Recurrent febrile seizures and serum cytokines: a controlled follow-up study.

BACKGROUND: The role of cytokines in the pathogenesis of febrile seizures (FSs) is unclear, and information regarding cytokine production outside of FS episodes is scarce. METHODS: In our controlled follow-up study of patients with FSs, we compared the levels of 12 serum cytokines after the patients' first FSs, during febrile episodes without FSs, after recurrent FSs, during healthy periods after FSs, and between patients and controls. RESULTS: Two-hundred fifty-one patients with first FS participated in the study, of whom 17 (mean age 1.6 years, SD 0.7) with recurrent FSs completed the protocol as required by the sample size calculations. The mean IL-1RA level was higher after the first FSs (2580 pg/mL, SD 1516) than during febrile episodes without FSs (1336 pg/mL, SD 1364, P = 0.006) and healthy periods after FSs (474 pg/mL, SD 901, P = 0.001). IL-1RA levels were also higher during first (2580 pg/mL) and recurrent FSs (2666 pg/mL, SD 1747) in comparison with febrile controls (746 pg/mL, SD 551) (P < 0.001 and P = 0.001, respectively), but there was no difference in the IL-1RA between febrile episodes without FSs and febrile controls. CONCLUSIONS: Patients with FSs produce stronger inflammatory reactions during febrile episodes with FSs compared with febrile episodes without FSs and febrile controls. IMPACT: In patients with FSs, IL-1RA was higher following first FS than during febrile episodes without FSs and healthy periods after FSs. IL-1RA was higher in patients with FSs following first and recurrent FSs than in febrile controls. There was no significant difference in IL-1RA between febrile episodes of patients without FSs and febrile controls. Using IL-1RA as a surrogate marker of IL-1 axis activity, our results indicate that patients with FSs produced stronger inflammatory reactions during FS episodes but not during other febrile episodes or healthy periods after FSs. Cytokines may play a role in pathogenesis of FSs.

Impaired gap junction formation and intercellular calcium signaling in urinary bladder cancer cells can be improved by Gö6976.

PURPOSE: Calcium wave propagation and connexin 26, 32 and 43 expression were studied in normal and malignant urothelial cells. MATERIALS AND METHODS: Human urothelial cell cultures were established from tissue biopsies obtained from three healthy control persons and compared to human transitional cell carcinoma (TCC) cell line 5637. Fluo-3 was used to study intercellular calcium signaling in urothelial cells. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in intercellular calcium signaling. In addition, Gö6976 was used to determine the effects of PKC alpha and betaI inhibition on intercellular calcium signaling. RESULTS: In normal urothelial cells, the primary pathway for intercellular calcium mediated cell signaling was gap junctional intercellular communication (GJIC), but the paracrine ATP-mediated signaling was also operative. In 5637 TCC cells, GJIC and ATP-mediated signaling routes were altered when compared to normal urothelial cells. More specifically, inhibition of GJIC resulted in a complete block of intercellular calcium signaling, while inhibition of ATP-mediated signaling decreased signal transduction in 5637 TCC cells. The results of the present study also demonstrated that connexin 26 was the most abundant gap junction plaque protein in cultured normal human urothelial cells and that it did not form gap junction plaques in 5637 TCC cell culture. Treatment with Gö6976 induced gap junction plaque formation by connexin 26 in 5637 TCC cells. In addition, the exposure to Gö6976 enhanced intercellular calcium mediated signaling in 5637 TCC cells, but not in normal cells. CONCLUSIONS: The results of the present study suggest that gap junctions play a major role in intercellular calcium signaling in urothelial cells. In addition, intercellular calcium signaling is altered in urinary bladder carcinoma cells, and it can be improved by PKC alpha and betaI inhibition. (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; Movie files of Fig. 2normal Gö6976-, normal Gö6976+, TCC Gö6976-, TCC Gö6976+ and image of Supplementary Figure 1).

The effect of extracellular calcium concentration on calcium-mediated cell signaling in NF1 tumor suppressor-deficient keratinocytes.

