 45 CsA-immunosuppressed rats. Eight out of nine tumour types grew and remained viable for 5 weeks or more in at least two of the transplanted rats. In 29 rats (64%) a distinct growth of primary human tumours was recorded. Five malignancies (intestinal-type gastric carcinoma, adenocarcinoma of the lung, lymph node metastasis of a testicular teratocarcinoma, soft tissue malignant fibrous histiocytoma (MFH), and small-cell sarcoma) showed invasive and progressive growth. In all five cases the largest tumours were 0.9 cm or over in diameter when the rats were killed 5-9 weeks after transplantation. In three cases (adenocarcinoma of the colon, hypernephroma, and a second MFH) the growth of the implants under the kidney capsule was slow, but small living tumour transplants were still found 3-6 weeks later. In every case the microscopic morphology of the xenograft tumour was identical with the original tumour. In two cases the primary xenografts (teratocarcinoma and small-cell sarcoma) were retransplanted into 11 CsA-immunosuppressed rats. In both types the second passage tumours grew, and the take-off and growth rates were comparable to the primary xenografts. Cyclosporin-treated laboratory rats are an alternative to immunodeficient nude and SCID mice for growing fresh human tumour transplants in vivo. Although a few infections were encountered, most of the rats survived the CsA treatment well for up to 2 months.

Increased expression of the matrix metalloproteinase 2 in differentiating Tera 2 human embryonal carcinoma cells.

Secretion of proteolytic enzymes by cells has been implicated in tissue remodeling during embryonic development as well as in invasive neoplastic diseases. We studied the regulation of type-IV-collagenase activity in Tera 2 human embryonal carcinoma cells, which in the undifferentiated state proliferate rapidly and are tumorigenic. The undifferentiated cells produced relatively low levels of matrix-metalloproteinase-2 (MMP-2) activity. This activity was not markedly affected by exogenous basic fibroblast growth factor (bFGF) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though the plasminogen activator activity of the cells was increased by these agents. Tera 2 cells can be induced by retinoic acid to differentiate into quiescent cells, of which many express neuronal characteristics. The type-IV-collagenase activity of the cells increased markedly during the differentiation. This increase was mainly due to increased expression of MMP-2. Expression of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) was not markedly affected by the differentiation of Tera 2 cells. The results show that in the Tera 2 cell system, increased expression of MMP-2 is characteristic of the differentiated derivatives. This is in contrast with many other model systems, where increased type-IV-collagenase activity is associated with the malignant phenotype. This pattern of regulation may reflect the facts that Tera 2 cells resemble early embryonic cells and that their differentiation mimics related cell-differentiation processes in the developing embryo.

Development of FGF-dependency in human embryonic carcinoma cells after retinoic acid-induced differentiation.

Rapidly growing human teratocarcinoma cells (Tera-2) can be induced to differentiate into quiescent, nontumorigenic cells expressing neuronal markers. To more closely mimic the in vivo conditions for tumor growth, we grew Tera-2 cells in three-dimensional collagen gel cultures. The undifferentiated cells proliferated in the gel, forming tight colonies. Addition of soluble fibroblast growth factor 1 or 2 (FGF1 or FGF2) into the gel resulted in scattering of single cells throughout the collagen gel. In a FGF gradient the cells moved rapidly toward a higher concentration. On the contrary, cells first differentiated for 8 days in retinoic acid died within a few days after transfer into the collagen gel. Alternatively, if retinoic acid was included in the collagen gel, the proliferating undifferentiated cells died after 4-5 days in the gel. This differentiation-related cell death was completely opposed by including FGF in the collagen gel. When placed in the FGF gradient, the fully differentiated cells survived at the areas of higher FGF concentration, but no more migrated. The survival of retinoic acid-differentiated Tera-2 cells in collagen was also mediated by direct contact with glioma cells or the heparan sulfate-rich portion of glioma or endothelial cell matrix. These effects on differentiated cells were sensitive to inhibition by affinity-purified anti-FGF2 IgG. Thus, FGF has the potential to act as a migration-inducing factor either in solution or, more likely, in vivo, as an immobilized, matrix-bound growth factor directing the movement of responsive cells. The development of differentiation-associated FGF dependency allows survival of the cells only at places where they are in close contact with either FGF-synthesizing cells or FGF-rich extracellular structures such as basement membranes.

Association of severe asthma attacks with weather, pollen, and air pollutants.

