Peter Hanfland

Allogeneic dendritic cells fused with tumor cells: preclinical results and outcome of a clinical phase I/II trial in patients with metastatic renal cell carcinoma.

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.

Therapeutic vaccination against metastatic renal cell carcinoma by autologous dendritic cells: preclinical results and outcome of a first clinical phase I/II trial

Abstract. In this study we have presented in vitro data and results of a preliminary clinical trial using dendritic cells (DC) in patients with progressive metastatic renal cell carcinoma. DC precursor cells were obtained from peripheral blood mononuclear cells (PBMC). DC were pulsed with autologous tumor cell lysate if available. In total, 15 patients were treated with a median of 3.95×106 DC administered and ultrasound-guided into a lymph node or into adjacent tissue. Seven patients remained with progressive disease (PD), 7 patients showed stable disease (SD), and one patient displayed a partial response (PR). Most interestingly, the patient who was treated with the highest number of DC (14.4×106 DC/vaccine) displayed a PR. Delayed-type hypersensitivity (DTH) reaction using autologous tumor lysate was positive in 3 out of 13 patients, including the patient with PR. Two out of 3 patients receiving additional treatment with keyhole limpet hemocyanin (KLH) showed reactivity to KLH after vaccination. CD3+CD4+ and CD3+CD28+ cells as well as the proliferation rate of peripheral blood lymphocytes (PBL) increased significantly in the blood of patients during therapy. In conclusion, our observations confirm the capability of tumor-lysate pulsed autologous DC vaccines to stimulate an immune response in patients with metastatic renal cell carcinoma even in the presence of a large tumor burden. The lack of adverse effects together with immunologic effects support further investigation of this novel therapeutic approach. Further studies are necessary to demonstrate clinical effectiveness in cancer patients, in particular in patients with less advanced disease.

Missense mutations at ALA-10 in the factor IX propeptide: an insignificant variant in normal life but a decisive cause of bleeding during oral anticoagulant therapy.

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 coumarin‐treated patients with an uncommon bleeding pattern.

Immunochemistry of the Lewis-blood-group system: Proton nuclear magnetic resonance study of plasmatic Lewis-blood-group-active glycosphingolipids and related substances☆☆☆

The Lea-, Leb-, and H-type 1 (LedH)-blood-group-active glycosphingolipids, as well as H-I-type 2 glycolipid, lactotetraosyl ceramide, and neo-lactotetraosyl ceramide were examined by 1H nuclear magnetic resonance at 360 MHz in dimethyl-d6 sulfoxide as solvent. The resonances of almost all protons of the sugar rings were assigned with the aid of spin decoupling and nuclear Overhauser difference spectroscopy. The latter technique was also applied to establish the sequences and sites of glycosidic linkage. This information, combined with the chemical shift-structure correlations established in our previous work, led to an independent identification of those six glycolipids. Type 1 (Galβ1 → 3GlcNAc) and type 2 (Galβ1 → 4GlcNAc) saccharide chains can be distinguished by this approach. Some deviations from additivity in chemical shifts, calculated for oligosaccharides from the data on their constituent sugar residues, furnished information on the conformational changes in crowded glycolipid molecules.

[4] Analysis of glycosphingolipids by high-resolution proton nuclear magnetic resonance spectroscopy

Publisher Summary Nuclear magnetic resonance (NMR) is a nondestructive method capable of furnishing many-sided structural information; it is therefore highly desirable fully to exploit its potential, aiming at complete structure determinations without resorting to data obtained by other methods. This chapter presents examples of such structure elucidation and discusses the prospects of proton NMR becoming a self-sufficient method in the field of structural research of glycosphingolipids. In this study spin decoupling difference spectroscopy is applied because it is reasonable balance between the measuring time invested and the spectral information obtained. In this way one can identify most of the signals of the sugar ring protons for several glycosphingolipids containing up to 10 sugar residues. Another successful approach is the use of the nuclear Overhauser effect. The second, decisive, part of the problem is to extract structural information from those data. This task is greatly facilitated if series of structurally related substances are available. The work on Forssman glycolipid and the pentasaccharide ceramide from rabbit erythrocytes with B-like blood group activity offers a good opportunity to illustrate the approach. The results obtained revealed regular changes of chemical shifts related to the structural features a-e listed in this chapter. These regularities are shown to be applicable to structure determination of a bifurcate ceramide decasaccharide and of a series of highly crowded fucose-containing glycosphingolipids of the Lewis blood-group system.

