Cultivation of hepatoma cell line HepG2 on nanoporous aluminum oxide membranes.

Nanoporous aluminum oxide membranes were prepared by anodic oxidation of aluminum for application as novel cell culture substrates. Self-supporting as well as mechanically stabilized nanoporous membranes were produced from aluminum plates and micro-imprinted aluminum foils, respectively. Membranes of two different pore sizes (70 and 260 nm) were selected to investigate cellular interactions with such nanoporous substrates using cells of hepatoma cell line HepG2. The membranes express excellent cell-growth conditions. As shown by scanning electron microscopy investigations, the cells could easily adhere to the membranes and proliferate during a 4 day cell culture period. The cells exhibit normal morphology and were able to penetrate into pores with a diameter of 260 nm by small extensions (filopodia). On mechanically stabilized aluminum oxide membranes it was observed that the cells even adhere to the walls of the small cavities. It was demonstrated experimentally that the nanoporous aluminum oxide membranes are well suited as substrates in cell culture model systems for metabolic, pharmacological/toxicological research, tissue engineering and studies on pathogens as well as bioartificial liver systems.

Treatment of non-small cell lung cancer with intensity-modulated radiation therapy in combination with cetuximab: the NEAR protocol (NCT00115518).

BACKGROUND: Even today, treatment of Stage III NSCLC still poses a serious challenge. So far, surgical resection is the treatment of choice. Patients whose tumour is not resectable or who are unfit to undergo surgery are usually referred to a combined radio-chemotherapy. However, combined radio-chemotherapeutic treatment is also associated with sometimes marked side effects but has been shown to be more efficient than radiation therapy alone. Nevertheless, there is a significant subset of patients whose overall condition does not permit administration of chemotherapy in a combined-modality treatment. It could be demonstrated though, that NSCLCs often exhibit over-expression of EGF-receptors hence providing an excellent target for the monoclonal EGFR-antagonist cetuximab (Erbitux) which has already been shown to be effective in colorectal as well as head-and-neck tumours with comparatively mild side-effects. METHODS/DESIGN: The NEAR trial is a prospective phase II feasibility study combining a monoclonal EGF-receptor antibody with loco-regional irradiation in patients with stage III NSCLC. This trial aims at testing the combination's efficacy and rate of development of distant metastases with an accrual of 30 patients. Patients receive weekly infusions of cetuximab (Erbitux) plus loco-regional radiation therapy as intensity-modulated radiation therapy. After conclusion of radiation treatment patients continue to receive weekly cetuximab for 13 more cycles. DISCUSSION: The primary objective of the NEAR trial is to evaluate toxicities and feasibility of the combined treatment with cetuximab (Erbitux) and IMRT loco-regional irradiation. Secondary objectives are remission rates, 3-year-survival and local/systemic progression-free survival.

Intensity-modulated radiotherapy with an integrated boost to the macroscopic tumor volume in the treatment of high-grade gliomas.

Integrated boost radiotherapy (IBRT) delivers a higher fraction size to the gross tumor volume and a conventional fraction size to the surrounding tissue of microscopic spread. We compared stereotactic conformal radiotherapy (SCRT) and intensity-modulated radiotherapy (IMRT) with regard to their suitability for IBRT in the treatment of high-grade gliomas. In 20 patients treated with conventional radiotherapy, an additional treatment plan for IBRT [planning target volume (PTV1) defined as contrast-enhancing lesion plus margin due to setup errors 75 Gy, PTV2 defined as edema plus margin due to microscopic spread and setup error 60 Gy] with 7 non-coplanar beams for IMRT and for SCRT was carried out and compared. The part of the PTV2 irradiated with more than 107% of the prescribed dose was 13.9% for IMRT and 30.9% for SCRT (P < 0.001). Dose coverage of PTV2 (volume above 95% of the prescribed dose) was improved with IMRT (88.4% vs. 75.3% with SCRT, P < 0.001). Dose coverage of PTV1 was slightly higher with SCRT (93.7% vs. 87.5% with IMRT), but the conformity to the boost shape was improved by IMRT [conformity index (COIN95) = 0.85 vs. 0.69 with SCRT]. Simultaneously the brain volume irradiated with > 50 Gy was reduced from 60 to 33 cc (P < 0.001). We conclude that IMRT is suitable for local dose escalation in the enhancing lesion and for delivering a homogeneous dose to the PTV2 outside the PTV1 at the same time. Our encouraging results justify application of IMRT for IBRT in the treatment of high-grade gliomas. For clinical evaluation a phase III study has been initiated.

High efficacy of fractionated stereotactic radiotherapy of large base-of-skull meningiomas: long-term results.

