Sublingual tacrolimus as an alternative to intravenous route in patients with thoracic transplant: a retrospective study.

Tacrolimus (TRL) is an immunosuppressive drug characterized by a narrow therapeutic index, low bioavailability, and pharmacokinetic variability. Intravenous (i.v.) TRL may be needed whenever the oral route is unavailable. The small amount of infusion formulation (5 mg/mL) results in a large dilution and need for careful technical management of the infusion. This study addressed the feasibility to provide sublingual (SL) as an alternative to i.v.. TRL for transplanted patients. In a substudy, we performed a retrospective analysis of 17 lung and heart transplant patients using SL TRL. It included therapeutic drug monitoring and 4 area under curve (AUC) measurements. Patients received SL TRL on a dose-to-dose basis from the oral formulation. The mean age of the subjects (14 male, 3 female) was 35.3 ± 15.6 years; 146 trough (C(0)) samples were collected during the SL period (15.8 ± 20.6 days) showing a conformity level of 90.4%. Mean dose, C(0), and AUC of SL tacrolimus were 0.116 ± 0.096 mg/kg, 12.9 ± 5 ng/mL, and 230 ± 74 ng·h/mL, respectively, with an average 1 hour time to peak concentration. Acute rejection episodes, renal toxicity, and drug interactions were not observed. This study supported the convenience of short-term SL TRL administration, even in unconscious patients. Further investigations are needed to validate the dose range of the SL route.

Valganciclovir prophylaxis for cytomegalovirus infection in thoracic transplant patients: retrospective study of efficacy, safety, and drug exposure.

Oral ganciclovir (GCV) was replaced by prodrug valganciclovir (vGCV) for cytomegalovirus (CMV) prophylaxis. We assessed retrospectively (2005-2007) vGCV effectiveness and safety during prophylaxis and 4 months after, in heart (HTx) and lung transplantation (LTx), including lung transplant for cystic fibrosis (CFTx). Patients with stable renal function received vGCV 900 mg daily during 3-6 and 8-12 months in HTx and LTx. Effectiveness was assessed by antigenemia (pp65Ag) and a GCV therapeutic drug monitoring to document exposure. A total of 32 patients (11 HTx, 7 LTx, and 14 CFTx) received vGCV for 106+/-67 days in HTx versus 270+/-85 days in LTx and CFTx. Doses were 700+/-225, 915+/-60, and 820+/-150 mg/24 h in HTx, LTx, and CFTx showing acceptable mean trough GCV 0.75+/-0.5 mg/L. Two of 9 cases of neutropenia were attributable to vGCV. Three CMV donor-positive/recipient-negative CFTx patients presented positive pp65Ag; 2 developed CMV disease (6%). We found that vGCV 900 mg, adapted to renal function, was effective and safe for long CMV prophylaxis together with efficient exposure in thoracic transplantation.

[Evaluation of flow rate regulators for intravenous infusion]. / Evaluation des régulateurs de débit passifs utilisés pour la perfusion intraveineuse.

OBJECTIVE: The aim of our study is to evaluate seven flow rate regulators (FRR) to assess the reliability of these devices compared to standard perfuser with roller clamp. STUDY DESIGN: Each FRR was tested with 5% dextrose and 0.9% sodium chloride combined with three different theoretical flow rates (30, 80 and 250 ml/h). Accuracy was compared with the theoretical value. Repeatability of flow rate was assessed thanks to variance break-up. RESULTS: Each FFR exhibits at least one combination "flow rate-solution" significantly different of the theoretical flow rate. Exadrop was the least successful of the FFR according to the accuracy. This FFR had for each combination a flow rate different of the theoretical (mean error: -24.0 ml/h). Tutodrop was the most successful of the FFR according to the accuracy with five combinations comparable to the theoretical value (mean error: -1.2 ml/h). The standard perfuser with roller clamp, used without FRR, reported two combinations comparable to the theoretical value and showed lowest rates for repeatability. CONCLUSION: Our study exhibits the poor performances of the FRR studied: according to expected flow regulation, the reported results demonstrate the lack of accuracy. Their only one value added compare to the roller clamp is to improve the repeatability of the flow rate.

High levels of p105 (NFKB1) and p100 (NFKB2) proteins in HPV16-transformed keratinocytes: role of E6 and E7 oncoproteins.

We have previously shown that functional components of the NF-kappaB signaling pathway are up-regulated and sequestered in the cytoplasm of human papillomavirus 16 (HPV16)-transformed cell lines leading to a reduced activity of NF-kappaB. In this study, we examined the expression of the NF-kappaB precursors p100 and p105 in keratinocytes transformed or not by HPV16. Western immunoblotting experiments demonstrated high levels of p100 and p105 proteins not only in HPV16+ cervical carcinoma-derived keratinocytes but also in keratinocytes stably transfected by HPV16 E6 or E7 oncogenes. Moreover, p100 and p105 proteins were predominantly cytoplasmic and nuclear in keratinocytes expressing E7 and E6, respectively. A predominantly cytoplasmic localization of E7 protein was also detected in all keratinocytes expressing E7. Our results suggest that HPV16 E6 and E7 proteins modulate the expression and the subcellular localization of p100 and p105 NF-kappaB precursors.

Differential production of cytokines and activation of NF-kappaB in HPV-transformed keratinocytes.

We have proposed that chronic infection of keratinocytes by HPV modifies the expression of potentially important cytokines by interfering with the NF-kappaB signal pathway. We evaluated the constitutive and IL-1beta-induced expression of GM-CSF and TNF-alpha and the expression/activity of NF-kappaB in HPV+ and HPV- cell lines. Despite the enhanced expression of the functional components of the NF-kappaB signaling pathway in HPV+ cell lines by a mechanism implicating the HPV oncoprotein E6, the constitutive activity of NF-kappaB and the expression of GM-CSF/TNF-alpha were significantly reduced relative to the HPV- cell line and normal keratinocytes. In contrast, we observed a superactivation of NF-kappaB activity after IL-1beta stimulation, a strong and transient induction of GM-CSF/TNF-alpha mRNA, but undetectable levels of secreted proteins in HPV+ cell lines. Our data demonstrate that E6 modulates the NF-kappaB signaling pathway and suggest that other HPV proteins also interfere with GM-CSF/TNF-alpha expression by transcriptional and/or posttranscriptional mechanisms.

The organotypic culture of HPV-transformed keratinocytes: an effective in vitro model for the development of new immunotherapeutic approaches for mucosal (pre)neoplastic lesions.

The purpose of this study is to develop a reliable in vitro human model to test new immunotherapeutic approaches for squamous cell carcinoma that develop on mucosal surfaces. The organotypic (raft) culture permits cells to proliferate and differentiate at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while human papillomavirus-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to (pre)neoplastic lesions seen in vivo. We have demonstrated the ability of these organotypic cultures to be manipulated by altering the epithelial stratification with cytokines (interferon-gamma and tumor necrosis factor-alpha) and by integrating activated lymphocytes or dendritic cells into the in vitro formed epithelial sheet. This model may provide a useful tool to investigate the factors contributing to the presence and function of immunocompetent cells within a neoplastic epithelium that develops on a mucosal surface.

Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity.

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.