Maternal intravenous immunoglobulin treatment does not prevent intracranial haemorrhage in fetal alloimmune thrombocytopenia

SUMMARY. 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 times 109/1. The fetal platelets were typed Zwa positive by DNA analysis. Despite 11 weeks of maternal ivIg treatment fetal platelet counts progressively declined to 6 times 10/1 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.

Characterization of B and H blood-group active glycosphingolipids from human B erythrocyte membranes

Two blood group B active glycosphingolipids (B-I and B-II) previously isolated and highly purified from human B erythrocytes [21] were analysed first by degradation with alpha-D-galactosidase from coffee beans, alpha-L-fucosidase from bovine kidney and with 0,1 N trichloracetic acid; the native B-glycolipids as well as their degradation products were then investigated by methylation analysis with combined gas chromatography-mass spectromety, by thin layer chromatography, two dimensional immunodiffusion and by the hemagglutination inhibition technique. Together with the results obtained by mass spectrometry of permethylated glycolipids [26] the following structures were elucidated: alpha-D-gakactpurampsu;-(1 leads to 3) [alpha-L-fucopyranosyl-(1 leads to 2)]-D-galactopyranosyl-(1 leads to 4)-N-acetyl-D-glucosaminosyl-(1 leads to 3)-D-galactopyranosyl-(1 leads to 4)-D-glucopyranosyl-(1 leads to 1)-ceramide for the B-I glycosphingolipid and alpha-D-galactopyransosyl-(1 leads to 3)-[alpha-L-fucopyranosyl-(1 leads to 2)]-D-galactopyranosyl-(1 leads to 4)-N-acetyl-D-glucosaminosyl-(1 leads to 3)-D-galactopyranosyl-(1 leads to 4)-N-acetyl-D-glucosaminosyl-(1 leads to 3)-D-galactopyranosyl-(1 leads to 4)-D-glucopyranosyl-(1 leads to 1)-ceramide for the B-II glycophingolipid. AH active glycolipid fraction from B erythrocytes further purified by thin layer chromatography was also investigated by methylation analysis. The pattern of its partially methylated alditol acetates was essentially the same as that of the alpha-galactosidase treated and permethylated B-I glycoliped. It is also exhibited strongly precipitating and hemagglutination inhibiting H properties as well as the two alpha-galactosidase treated B-I and B-II glycosphingolipids. Based upon these data the following tentative structure was proposed: alpha-L-fucopyranosyl-(1 leads to 2)-D-galactopyranosyl-(1 leads to 4)-N-acetyl-D-glucosaminosyl-(1 leads to 3)-D-Galactopyranosyl-(1 leads to 4)-D-glucopyranosyl-( 1 leads to 1)-ceramide. Gas chromatographic analysis revealed sphingosine and lignoceric, nervonic and behenic acids to be the main components of the ceramide residues of the three glycophingolipids. From the data presented the H active substance very probably can be regarded as the immediate precursor of the B-I gly cosphingolipid from human B erythrocyte membranes.

