Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance graft-versus-host disease via tissue damage.

Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.

Kinetic targeting of pegylated liposomal doxorubicin: a new approach to reduce toxicity during chemotherapy (CARL-trial).

BACKGROUND: The therapeutic success of chemotherapeutic agents is often limited by severe adverse effects. To reduce toxicity of these drugs, nanoscale particle-based drug delivery systems (DDS) are used. DDS accumulate to some extent in tumor tissues, but only a very small portion of a given dose reaches this target. Accumulation of DDS in tumor tissues is supposed to be much faster than in certain other tissues in which side effects occur ("Kinetic Targeting"). Once saturation in tumor tissue is achieved, most of the administered DDS still circulate in the plasma. The extracorporeal elimination of these circulating nanoparticles would probably reduce toxicity. METHODS: For the CARL-trial (Controlled Application and Removal of Liposomal chemotherapeutics), pegylated liposomal doxorubicin (PLD) was used as chemotherapeutic agent and double filtration plasmapheresis (DFPP) was performed for extracorporeal elimination of liposomes. PLD was given as 40 mg/m2 every 3 weeks in combination with vinorelbine 2 × 25 mg/m2 (neoadjuvant treatment of breast cancer, 12 patients), or as 40 mg/m2 every 4 weeks (recurrent ovarian cancer, 3 patients). Primary endpoints were the efficiency and safety profile of DFPP, and secondary endpoints were side effects and tumor response. RESULTS: DFPP eliminated ~62% of circulating PLD, corresponding to ~45% of the total dose (n = 57 cycles). AUC of doxorubicin was reduced by 50%. No leakage of doxorubicin was detected during elimination, and no relevant DFPP-related side effects occurred. Reduction in tumor size > 30% occurred in 10/12 (neoadjuvant) and in 1/3 patients (recurrent). Only five grade 2 events and one grade 3 event (mucositis, neutropenia or leucopenia) and a single palmar-plantar erythrodysesthesia grade 2 were reported. CONCLUSION: Extracorporeal elimination of PLD by DFPP is safe and efficient. CARL can diminish the main dose-limiting side effects of PLD, and probably many different DDS alike. TRIAL REGISTRATION: DRKS00000163.

Controlled application and removal of liposomal therapeutics: effective elimination of pegylated liposomal doxorubicin by double-filtration plasmapheresis in vitro.

INTRODUCTION: Nanoscale particle-based drug delivery systems like long circulating liposomal doxorubicin show unique pharmacokinetic properties and improved toxicity profiles. Liposomal doxorubicin accumulates in tumor tissue due to the enhanced permeation and retention effect, but only a small fraction of a total dose reaches the tumor site. Accumulation of liposomal doxorubicin is much faster in tumor sites than in certain organs where dose limiting adverse effects occur. Finding a way to detoxify the predominant part of a given dose, circulating in the blood after accumulation is completed, will presumably reduce severe side effects during chemotherapy. METHODS: Elimination properties of therapeutic used pegylated liposomal doxorubicin (Doxil/Caelyx) and therapeutic used double-filtration plasmapheresis systems were evaluated in vitro and in reconstituted human blood. RESULTS: Liposomes can be filtered by appropriate membranes without leakage of doxorubicin up to a pressure of 1 bar. At higher pressures, liposomes ( approximately 85 nm) may squeeze through much smaller pores without significant leakage of doxorubicin, whereas decreasing pore size to approximately 8 nm leads to increased leakage of doxorubicin. With therapeutic used apheresis systems, liposomal doxorubicin can be efficiently eliminated out of buffer medium and reconstituted human blood. No leakage of doxorubicin was detected, even when liposomes were circulating for 48 h in human plasma before apheresis. CONCLUSIONS: Convenient apheresis techniques are capable of a safe and efficient elimination of therapeutic used liposomal doxorubicin in an experimental model system.

Controlled application and scheduled removal of nanoparticle based chemotherapeutics (CARL) will reduce dose limiting adverse events in anticancer chemotherapy.

