SummaryPresent therapies cannot cure the large majority of patients with multiple myeloma (MM) and therefore new treatment strategies are imperative. This study analysed the different glycosylation profiles of Mucin-1 (MUC1) on MM and acute myeloid leukaemia (AML) cells using a series of anti-MUC1 antibodies. Seventy-three per cent of the MM patients had plasma cells that expressed the fully glycosylated forms of MUC1. In contrast to controls, normal bone marrow cells and AML cells, the differentiation-dependent and cancer-associated glycoforms of MUC1 were present on 59% and 36% MM tumour cells respectively. This indicated that aberrantly glycosylated MUC1 is a potential immunotherapeutic target in MM patients.Keywords: multiple myeloma, Mucin-1, glycoforms, immunotherapy, tumour-associated antigens. Flow cytometryBM samples were prepared for flow cytometry according to standard procedures. All incubations were at room temperature and samples were washed in between. Briefly: following lysis of erythrocytes, cells were incubated with a series of anti-MUC1 mAbs (van Leeuwen et al, 2006) or isotype control (BD Biosciences, San Diego, CA, USA). Subsequently, cells were incubated with a PE-conjugated rabbit anti-mouse IgG antibody (Dako, Glostrup, Denmark), after which unoccupied binding sites were blocked by incubation with normal mouse serum. Finally, cells were stained with CD38-fluorescein isothiocyanate (FITC), CD45-peridinin chlorophyll (PerCP), CD34-allophycocyanin (APC) or CD11c-APC, CD33-FITC (all: BD Biosciences), followed by fixation, and acquisition of 10 6 events for each sample using a FACSsort cytometer (BD Biosciences). Staining intensities for MUC1 were defined by the mean fluorescence intensity (MFI) ratios (MFI specific antibody/MFI isotype control). An MFI ratio that was greater than the average MFI ratio of control cells (CD34 + cells present in BM from MM patients) + 2 standard deviations (SD) was considered as positive for MUC1 expression. Results and discussionThe monoclonal PC causing MM can have an immature and mature phenotype based on their differential expression of CD45 (Schneider et al, 1997;Kumar et al, 2003). As demonstrated in Fig 1A, et al, 1996). BM from all MM patients had a MPC population, while 19 patients also had an IPC subset. The histograms in Fig 1C show that the different glycoforms of MUC1 were detected on IPC as well as MPC, with a similar expression profile. None of the groups 2 and 3 mAbs reacted with control cells or BM cells from normal donors, including non-malignant PC, although a subset of these cells were stained occasionally by antibodies from group 1 (data not shown). Figure 2A demonstrates that MPC from 59% and 36% of the MM patients bound antibodies from groups 2 and 3, respectively, while 73% were positive for group 1 antibodies. A similar trend was observed for IPC although less samples reacted with antibodies from groups 2 and 3 (Fig 2B). Staining of PC by group 2 and/or 3 antibodies was always accompanied by intense recognition by antibodies from grou...

Members of the Sox gene family of transcription factors are defined by the presence of an 80 amino acid homology domain, the High Mobility Group (HMG) box. Here we report the cloning and initial analysis of murine Sox-13 . The 984 amino acids Sox-13 protein contains a single HMG box, a leucine zipper motif and a glutamine-rich stretch. These characteristics are shared with another member of the Sox gene family, Sox-6. High level embryonic expression of Sox-13 occurs uniquely in the arterial walls of 13.5 days post coitum (dpc) mice and later. Low level expression was observed in the inner ear of 13.5 dpc mice and in a limited number of cells in the thymus of 16.5 dpc mice, from which Sox-13 was originally cloned. At 18.5 dpc, Sox-13 is expressed in the tracheal epithelium below the vocal cord and in the hair follicles. The Sox-13 protein binds to the consensus HMG box motif, AACAAAG, but does not transactivate transcription through a concatamer of this motif. Sox-13, like other members of the Sox family likely plays an important role in development.