BACKGROUND: The association between exacerbations of asthma and weather or air pollution is not well understood. The relationships between visits to the emergency room for asthma attacks and the meteorological, aerobiological, and chemical characteristics of the outdoor air have been evaluated. METHODS: The number of daily attendances for asthma attacks at the emergency room of Oulu University Central Hospital was recorded over one year together with daily meteorological readings (temperature, humidity, barometric pressure, rainfall), levels of air pollutants (nitrogen dioxide (NO2), sulphur dioxide (SO2), hydrogen sulphide (H2S), total suspended particles (TSP)), and pollen counts (birch, alder, pine, willow, total pollen). The relationship between the number of attendances and the measured variables was then analysed by multiple regression and stepwise discriminant analysis. RESULTS: The total number of attendances during the year was 232, with lower figures in summer and higher in winter. No association was found between visits for asthma attacks and airborne pollen levels or meteorological factors except for temperature, which had a low inverse correlation with attendance. The most significant correlations were found between asthma visits and the levels of NO2; those for SO2, TSP, and H2S were also significant. Intercorrelations between SO2 and temperature or NO2 and between temperature and TSP or NO2 were also found, but only NO2 correlated significantly with attendances after standardisation for temperature. CONCLUSIONS: Increased levels of pollutants, especially NO2, were associated with attacks of asthma, but the explanation for this is unclear. Air pollen levels were not associated with asthma attacks and only temperature among the meteorological factors had a small association with asthma.

Modulation of fibroblast growth factor receptor expression and signalling during retinoic acid-induced differentiation of Tera-2 teratocarcinoma cells.

We have analyzed the regulation of fibroblast growth factor receptors (FGFRs) during retinoic acid (RA) induced differentiation of Tera-2 human embryonal carcinoma cells. Undifferentiated Tera-2 cells expressed mRNAs for all four known FGFRs. Their differentiation led to loss of FGFR-4 mRNA expression and mRNA levels for FGFR-2 and FGFR-3 were considerably downregulated, whereas the mRNA levels for FGFR-1 remained unaltered. A substantial decrease in binding of K-FGF was found to occur upon RA-induced differentiation of the cells. In undifferentiated Tera-2 cells FGF stimulation caused an increase of c-fos mRNA, and c-jun mRNAs, but no increase of junB mRNA, whereas in the differentiated cells, FGFs strongly stimulated the expression of all three genes. Thus differentiation of the Tera-2 cells leads to marked changes in FGFR gene expression as well as to complex alterations in their responses to exogenous FGFs.

The expression and localization of urokinase-type plasminogen activator and its type 1 inhibitor are regulated by retinoic acid and fibroblast growth factor in human teratocarcinoma cells.

Human Tera 2 embryonal carcinoma cells switch gradually from rapidly growing undifferentiated cells to almost nonproliferating cells during retinoic acid (RA)-induced neuronal differentiation. This process is associated with the increased expression of type 1 plasminogen activator inhibitor (PAI 1) mRNA, and the secreted inhibitor is immobilized to the pericellular area. Furthermore, the differentiation is accompanied by a decrease in the amount of both the secreted tissue-type PA (tPA) and the mainly cell-associated urokinase-type PA (uPA) activity. In RA-differentiated cells, uPA becomes localized at the vinculin-rich cell-substratum adhesion sites. Fibroblast growth factor activity has been associated with various events during embryonal growth and with the regulation of proteolytic enzymes. A short-term treatment of the undifferentiated Tera 2 cells with basic fibroblast growth factor (bFGF) increases uPA mRNA levels and the cell-associated uPA activity, whereas the secretory tPA activity decreases. bFGF induces PAI 1 mRNA expression in the undifferentiated cells, but unlike PAI 1 protein after RA-treatment, the inhibitor does not accumulate around the cells but is released in the medium. A similar exposure to bFGF has less effect on the RA-differentiated Tera 2 cells. Under these conditions bFGF treatment leads to an increase in the amounts of PAI 1 and uPA mRNAs, but no changes in the localization of these components can be seen. Differentiation of human embryonal carcinoma cells is thus connected with an altered response to bFGF.

Actin cytoskeleton of extraembryonic endoderm and teratocarcinoma-derived endoderm cells.