PURPOSE: Large skull-base meningiomas are difficult to treat due to their proximity or adherence to critical structures. We analyzed the long-term results of patients with skull-base meningiomas treated by a new approach with high-precision fractionated stereotactic radiotherapy. PATIENTS AND METHODS: One hundred eighty-nine patients with benign meningiomas were treated with conformal fractionated stereotactic radiotherapy between 1985 and 1998. Patients were undergoing a course of radiotherapy either as primary treatment, following subtotal resection, or for recurrent disease. The median target volume was 52.5 mL (range, 5.2 to 370 mL). The mean radiation dose was 56.8 Gy (+/- 4.4 Gy). Follow-up examinations, including magnetic resonance imaging, were performed at 6-month intervals thereafter. RESULTS: The median follow-up period was 35 months (range, 3 months to 12 years). Overall actuarial survival for patients with World Health Organization (WHO) grade I meningiomas was 97% after 5 years and 96% after 10 years. Local tumor failure was observed in three of 180 patients with WHO grade I tumors and was significantly higher in two of nine patients with WHO grade II tumors. A volume reduction of more than 50% was observed in 26 patients (14%). Preexisting cranial nerve symptoms resolved completely in 28% of the patients. Clinically significant treatment-induced toxicity was seen in 1.6% of the patients. No treatment-related deaths occurred. CONCLUSION: The results of this study demonstrate that fractionated stereotactic radiotherapy is safe and effective in the therapy of subtotally resected or unresectable meningiomas. The overall morbidity and incidence subacute and late side effects of this conformal radiotherapy approach were low.

The role of proton therapy in the treatment of large irradiation volumes: a comparative planning study of pancreatic and biliary tumors.

PURPOSE: The purpose of this study was to examine the potential benefit of proton therapy for abdominal tumors. Extensive comparative planning was conducted investigating the most up-to-date photon and proton irradiation technologies. METHODS AND MATERIALS: A number of rival plans were generated for four patients: two inoperable pancreatic tumors, one inoperable and one postoperative biliary duct tumor. The dose prescription goal for these large targets was 50 Gy, followed by a boost dose up to 20 Gy to a smaller planning target volume (PTV). Photon plans were developed using "forward" planning of coplanar and noncoplanar conformal fields and "inverse" planning of intensity-modulated (IM) fields. Proton planning was simulated as administered using the so called spot-scanning technique. Plans were evaluated on the basis of normal tissues' dose-volume constraints (Emami B, Lyman J, Brown A, et al. Tolerance of normal tissue to therapeutic irradiation. Int J Radiat Oncol Biol Phys 1990;21:109-122) and coverage of treatment volumes with prescribed doses. RESULTS: For all cases, none of the forward calculated photon plans was able to deliver 50 Gy to large PTVs at the same time respecting the dose-volume constraints on all critical organs. Nine evenly spaced IM fields achieved or nearly achieved all maximum dose constraints to critical structures for two out of three inoperable patients. IM plans also obtained good results for the postoperative patient, even though the dose to the liver was very close to the maximum allowed. In all cases, photon irradiation of large PTV1s to 50 Gy followed by a 20 Gy boost entailed a risk very close to or higher than 5% for serious complications to the kidneys, liver, or bowel. Simple arrangements of 2, 3, and 4 proton fields obtained better dose conformation to the target, allowing the delivery of planned doses including the boost to all patients, without excessive risk of morbidity. Dose homogeneity inside the targets was also superior with protons. CONCLUSION: For the irradiation of large PTVs located in the abdominal cavity, where multiple, parallel structured organs surround the target volumes, proton therapy, delivered with a sophisticated isocentric technique, has the potential to achieve superior dose distributions compared with state-of-the-art photon irradiation techniques. IM photon plans obtain better results in the postoperative case, because the reduced volume lessens the effect of the unavoidable increase of integral dose to surrounding tissues.

Synthetic endotoxin-binding peptides block endotoxin-triggered TNF-alpha production by macrophages in vitro and in vivo and prevent endotoxin-mediated toxic shock.

Lipid A, the conserved portion of endotoxin, is the major mediator of septic shock; therefore, endotoxin-neutralizing molecules could have important clinical applications. Here we show that peptides derived from Limulus anti-LPS factor (LALF), bactericidal/permeability increasing protein (BPI) and endotoxin-binding protein, bind to lipid A and block the recombinant LALF/lipid A interaction in vitro. Because their neutralizing capacity in vitro as well as in vivo has been limited, we created hybrid peptides comprising two endotoxin-binding domains. The hybrid molecule LL-10-H-14, containing endotoxin-binding domains from LALF and endotoxin-binding protein, turned out to be the most active peptide within the series of peptides tested here to inhibit the CD14/lipid A interaction and is able in vitro to block the endotoxin-induced TNF-alpha release of murine macrophages up to 90%. Furthermore, LL-10-H-14 not only reduced peak serum levels of TNF-alpha of mice when preinjected but also reduced TNF-alpha levels when given 15 min after the endotoxin challenge. As compared with other peptides, only LL-10-H-14 is able to strongly decrease endotoxin-stimulated TNF-alpha release by human macrophage cell lines as well as by PBMC. Furthermore, the hybrid peptide is protective against endotoxin-provoked lethal shock. As such, LL-10-H-14 could have prophylactic and/or therapeutic properties in humans for the management of septic shock.

Methods for transferring patient and plan data between radiotherapy treatment planning systems.