Summary:Allogeneic stem cell transplantations (SCT) are currently being used as a therapy for hematological malignancies, some solid tumors and nonmalignant bone marrow deficiencies. Nevertheless, clinical applicability is limited due to toxicity of conditioning regimens, graft-versus-host disease (GVHD) and the scarcity of HLA-identical family donors. New concepts are based on nonmyeloablative conditioning to reduce toxicity, prevention or amelioration of GVHD and the use of haploidentical donors to increase donor availability. To combine these requirements, we have developed a nonmyeloablative conditioning regimen, consisting of low-dose total body irradiation and cyclophosphamide-based chemotherapy. In a haploidentical F1-F1 mouse model, this nonmyeloablative transplantation protocol resulted in stable full donor chimerism, but also in the development of severe GVHD. Administration of keratinocyte growth factor (KGF) reduced GVHD, evident as reduced weight loss and a lesser degree of dermatitis, compared to saline-treated controls. KGF preserved plasma citrulline and tumor necrosis factor-a levels, both indicative for reduced injury to the gastrointestinal tract. This was confirmed by histological findings. At 6 months after transplantation, survival rates were significantly higher in KGF-treated animals as compared to phosphate buffered saline-treated controls. These results indicate that KGF preserves gut integrity and might therefore contribute substantially to reduction of lethal GVHD in (nonmyeloablative) haploidentical transplantation. Bone Marrow Transplantation (2005) 36, 907-915.

Dendritic cells (DCs) are the best professional antigen-presenting cells to stimulate cytotoxic as well as T helper cells and are therefore appropriate candidates for establishing immunotherapy. The concept of our vaccination program is to introduce the tumor-associated antigen mucin-1 (MUC1) into DCs. Analysis of immature and mature DCs--before transducing the antigen MUC1--already demonstrated expression of MUC1 on in vitro monocyte-derived DCs upon maturation. Different culture methods as well as maturation cocktails showed similar results concerning the upregulation of MUC1 expression. Furthermore, we studied the expression of MUC1 on DCs in vivo. No MUC1 expression was found on blood DCs, or on thymic or tonsil DCs. On the other hand, synovial fluid from patients with arthritis contained DCs that were found to express MUC1. This study shows for the first time that the tumor-associated antigen MUC1 is expressed on in vivo DCs. We further show that MUC1 is also expressed on in vitro cultured bone marrow-derived DCs of human MUC1 transgenic mice, supporting the relevance of this mouse model to the human situation. The observation that MUC1 is present on in vivo DCs suggests a functional role, but this physiological function remains to be elucidated.

Allogeneic stem cell transplantations (SCT) are currently being used as a therapy for hematological malignancies, some solid tumors and non-malignant bone marrow deficiencies. Nevertheless, clinical applicability is limited due to toxicity of conditioning regimens, graft-versus-host disease (GVHD) and the scarcity of HLA-identical family donors. New concepts are based on nonmyeloablative conditioning to reduce toxicity, prevention or amelioration of GVHD and the use of haploidentical donors to increase donor availability. To meet these requirements, we have developed a nonmyeloablative conditioning regimen in a haploidentical F1 → F1 mouse model. These mini-transplantations, consisting of low dose total body irradiation and cyclophosphamide-based chemotherapy, resulted in stable full donor chimerism, but also in the development of GVHD. Administration of keratinocyte growth factor (KGF) before and after SCT resulted in reduced GVHD, evident as reduced weight loss and a lesser degree of dermatitis than in saline-treated controls. KGF preserved plasma citrulline and TNF-a levels, both indicative for reduced radiation-induced injury to the gastrointestinal tract. Citruline has recently been described as a new marker for gastrointestinal toxicity. Six months after transplantation, survival rates were significantly higher in KGF-treated animals (57% as compared to PBS-treated controls (5.3%). Figure Figure These data indicate that nonmyeloablative haploidentical transplantations might be feasible and that KGF might contribute substantially to reduction of lethal GVHD, also after nonmyeloablative procedures. For clinical transplantation such an effect would be significant if intensive T cell depletion might be prevented in haploidentical transplantation protocols and therefore reduced infection rates can be obtained.