Immunochemistry of the Lewis-blood-group system: Partial characterization of Lea-, Leb-, and H-type 1 (LedH)-blood-group active glycosphingolipids from human plasma

Abstract Four different H-type 1 (Le dH ) blood-group-active glycosphingolipids (Le dH -I–IV) have been isolated from the plasma of blood-group O Le(a−b−) secretors. The agglutination of O Le(a−b−) erythrocytes from secretors by 50 μl of 4 hemagglutinating units of caprine anti-Le dH (anti-H-type 1) serum was inhibited by 0.02 μg of each of all four glycolipids. No Le a or Le b activities or reaction against Ulex europaeus lectin could be found. Le dH -I, -II, -III, and -IV at 0.05, 0.01, 0.01, and 0.02 μg each are sufficient for incubation in order to convert 9 × 10 7 O Le(a−b−) erythrocytes from nonsecretors into H-type 1 (Le dH )-positive cells. Structural analysis of the H-type 1 glycolipids was performed in comparison to that of Le a - and Le b -blood-group-active glycolipids from human plasma isolated previously: Gas chromatography of peracetylated alditols revealed sugar composition. Combined gas chromatography-mass spectrometry established the glycosidic linkages. Together with the results obtained by direct inlet mass spectrometry of permethylated glycosphingolipids and by 360-MHz 1 H nuclear magnetic resonance spectroscopy (Egge, H., and Hanfland, P., 1981, Arch. Biochem. Biophys. , 210 , 396–404; Dabrowski, J., Hanfland, P., Egge, H., and Dabrowski, U., 1981, Arch. Biochem. Biophys. , 210 , 405–411) the complete structures of the oligosaccharide chains of the Le a -, Le b -, and H-type 1-active glycolipids were established: Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le a antigens; Fucα1 → 2Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the Le b antigens; and Fucα1 → 2Galβ1 → 3GlcNAcβ1 → 3Galβ1 → 4Glcβ1 → 1 Cer for the H-type 1 (Le dH ) glycolipids. The diverse antigens of the same blood-group specificity obviously differ from one another in their lipid residue. In addition, plasmatic neolactotetraosylceramide could be identified, differing from that of human erythrocytes by a slower migration behavior in thin-layer chromatography.

Assay discrepancy in mild haemophilia A due to a factor VIII missense mutation (Asn694Ile) in a large Danish family

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–Jürgens disease. However, the current study rules out the co‐existence of either von Willebrands disease or the presence of the Normandy variant of von Willebrand factor (type 2N).

Studies of the binding specificity of the soluble 14 000-dalton bovine heart muscle lectin using immobilised glycolipids and neoglycolipids

The aim of the present study has been to investigate the binding specificity of the soluble 14,000-dalton lectin of bovine heart muscle towards immobilised oligosaccharides in clustered form. To this end, chromatogram overlay assays and quantitative plastic-microwell-binding assays have been performed using several natural glycolipids and neoglycolipids containing one or more of the disaccharide units, beta-D-Galp-(1----4 or 3)-D-GlcNAc or beta-D-Galp-(1----4)-D-Glc and related structures. The microwell assay gave the most consistent results. It was observed that for binding by the soluble lectin the optimal sequence, which is beta-D-Galp-(1----4 or 3)-D-GlcNAc, must occur at the nonreducing end of longer oligosaccharides when linked to lipid. These oligosaccharides may be of poly(N-acetyllactosamine) type or they may be mono- or multi-antennary, complex-type chains in which the disaccharide is joined directly to a trimannosyl core. The lectin bound to such immobilised lipid-linked oligosaccharides on which the terminal D-galactosyl groups are substituted with alpha-L-Fucp-(1----2), alpha-D-Galp-(1----3), or alpha-NeuAc-(2----3) groups. However, no binding was detected if the terminal D-galactosyl groups were substituted with an alpha-NeuAc-(2----6) group or the subterminal N-acetylglucosamine units with an alpha-L-Fucp-(1----3 or -4) group. Internally located N-acetyllactosamine units where the D-galactose units are disubstituted by beta-D-GlcNacp-(1----3) and -(1----6) units, as in branched poly(N-acetyllactosamine) backbones were not bound by the bovine lectin. These results are in accord with previous observations on the bovine lectin and the corresponding human and rat lectins, using structurally defined oligosaccharides as inhibitors of binding. The results of comparative binding experiments using paragloboside and ceramide hexasaccharide which contain one and two N-acetyllactosamine units, respectively, joined in linear sequence to the lactosylceramide core, were equivocal with respect to the availability of the internal N-acetyllactosamine units for binding by the bovine lectin.