Clinical success of cancer chemotherapy is impaired by dose limiting toxicities. Nanoscale particle based drug delivery systems (DDS) like long circulating liposomes show improved toxicity profiles. Nevertheless, their unique pharmacokinetic properties lead to new dose limiting adverse events such as elevated skin toxicity. Though DDS accumulate in tumor tissue, only a very small fraction of the total dose reaches the target site. The overwhelming amount of a given dose is needed only to build up a diffusion gradient for effective accumulation at the target site. Due to the altered endothelial barrier, accumulation of DDS in tumor tissue is much faster than accumulation in other tissues, where dose limiting side effects occur. On the basis of these pharmakinetic data we hypothesize, that once accumulation in the tumor tissue is completed, rapid elimination of the DDS fraction still circulating in the plasma may diminish otherwise dose limiting toxicities. Rapid elimination of circulating DDS might be performed by extracorporeal apheresis treatment. Within this paper the principle of kinetic targeting by scheduled extracorporeal elimination of long circulating DDS is presented in detail. Benefits for patients are as well discussed as possible criticisms and future developments. In conclusion, the combination of DDS and scheduled apheresis may allow the development of new chemotherapy regiments with higher impact and/or less toxicity.

Elimination of liposomes by different separation principles used in low-density lipoprotein apheresis.

Clinical success of many therapies is impaired by dose limiting toxicities. Nanoscale particle-based drug delivery systems such as liposomes show unique pharmacokinetic properties and improved toxicity profiles. Liposomes accumulate in tumor tissue, but only a small fraction of a total dose reaches the target site. The overwhelming amount of a given dose is needed only to build up a diffusion gradient for effective accumulation at the target site. In order to find a way to detoxify this predominant fraction after accumulation is completed, the different separation principles used for the apheresis of lipoproteins were evaluated for the extracorporeal elimination of liposomes. Appropriate radiolabeled model liposomes were prepared by extrusion. Separation efficacy, leakage of liposomal content and influence of plasma contact were measured. Membranes with pore sizes between 25 and 400 nm were used to investigate filtration properties of liposomes. Liposomes were precipitated by adding heparin and Ca(2+). Adsorption chromatography was investigated using dextran sulfate, heparin sepharose and functionalized polyacrylamide beads. Membrane filtration allowed the elimination of various liposomes, while precipitation and adsorption were only useful for positively charged liposomes. Leakage of liposomal content was not induced by adsorption, but precipitation induced leakage. Leakage during filtration was dependent on liposomal membrane lipids. Plasma contact reduced precipitation and adsorption efficacy of positively charged liposomes, while filtration properties of liposomes remained unchanged. For extracorporeal elimination of liposomal drug delivery systems, filtration-based techniques are presumably more convenient and versatile than precipitation- or adsorption-based apheresis technologies.

Immortalized fibroblasts from NF-kappaB RelA knockout mice show phenotypic heterogeneity and maintain increased sensitivity to tumor necrosis factor alpha after transformation by v-Ras.

Activation of the NF-kappaB pathway can either promote or block apoptosis and oncogenesis in different cell types and circumstances. In this report, we show that independently derived immortalized mouse embryonic fibroblast cell lines prepared from RelA knockout mice have different phenotypes, based on their sensitivity to tumor necrosis factor alpha (TNFalpha)-induced apoptosis, morphology, ability to form colonies in soft agar, and the presence of distinct kappaB site-binding complexes. In addition, these RelA-deficient cell lines appear to have distinct alterations in the p53 pathway, which correlate with the normal vs transformed status of individual cell lines. We have also infected mouse embryonic fibroblasts lacking RelA, c-Rel or p50 with a retrovirus for the expression of v-Ha-Ras to determine whether individual NF-kappaB family members are required for Ras-mediated transformation. All three NF-kappaB-deficient cell types could be transformed by v-Ha-Ras. However, v-Ras-infected RelA-deficient cells formed colonies in soft agar at an approximately fourfold reduced efficiency compared to v-Ras-transformed control mouse 3T3 and p50-deficient cells. Ras transformation did not alter the sensitivity of RelA-deficient cells to TNFalpha-induced apoptosis, and Ras transformation did not affect the general resistance of 3T3, c-Rel-deficient, and p50-deficient cells to TNFalpha-induced apoptosis. However, TNFalpha specifically and dose-dependently decreased the ability of v-Ras-transformed RelA-deficient cells to form colonies in soft agar. These results suggest that RelA is a potential protein target for human tumors driven by oncogenic Ras mutations, but caution that inhibition of RelA may promote tumorigenesis in some circumstances.