Distinct F-actin- and myosin-containing stress fibers were observed in situ in many endoderm cells of parietal yolk sacs from 11-day mouse embryos. In visceral endoderm (VE) such fibers were not seen, and F-actin was concentrated in the cell periphery. Correspondingly, in electron microscopy ventral cell membrane-associated bundles of microfilaments were revealed in the periphery of parietal endoderm (PE) cells but not in VE cells. Both PE and VE cells formed stress fibers in primary cultures. Undifferentiated F9 embryonal carcinoma cells formed only short actin spikes and fibrils irrespective of growth substratum. In PE-like derivatives of F9 cells, on the other hand, distribution of F-actin was markedly affected by the growth substratum: They formed distinct stress fibers when plated on fibronectin but did not when plated on gelatin. Similarly, in teratocarcinoma-derived PE cells (PYS-2) adhesion to fibronectin induced the formation of distinct bundles of F-actin and plaques of vinculin. The results suggest that the susceptibility of teratocarcinoma cell actin cytoskeleton to the influence of molecular composition of surrounding matrix is developmentally regulated. On the other hand, the reason for the presence of stress fibers in PE cells and for their absence in VE cells is unclear.

Embryonal carcinoma cells adhere preferentially to fibronectin and laminin but their endodermal differentiation leads to a reduced adherence to laminin.

F9 and PC13 embryonal carcinoma (EC) cells adhered rapidly to growth substrata coated with fibronectin or laminin. When F9 cells were induced to differentiate into visceral or parietal endoderm-like cells, their ability to adhere to laminin diminished, but their adherence to fibronectin remained unchanged. Correspondingly, permanently differentiated teratocarcinoma-derived endoderm cells (PYS-2 and PSA-5e) adhered markedly less efficiently to laminin than to fibronectin. F9 cells adhered to proteolytic fibronectin fragments containing the cell-binding site but not to fragments containing gelatin- or heparin-binding sites. They also adhered slowly to gelatin, but this adhesion was completely blocked by cycloheximide. The results show that the teratocarcinoma stem cells may have specific mechanisms mediating adhesion to fibronectin and laminin and that endodermal differentiation leads to a reduction in their capacity to adhere to laminin but not to fibronectin.

Lectin binding of F9 embryonal carcinoma cells: evidence for population heterogeneity and developmentally regulated high-Mr cell surface proteins.

Undifferentiated F9 embryonal carcinoma (EC) cells bound fluorochrome-coupled Helix pomatia agglutinins (HPA) and peanut agglutinins (PNA) homogeneously, but were distinctly heterogeneous in their binding of Dolichos biflorus agglutinin (DBA) conjugates. Upon chemically induced differentiation the proportion of cells binding the DBA conjugates increased, but a distinct heterogeneity in the intensity of binding remained among the parietal endoderm (PE)-like F9 derivatives. These cells were heterogeneous in their binding of HPA conjugates as well, and many of them failed to bind PNA conjugates, apparently due to sialylation of the PNA-binding sites. Electrophoretic analysis of lectin-binding glycoproteins in the detergent-soluble fraction of the cells revealed the appearance of a doublet of polypeptides of Mr 300,000-400,000 upon differentiation induced by retinoic acid (RA). In addition, an Mr 220,000 polypeptide appeared upon differentiation induced by RA and dibutyryl cyclic AMP (dbcAMP). These polypeptides were obtained from both metabolically labelled and surface-labelled cells. A major secreted glycoprotein, which comigrated with laminin, bound to DBA. This suggests that laminin secreted by the differentiated F9 derivatives contains O-glycosidic saccharides. The results show that even though differentiation of F9 cells leads to changes in their binding of fluorochrome-coupled lectins, these lectin conjugates reveal distinct population heterogeneity among undifferentiated and differentiated F9 cells and are hence likely to be of limited value in the characterization of individual cells. At the whole cell population level, on the other hand, affinity binding to lectins reveals the appearance of high-Mr cell surface proteins in differentiating F9 cells.

Teratocarcinoma stem cells as a model for differentiation in the mouse embryo.

Embryonal carcinoma (EC) cells, which are the malignant stem cells of teratocarcinomas, are considered similar to early embryo cells. The EC cells can be grown in vitro, and many of them can be experimentally induced to differentiate; upon differentiation, the cells become benign. Here we review some of the changes that take place in the cellular and molecular characteristics of murine F9 EC cells as they differentiate into endodermal cells. Upon differentiation of F9 cells, distinct changes occur in their cell surface molecules, cytoskeleton-associated proteins and cell adhesion properties. Simultaneously, the rate of cell proliferation decreases due to a dramatic increase in duration of G1 and S phases of the cell cycle. The changes in gene expression and cell behavior occurring during endodermal differentiation of EC cells closely resemble those occurring when the endoderm differentiates in the embryo. Teratocarcinoma stem cell lines may thus be exploited to enhance understanding of both teratoma-type neoplasms and embryonic development.