Capacitative calcium entry and calcium wave propagation were studied in keratinocytes from healthy volunteers and patients with type 1 neurofibromatosis (NF1) in calcium-depleted and in low calcium culture medium. In previous studies, we found evidence that mutations of the NF1 tumor suppressor gene can lead to altered calcium-mediated cell signaling in keratinocytes cultured in the presence of a high extracellular calcium concentration. The present study demonstrated that the differences between normal and NF1 keratinocytes were dependent on extracellular calcium concentration. Specifically, when keratinocytes were exposed to thapsigargin under calcium-depleted culture conditions the subsequent increase in free intracellular calcium concentration was moderate in NF1 keratinocytes compared to controls. The finding indicates lowered endoplasmic calcium stores in NF1 which may also in part explain the reduced activation signal for capacitative calcium influx and the wound-induced intracellular Ca2+ transient observed in NF1 keratinocytes maintained in culture medium containing 0.05 mM calcium. The differences between control and NF1 keratinocytes were most pronounced when the cells were cultured in the presence of a high (1.8 mM) calcium concentration. Since elevated extracellular calcium levels induce keratinocytes to form cellular contacts and lead to terminal differentiation, markedly aberrant responses of NF1 keratinocytes in the presence of a high calcium concentration may help to explain previous findings on impaired formation of cellular junctions and differentiation in NF1 deficient cells.

Reevaluation of the normal epidermal calcium gradient, and analysis of calcium levels and ATP receptors in Hailey-Hailey and Darier epidermis.

Electron probe microanalysis was used to analyze elemental content of human epidermis. The results revealed that the calcium content of the basal keratinocyte layer was higher than that of the lowest spinous cell layer in normal epidermis. This was surprising, as it is generally accepted that the calcium level increases with cellular differentiation from the proliferative basal layer to the stratum corneum. Hailey-Hailey disease (HHD) and Darier disease (DD) are caused by mutations in Ca(2+)-ATPases with the end result of desmosomal disruption and suprabasal acantholysis. The results demonstrated three major aberrations in HHD and DD lesions. First, in HHD and DD lesions the calcium content in the basal layer was lower than in the normal skin. Second, adenosine triphosphate (ATP) receptor P2Y2 was not localized to plasma membrane in acantholytic cells, whereas P2X7 appeared in the plasma membrane, potentially mediating apoptosis. Third, transition of keratin 14 to keratin 10 was abnormal as demonstrated by the presence of keratinocytes expressing both cytokeratins, which are usually exclusive in normal epidermis. Our results provide to our knowledge previously unreported elements for understanding how the disturbed calcium gradient is linked to the alterations in ATP receptors and keratin expression, leading to the clinical findings in HHD and DD.

Collagenase-2 (MMP-8) and matrilysin-2 (MMP-26) expression in human wounds of different etiologies.

Wound healing involves highly controlled events including reepithelialization, neoangiogenesis, and reformation of the stromal compartment. Matrix metalloproteinases (MMPs) are a family of neutral zinc-dependent endopeptidases known to be essential for the wound-healing process. MMP-8 (collagenase-2) is a neutrophil-derived highly effective type I collagenase, recently indicated to be important for acute wound healing. MMP-26 is a more recent and less well-studied member of the MMP family. Our aim was to study the expression of MMP-8 and MMP-26 in human cutaneous wound repair and chronic wounds using histological methods and cell culture. MMP-8 expression was associated with epithelial cells, neutrophils, and other inflammatory cells in chronic human wounds. MMP-26 was prominently expressed in the extracellular compartment of most chronic wounds in close vicinity to the basement membrane area. MMP-26 was also expressed in acute day 1 wounds with declining expression thereafter. In vitro wound experiments showed that both MMP-8 and MMP-26 were expressed by migrating human mucosal keratinocytes. Inhibiting MMP-26 resulted in aberrant keratinocyte migration and proliferation. We conclude that MMP-8 and MMP-26 are differentially expressed in acute and chronic wounds.

An intact actin-containing cytoskeleton is required for capacitative calcium entry, but not for ATP-induced calcium-mediated cell signaling in cultured human keratinocytes.

