A phase I/II minor histocompatibility antigen-loaded dendritic cell vaccination trial to safely improve the efficacy of donor lymphocyte infusions in myeloma.

Allogeneic stem cell transplantation (allo-SCT) with or without donor lymphocyte infusions (DLI) is the only curative option for several hematological malignancies. Unfortunately, allo-SCT is often associated with GvHD, and patients often relapse. We therefore aim to improve the graft-versus-tumor effect, without increasing the risk of GvHD, by targeting hematopoietic lineage-restricted and tumor-associated minor histocompatibility antigens using peptide-loaded dendritic cell (DC) vaccinations. In the present multicenter study, we report the feasibility, safety and efficacy of this concept. We treated nine multiple myeloma patients with persistent or relapsed disease after allo-SCT and a previous DLI, with donor monocyte-derived mHag-peptide-loaded DC vaccinations combined with a second DLI. Vaccinations were well tolerated and no occurrence of GvHD was observed. In five out of nine patients, we were able to show the induction of mHag-specific CD8+ T cells in peripheral blood. Five out of nine patients, of which four developed mHag-specific T cells, showed stable disease (SD) for 3.5-10 months. This study shows that mHag-based donor monocyte-derived DC vaccination combined with DLI is safe, feasible and capable of inducing objective mHag-specific T-cell responses. Future research should focus on further improvement of the vaccination strategy, toward translating the observed T-cell responses into robust clinical responses.

Efficacy of host-dendritic cell vaccinations with or without minor histocompatibility antigen loading, combined with donor lymphocyte infusion in multiple myeloma patients.

Donor lymphocyte infusions (DLI) can induce durable remissions in multiple myeloma (MM) patients, but this occurs rather infrequently. As the graft-versus-tumor (GvT) effect of DLI depends on the presence of host-dendritic cells (DCs), we tested in a phase I/II trial whether the efficacy of DLI could be improved by simultaneous vaccination with host-DCs. We also analyzed the possibility of further improving the GvT effect by loading the DCs with peptides of mismatched hematopoietic cell-specific minor histocompatibility antigens (mHags). Fifteen MM patients not responding to a first DLI were included. Eleven patients could be treated with a second equivalent dose DLI combined with DC vaccinations, generated from host monocytes (moDC). For four patients, the DC products did not meet the quality criteria. In four of the treated patients the DCs were loaded with host mHag peptides. Toxicity was limited and no acute GvHD occurred. Most patients developed objective anti-host T-cell responses and in one patient a distinct mHag-specific T-cell response accompanied a temporary clinical response. These findings confirm that DLI combined with host-DC vaccination, either unloaded or loaded with mHag peptides, is feasible, safe and capable of inducing host-specific T-cell responses. The limited clinical effects may be improved by developing more immunogenic DC products or by combining this therapy with immune potentiating modalities like checkpoint inhibitors.

The impact of circulating suppressor cells in multiple myeloma patients on clinical outcome of DLIs.

Allo-SCT followed by DLIs can establish long-term remissions in multiple myeloma (MM) patients. In many patients, however, the immunotherapeutic graft-versus-tumor (GVT) effect is moderate and not sustained, implying that immune suppression is mediated, among other factors, by regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs). Towards a better understanding and, eventually, manipulation of the immune-regulatory mechanisms in transplanted MM patients, we retrospectively sought a correlation between DLI outcome and circulating CD14(+) MDSCs, CD14(-) MDSCs and Tregs in 53 MM patients before their first DLI. We found significantly elevated frequencies of highly suppressive CD14(+) MDSCs, CD14(-) MDSCs and Tregs in pre-DLI samples from patients. Higher frequencies of Tregs, but not of MDSCs, were significantly associated with non-responsiveness to DLI. Furthermore, a lower frequency of Tregs predicted the development of chronic GVHD, which, in turn, displayed a high association with GVT. Elevated Treg frequencies before DLI were also associated with significantly shorter PFS and OS. Hence, our data reinforce the idea of active suppression of antitumor responses by Tregs in MM patients and therefore suggest that targeting patient Tregs before DLI may improve outcome of DLI.

Measurement of stray light and glare: comparison of Nyktotest, Mesotest, stray light meter, and computer implemented stray light meter.

AIM: To evaluate the properties of devices for measuring stray light and glare: the Nyktotest, Mesotest, "conventional" stray light meter and a new, computer implemented version of the stray light meter. METHODS: 112 subjects, divided in three groups: (1) young subjects without any eye disease; (2) elderly subjects without any eye disease, and (3) subjects with (early) cataract in at least one eye. All subjects underwent a battery of glare and stray light tests, measurement of visual acuity, contrast sensitivity, refraction, and LOCS III cataract classification. Subjects answered a questionnaire on perceived disability during driving. RESULTS: Repeatability values were similar for all glare/stray light tests. Validity (correlation with LOCS III and questionnaire scores), discriminative ability (ability to discriminate between the three groups), and added value (to measurement of visual acuity and contrast sensitivity) were all superior for both stray light meters. Results of successive measurements are interrelated for the conventional but not the new stray light meter. This indicates a better resistance to fraud for the latter device. CONCLUSIONS: The new computer implemented stray light meter is the most promising device for future stray light measurements.

Quality assessment in general practice trainers.

INTRODUCTION: General practice trainers hold a key position in general practice training, especially through their provision of a role model. Their own competence in general practice care is important in this regard. The purpose of the study was to evaluate whether a quality assessment programme could identify the strengths and weaknesses of GP trainers in four main domains of general practice care. METHODS: The quality assessment programme comprised validated tests on four domains of general practice: general medical knowledge, knowledge of medical-technical skills, consultation skills and practice management. The criterion for the identification of relative strengths and weaknesses of GP trainers was a variation in the scores of trainers indicating higher and lower scores (strengths and weaknesses) within each domain. RESULTS: GP trainers (n=105) were invited to participate in the study and 90% (n=94) did so. The variation in scores allowed the indication of strengths and weaknesses. Main strengths were: general medical knowledge of the digestive system; knowledge of medical skills relating to the skin; consultation skills concerning empathy; practice management with regard to accessibility. Main weaknesses were: general medical knowledge of the neurological system; knowledge of the medical/technical skills relating to the endocrine metabolic and nutritional system; consultation skills regarding shared decision making; practice management involving cooperation with staff and other care providers. DISCUSSION: This first systematic evaluation of GP trainers identified their strengths and weaknesses. The weaknesses identified will be used in the improvement process as topics for collective improvement in the GP trainers' general curriculum and in individual learning plans.

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae.

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae. Fungal Genetics and Biology 29, 28-37. Restriction enzyme-mediated integration (REMI) has been employed as a mutagen to generate two insertion libraries in an Aspergillus oryzae strain expressing a Thermomyces lanuginosus lipase. The REMI libraries were created using linearized plasmid containing the A. oryzae pyrG and either BamHI or EcoRI enzyme. The libraries were screened for lipase production, and mutants with increased production were isolated. The genomic DNA flanking the integration event was cloned from one of the mutants with increased lipase titers (DEBY10.3). Nucleotide sequence of the flanking DNA revealed similarity to the Aspergillus nidulans palB gene. Disruption of the palB gene in a strain producing lipase resulted in increased lipase expression. Additionally, complementation of the palB phenotype of DEBY10.3 led to a decrease in lipase production. These lines of evidence demonstrate that the increase in lipase yield in DEBY10.3 is linked to the palB phenotype generated by the integration of the pyrG gene into the palB gene. The results also demonstrated that tagged mutagenesis with REMI can be used to identify genes that influence expression of heterologous proteins.