These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.

Therapeutic vaccination with dendritic cells (DC) can lead to tumor regression in animal models and has shown promising results in the first clinical trials of metastatic renal cell carcinoma and malignant melanoma. In vitro data and results of a clinical phase I/II trial using DC tumor fusions in patients with progressive metastatic renal cell carcinoma are presented here. In addition to toxicity and feasibility, complex immune monitoring was a point of interest. DC precursor cells were obtained from the peripheral blood mononuclear cells (PBMCs) of healthy donors and were fused with either allogeneic (8 patients) or autologous (4 patients) renal tumor cells. In total, 12 patients with progressive metastatic renal cell carcinoma were treated with an average of 2.8 x 10(7) tumor cells fused with 1.8 x 10(7) DC each administered on days 0, 28, and 56 intradermally. Fusion efficacy for the tumor cells used was 14.3% +/- 7.8%. Cell viability was 59.8% +/- 6.8% after fusion and irradiation. We observed no adverse effects and no difference in clinical outcome between the allogeneic and the autologous treatment. Eight patients remained in a progressive disease state and four patients in a stable disease state. T-cell immunity was carefully monitored before, during, and after treatment. Delayed-type hypersensitivity (DTH) reaction using tumor cells was positive after treatment in 7 of 12 patients, 2 of whom were found to have stable disease. An increase in the reactivity against recall antigens was seen in most patients. Interestingly, cytotoxicity of peripheral blood lymphocytes (PBLs) against renal cell carcinoma cells increased during treatment as well as the percentage of interferon-gamma-secreting cells. This effect was significantly enhanced within the group that had stable disease. The lack of adverse effects together with positive immunologic signs justifies further investigation of this novel therapeutic approach. Further studies are necessary to test for clinical effectiveness in patients with tumors, especially those with less advanced disease.

Summary. Bleeding complications are the most common and unwanted side-effect of oral anticoagulant therapy. We report three patients in whom mutations in the factor IX (FIX) propeptide were found to cause severe bleeding during coumarin therapy. Strikingly, the bleeding occurred within the therapeutic ranges of the prothrombin time (PT) and international normalized ratio (INR). In all three patients coumarin therapy caused an unusually selective decrease of FIX activity (FIX:C) to levels below 1-3%. Upon withdrawal of coumarin, FIX:C increased to subnormal or normal values of 55%, 85% and 125%, respectively. Analysis of the FIX gene revealed two different missense mutations affecting the Ala-10 residue in the propeptide coding region: Ala[GCC] to Val [GTC] in two patients and Ala[GCC] to Thr[ACC] in one patient. No further mutation was detected by screening 195 random blood donors for mutations at Ala-10, thus excluding a frequent polymorphism at this position. The mutation in the FIX propeptide at a position which is essential for the carboxylase recognition site causes a reduced affinity of the carboxylase enzyme to the propeptide. This effect leads to an impaired carboxylase epoxidase reaction which is decisively triggered by the vitamin K concentration. Determination of FIX and APTT in addition to PT and INR is therefore recommended in coumarintreated patients with an uncommon bleeding pattern.

Background: F77 is a previously uncharacterized prostate cancer-associated antigen.Results: Using a microarray of sequence-defined glycolipids and neoglycolipids, mucin-O-glycan designer arrays, and mass spectrometry, we demonstrate that F77 antibody recognizes blood group H on 6-linked branches of poly-N-acetyllactosamine backbones.Conclusion: F77 antigen is expressed on glycolipids and on glycoprotein O-glycans.Significance: F77 antigen can now be explored rationally as a cancer biomarker.

Summary. Factor VIII gene analysis in a large consanguinous Danish family comprising 24 affected males and four homozygously affected females revealed an Asn694Ile mutation within the A2 domain. The factor VIII gene mutation led to a mild haemophilia A phenotype with factor VIII function displaying discordance between one-stage clotting and chromogenic two-stage assays. In one-stage assays, values ranged from 0´05 to 0´30 IU/ml (males) and from 0´19 to 0´29 IU/ml (homozygous affected females), whereas the chromogenic two-stage assay produced values of around only 50% of the one-stage result [0´02±0´12 IU/ ml (males); 0´06±0´10 IU/ml (females)]. The differences are suggested to be caused by the effect of the mutation on the active cleaved form of the factor (F)VIII protein. As the original amino acid (Asn) is conserved in all known FVIII A2 sequences, but not in ceruloplasmin, we suggest that Asn694 is involved in an A2-specific functional role. Examination of a homology model of the A domains predicts that the Asn694Ile mutation (i) results in the loss of two potential hydrogen±bonding interactions and (ii) hampers the integration of the bulky side-chain of Ile into the A2 domain core, probably causing an altered stability and/or folding of the protein. Interestingly, the disease in this Danish family was originally proposed to be von Willebrand±Ju È rgens disease. However, the current study rules out the co-existence of either von Willebrand's disease or the presence of the Normandy variant of von Willebrand factor (type 2N).

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The carbohydrate composition of eight glycosphingolipids--glucosylceramide, lactosylceramide, lacto-N-triaosylceramide, neolactotetraosylceramide, IV3Gal alpha neolactotetraosylceramide, globotriaosylceramide, globoside (P-antigen), and Forssman glycolipid (IV3GalNAc alpha GbOse4Cer)--derived from different sources was analyzed by 1H nuclear magnetic resonance at 360 MHz in dimethyl-d6 sulfoxide as solvent. The resonances of all H-1 and H-2 protons as well as those of most of the H-3 and H-4, and of some H-5 protons, of the sugar rings were assigned with the aid of spin decoupling difference spectroscopy. They show regularities related to the type, anomeric configuration, site of glycosidic linkage, and sequence of the component sugars glucose, galactose, glucosamine, and galactosamine. These regularities are thus suited for elucidation of hitherto unknown structure of more complex glycosphingolipids.

In fetal alloimmune thrombocytopenia (FAIT) the fetus is threatened by intracranial haemorrhage (ICH); therefore early diagnostic and therapeutic intervention is required. We followed the clinical course of a 30-year-old woman during her fifth pregnancy after she had given birth to a child with alloimmune thrombocytopenia due to anti-Zwa. The fetus was monitored by 13 fetal blood samplings (FBS) always followed by transfusion of either maternal or compatible donor platelets. Intravenous immunoglobulin (ivIg) treatment of the mother was begun at 20 weeks of gestation when the fetal platelet count was 36 x 10(9)/l. The fetal platelets were typed Zwa positive by DNA analysis. Despite 11 weeks of maternal ivIg treatment fetal platelet counts progressively declined to 6 x 10(9)/l and ICH occurred. Subsequently, the fetus was successfully managed by intrauterine platelet transfusions at shorter intervals (3-5 days) and elective Cesarean section was carried out at 35 weeks of gestation. We conclude that maternal ivIg treatment does not prevent ICH in FAIT. The treatment of choice for severely affected cases is serial FBS combined with transfusion of compatible platelets.