Chronic myelogenous leukemia (CML) is characterized by the presence of a BcrAbl fusion protein with deregulated tyrosine kinase activity that is required for maintaining the malignant phenotype. Imatinib, a selective inhibitor of Bcr-Abl, induces major cytogenetic remission (MCR) or complete cytogenetic remission (CCR) in the majority of patients with CML in first chronic phase. However, thorough re-evaluation of cytogenetics in a cohort of patients in MCR or CCR demonstrated clonal karyotypic abnormalities in more than 10% of cases, some of which were clinically associated with a myelodysplastic syndrome (MDS). Further analysis identified previous exposure to cytarabine and idarubicin as significant risk factors for the subsequent occurrence of abnormalities in Philadelphia chromosome-negative (Ph ؊ ) cells. To investigate if cytogenetically normal but clonal hematopoiesis might be present in other patients in cytogenetic remission, we studied X-chromosome inactivation as a marker of clonality by polymerase chain reaction analysis of the human androgen receptor (HUMARA). We find that imatinib restores a polyclonal pattern in most patients in CCR and MCR. Nonetheless, our results are consistent with the notion that targeted therapy of CML with imatinib favors the manifestation of Ph ؊ clonal disorders in some patients. They indicate that patients on imatinib should be followed with conventional cytogenetics, even after induction of

For patients with chronic diseases, especially those with chronic low back pain, the patient-physician relationship is significant for treatment adherence. In a sample of N = 688 low back pain patients, we examined the hypothesis that aspects of the patient-physician relationship (e.g. satisfaction with care, trust in the physician, patient participation) have a significant association with outcomes (pain, disability, quality of life, pain-related psychological impairment) after a multimodal treatment program (rehabilitation) after adjusting for a number of sociodemographic, medical, and psychological factors. Results show that the patient-physician relationship is significantly associated with the outcome. In the medium term (6 months after rehabilitation), the effect of the patient-physician relationship is clearer than in the short term (end of rehabilitation). In addition, risk factors for less improvement are female gender, higher age, low income, comorbidity, low treatment motivation, fear avoidance beliefs, and external locus of control. Future studies should examine the causal paths between the relationship variables and the outcome variables.

Numerous attempts to target viral gene therapy vectors to specific cells have met with limited success. Here we describe a novel virus vector-targeting platform based on a unique combination of genetic and chemical vector particle modifications to overcome typical restrictions in virus vector targeting. We genetically introduced cysteines at solvent-exposed positions of the adenovirus capsid. The corresponding thiol groups were highly reactive, and we established procedures for controlled covalent coupling to them of protein and nonprotein ligands. After the coupling of transferrin, the particles were efficiently targeted to the transferrin receptor pathway. Depending on the chemistry used, ligands could be coupled under the formation of thioether or disulfide bonds, the latter allowing for separation of ligand and particle after cell entry in the endosome. Furthermore, this technology could be efficiently combined with vector shielding for true retargeting: after amino-PEGylation of the vector particles the genetically introduced thiols were still accessible for ligand coupling, and particles could be retargeted to the transferrin receptor. Since this platform is robust, can be scaled, is compatible with industrial standards, and can integrate chemically diverse molecules as ligands, it may be used for clinical gene therapy and, potentially, also for vaccination.

In vivo gene transfer with adenovirus vectors would significantly benefit from a tight control of the adenovirus-inherent liver tropism. For efficient hepatocyte transduction, adenovirus vectors need to evade from Kupffer cell scavenging while delivery to peripheral tissues or tumors could be improved if both scavenging by Kupffer cells and uptake by hepatocytes were blocked. Here, we provide evidence that a single point mutation in the hexon capsomere designed to enable defined chemical capsid modifications may permit both detargeting from and targeting to hepatocytes with evasion from Kupffer cell scavenging. Vector particles modified with small polyethylene glycol (PEG) moieties specifically on hexon exhibited decreased transduction of hepatocytes by shielding from blood coagulation factor binding. Vector particles modified with transferrin or, surprisingly, 5,000 Da PEG or dextran increased hepatocyte transduction up to 18-fold independent of the presence of Kupffer cells. We further show that our strategy can be used to target high-capacity adenovirus vectors to hepatocytes emphasizing the potential for therapeutic liver-directed gene transfer. Our approach may lead to a detailed understanding of the interactions between adenovirus vectors and Kupffer cells, one of the most important barriers for adenovirus-mediated gene delivery.

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Heat shock proteins (Hsp) of the Hsp70/90 families facilitate cellular immune responses to antigenic peptides or proteins bound to them and have therefore been used as vaccine vehicles. We developed an expression system in which chimeric proteins with an Hsp-capturing, viral J domain fused to diverse antigen-encoding sequences form stable complexes with eukaryotic (Hsp70, Hsp73) or bacterial (DnaK) stress proteins and accumulate to high steady-state levels. J domains from different species (viruses/SV40, bacteria/Chlamydia trachomatis or plants/Arabidopsis thaliana) efficiently capture murine or human stress proteins in this system, thus making different J domains available for vaccine production. A novel expression and purification method was developed to produce native Hsp/antigen complexes in transfectants. These purified Hsp/antigen complexes efficiently elicited antigen-specific CD8 T cell responses in mice when delivered as vaccines without adjuvants. In situ complex formation of antigen with Hsp was critical for CD8 T cell priming. Because the described expression system supports the flexible design of multivalent vaccines, it is an attractive strategy to elicit CD8 T cell responses either to recombinant proteins or to selected antigenic domains of these molecules.