The effectiveness of conformal radiotherapy can ultimately only be assessed by the use of clinical trials. As large multicentre clinical trials become more widespread, methods of transferring patient and plan data between radiotherapy treatment planning systems become increasingly important. In this paper, the general strategy for the transfer of data is discussed, and also illustrated with reference to two specific systems: TARGET 2 (GE Medical Systems) and VOXELPLAN (DKFZ-Heidelberg). The transfer method involves using a computer program to translate the data formats used by each of the two systems for CT scans, patient outlines, plan information and block descriptions. This paper does not address the question of transferring beam data between systems: beam data must first be entered separately into both machines. The physical concepts encountered when transferring plans are described, with specific reference to the two planning systems used. Differences in the strategies used by the two planning systems for definition of irregular field shapes are compared. The dose calculations used by the two systems are also briefly evaluated. Isodoses produced by VOXELPLAN around a circular target volume are found to be up to 3 mm different in location to those produced by TARGET 2, owing to the use of a smooth field shape contour as opposed to a stepped field shape which closely models the leaves of a multileaf collimator. In general, dose distributions generated by both systems are comparable, but some differences are found in the presence of large tissue inhomogeneities. It is concluded that the transfer of patient and plan data between two different treatment planning systems is feasible, provided that any differences in field shape definition methods or dose calculation methods between the two systems are understood.

High affinity endotoxin-binding and neutralizing peptides based on the crystal structure of recombinant Limulus anti-lipopolysaccharide factor.

Lipid A, the conserved portion of endotoxin or lipopolysaccharide, is the major mediator of septic shock, and therefore endotoxin-neutralizing molecules could have important clinical applications. The crystal structure of recombinant Limulus anti-lipopolysaccharide factor (rLALF) (Hoess, A., Watson, S., Siber, G. R., and Liddington, R. (1993) EMBO J. 12, 3351-3356), has been used to design synthetic peptides comprising different parts of the exposed amphipathic loop in the proposed endotoxin-binding domain of rLALF. We investigated the minimal requirements of rLALF for endotoxin and lipid A binding with linear 10-mer peptides. Only one linear peptide, corresponding to amino acids 36-45 of rLALF, was able to bind lipid A and endotoxin above background levels. Cyclic peptides, however, bind lipid A and endotoxin with high affinity, presumably by mimicking the three dimensional characteristics of the exposed hairpin loop. The cyclic peptide including amino acids 36-47, LALF-14, has a lipid A binding activity comparable to the high affinity endotoxin-binding peptide polymyxin B. LALF-14 has an improved serum half-life compared with its linear counterpart, and it is not toxic for cultured human monocytes or red blood cells. In mice, it blocks tumor necrosis factor-alpha induction after endotoxin challenge. The characterization of the minimal endotoxin-binding domain of rLALF and, importantly, its structure provided a basis for designing small molecules that could have prophylactic and/or therapeutic properties in humans for the management of septic shock.

Multivalent antibody fragments with high functional affinity for a tumor-associated carbohydrate antigen.

We report in this work a human-derived self-assembling polypeptide based on the tetramerization domain of the human transcription factor p53, which can be fused to single-chain Fv Ab (scFv) fragments via a long and flexible hinge sequence of human origin, allowing exploitation of the functional affinity increase of binding to a ligand or cell surface with multimeric binding sites. We have demonstrated the use of this polypeptide by applying it to the construction of a tetrameric scFv against the tumor-associated carbohydrate Ag Lewis Y (Fuc alpha 1-->2Gal beta 1-->4[Fuc alpha 1-->3] GlcNAc beta 1-->3R). For comparison purposes, the corresponding scFv and dimeric mini-antibody, comprising the scFv fused via a flexible murine hinge to an artificial dimerization domain, were also created. The recombinant mini-antibody proteins were expressed in functional form in Escherichia coli and showed the expected m.w. of a dimer and tetramer, respectively. Analysis of Lewis Y-binding behavior by surface plasmon resonance revealed specific but very weak binding of the scFv fragment. In contrast, both dimeric and tetrameric scFv fusion proteins exhibited an enormous gain in functional affinity that was greatest in the case of the tetrameric mini-antibody.

Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A resolution.

Lipopolysaccharide (LPS), or endotoxin, is the major mediator of septic shock, a serious complication of Gram-negative bacterial infections in humans. Molecules that bind LPS and neutralize its biological effects or enhance its clearance could have important clinical applications. Limulus anti-LPS factor (LALF) binds LPS tightly, and, in animal models, reduces mortality when administered before or after LPS challenge or bacterial infection. Here we present the high resolution structure of a recombinant LALF. It has a single domain consisting of three alpha-helices packed against a four-stranded beta-sheet. The wedge-shaped molecule has a striking charge distribution and amphipathicity that suggest how it can insert into membranes. The binding site for LPS probably involves an extended amphipathic loop, and we propose that two mammalian LPS-binding proteins will have a similar loop. The amphipathic loop structure may be used in the design of molecules with therapeutic properties against septic shock.