TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK.

Occludin is a key tight junction (TJ) protein in cerebral endothelial cells (CECs) playing an important role in modulating blood-brain barrier (BBB) functions. This protein (65kDa) has been shown to engage in many signaling pathways and phosphorylation by both tyrosine and threonine kinases. Despite yet unknown mechanisms, pro-inflammatory cytokines and endotoxin (lipopolysaccharides, LPS) may alter TJ proteins in CECs and BBB functions. Here we demonstrate the responses of occludin in an immortalized human cerebral endothelial cell line (hCMEC/D3) to stimulation by TNFα (10 ng/mL), IL-1ß (10 ng/mL) and LPS (100 ng/mL). Exposing cells to TNFα resulted in a rapid and transient upward band-shift of occludin, suggesting of an increase in phosphorylation. Exposure to IL-1ß produced significantly smaller effects and LPS produced almost no effects on occludin band-shift. TNFα also caused transient stimulation of p38MAPK and ERK1/2 in hCMEC/D3 cells, and the occludin band-shift induced by TNFα was suppressed by SB202190, an inhibitor for p38MAPK, and partly by U0126, the MEK1/2-ERK1/2 inhibitor. Cells treated with TNFα and IL-1ß but not LPS for 24 h resulted in a significant (p < 0.001) decrease in the expression of occludin, and the decrease could be partially blocked by SB202190, the inhibitor for p38MAPK. Treatment with TNFα also altered cell morphology and enhanced permeability of the CEC layer as measured by the FITC-dextran assay and the trans-endothelial electrical resistances (TEER). However, treatment with SB202190 alone could not effectively reverse the TNFα -induced morphology changes or the enhanced permeability changes. These results suggest that despite effects of TNFα on p38MAPK-mediated occludin phosphorylation and expression, these changes are not sufficient to avert the TNFα-induced alterations on cell morphology and permeability.

Enhanced delivery of human growth hormone across cell membrane by Tat-PTD.

In this study, we utilized the HIV protein Tat protein transduction domain (Tat-PTD) to enhance the intestinal absorption of recombinant human growth hormone (rhGH) delivered by oral administration. Insulin-like growth factor 1 (IGF-1), the key factor in the GH signal transduction pathway, was differentially affected at the mRNA level by various concentrations of rhGH. At high rhGH concentrations (500 ng/ml), IGF-1 was downregulated, while low concentrations (5 ng/ml) caused IGF-1 upregulation. The addition of Tat-PTD had a significant facilitating effect on rhGH. Frozen tissue sections visualized with fluorescence microscopy, cultured cells visualized by confocal microscopy and flow cytometry all confirmed that rhGH fused to Tat-PTD demonstrated more intracellular fluorescent signal when compared to rhGH alone. ELISA showed that after 2 h of incubation, Tat-rhGH levels in the rat intestinal cavity were 1.38-fold higher than rhGH. These data indicated that Tat-PTD effectively improved the internalization of rhGH and enhance the signal transduction of rhGH, possibly laying a solid foundation for the novel oral administration of rhGH.

Caspofungin-induced severe toxic epidermal necrolysis.

OBJECTIVE: To report a case of suspected caspofungin-induced toxic epidermal necrolysis (TEN). CASE SUMMARY: An 86-year-old man had a right femur intertrochanteric fracture and right proximal humerus fracture due to an accidental fall. Disseminated Candida krusei infection complicated the postoperative course. Candidemia persisted despite 25 days of treatment with parenteral fluconazole. The antifungal agent was changed to intravenous caspofungin. Immediately after administration of a caspofungin 70-mg loading dose, a transient skin rash developed, which resolved after discontinuation of the drug and immediate treatment with intravenous diphenhydramine 30 mg and methylprednisolone 40 mg. Six days later, a caspofungin 70-mg loading dose was given again due to increasing sepsis. Erythematous and purpuric macules and plaques developed the next day and rapidly progressed to extensive erythema, exfoliation, blisters, and skin erosions. A dermatologist was consulted and TEN was diagnosed. The patient was treated with intravenous hydrocortisone 100 mg every 8 hours and diphenhydramine 30 mg every 8 hours. The skin lesions progressed unrelentingly and the patient died of refractory shock 6 days later. DISCUSSION: TEN is a rare but life-threatening systemic and cutaneous disease that is most often the result of an adverse drug reaction. It usually manifests as fever and influenza-like symptoms 1-3 days before the development of mucocutaneous lesions, namely erythema and erosions of the buccal, ocular, and genital mucosa and characteristic epidermal detachment. The precise pathogenesis is not fully understood. Several models have been described, including immunopathology, genetic susceptibility, Fas-Fas ligand, perforin/granzyme, and cytokine dysregulation. Use of the Naranjo probability scale indicated a probable relationship between caspofungin and the development of TEN in this patient. CONCLUSIONS: This is the first report of caspofungin-induced TEN. Health-care professionals are advised to be aware of the early presentations of TEN in patients receiving caspofungin.