BACKGROUND: The present study was focused on structural relationship between intracellular calcium stores and plasma membrane store-operated calcium channels in cultured normal and NF1 keratinocytes. MATERIAL/METHODS: Calcium mobilization induced by thapsigargin or extracellular ATP was studied in control and cytochalasin D-treated human keratinocytes. RESULTS: Treatment of keratinocytes with cytochalasin D disrupted the actin cytoskeleton and changed the cells from a planar, extended morphology, to a rounded shape. In normal control keratinocytes, thapsigargin induced a marked increase in intracellular calcium concentration ([Ca2+]i). The capacitative calcium influx of cytochalasin D-treated normal keratinocytes was significantly weaker compared to normal control cells. In normal keratinocytes, ATP induced a rapid and transient increase in [Ca2+]i. Thus disruption of the cytoskeleton blocked thapsigargin-induced calcium mobilization, but had no effect on ATP-induced [Ca2+]i mobilization in keratinocytes. The results suggest that microfilaments play crucial role for functional capacitative Ca2+ entry in cultured keratinocytes. The cytoskeleton and calcium mediated cell signaling have been demonstrated to be abnormal in keratinocytes cultured from patients with neurofibromatosis type 1 (NF1). In NF1 keratinocytes, thapsigargin induced a slow and moderate increase in [Ca2+]i. The effect of cytochalasin D on NF1 keratinocytes was less pronounced compared to normal keratinocytes. In NF1 keratinocytes, ATP induced a rapid and transient increase in [Ca2+]i. CONCLUSIONS: The actin microfilaments play a crucial role for functional capacitative Ca2+ entry in cultured keratinocytes, and that aberrant organization of cytoskeleton may partly explain altered calcium-mediated cell signaling in NF1.

Altered calcium-mediated cell signaling in keratinocytes cultured from patients with neurofibromatosis type 1.

Capacitative calcium entry and calcium wave propagation were studied in keratinocytes cultured from control persons and patients with type 1 neurofibromatosis. The cells were stimulated mechanically in the presence of inhibitors of gap-junctional or ATP-mediated communication to determine which pathways are operative in Ca(2+) signaling between these cells. Keratinocytes cultured from patients with type 1 neurofibromatosis (NF1) had a tendency to form cultures with markedly altered calcium-related signaling characteristics. Specifically, the resting Ca(2+) levels, intracellular Ca(2+) stores, capacitative calcium influx, and gap-junctional signal transduction were defective in NF1 keratinocytes. Western transfer analysis revealed apparently equal connexin 43 protein levels in normal control and in NF1 keratinocytes. Indirect immunofluorescence, however, demonstrated that connexin 43 was relatively evenly distributed in NF1 cells and did not form typical gap-junctional plaques between keratinocytes. Furthermore, the speed of the calcium wave was reduced in NF1 cells compared to normal keratinocytes. The results demonstrate that keratinocytes cultured from patients with NF1 display altered calcium-mediated signaling between cells.

NF1 tumor suppressor mRNA is targeted to the cell-cell contact zone in Ca(2+)-induced keratinocyte differentiation.

SUMMARY: We have previously shown that NF1 (type 1 neurofibromatosis) p21ras GTPase-activating tumor suppressor protein undergoes major relocalization during the formation of cell-cell junctions in differentiating keratinocytes in vitro. This prompted us to study the distribution of NF1 mRNA under the same conditions by in situ hybridization. In differentiating keratinocytes, the NF1 mRNA signal intensified within the cell cytoplasm within the first 0.5 to 2 hours after induction of cellular differentiation. First, the hybridization signal was evenly distributed throughout the cytoplasm. Subsequently, NF1 mRNA was gradually polarized to the cellular periphery at the side of cell-cell junctions and finally disappeared. Reappearance of NF1 mRNA was found in migrating keratinocytes forming a bilayered culture. Disruption of microfibrillar cytoskeleton, but not microtubules, caused a marked change in the subcellular distribution of NF1 mRNA. This data may suggest that intact actin microfilaments are essential for transport of NF1 mRNA to the cell periphery. This is the first study demonstrating that NF1, or any tumor suppressor mRNA, belongs to a rare group of mRNAs not targeted to free polysomes or ribosomes of the rough endoplasmic reticulum. This finding recognizes a potential way for post-transcriptional modification of NF1 expression.