Modifying antibody specificity by chain shuffling of V / V between antibodies with related specificities.

Histo-blood group antigens are important markers of developmental stages and as such also often of tumours. Generation of antibodies towards these carbohydrate structures is still a challenging task as they may lack specificity, affinity or are only of the IgM class. We have examined four own antibodies to Lewis Y/H type 2 for their fine specificities using a large panel of mono- and oligosaccharides. Sequence alignment to other antibodies with similar specificity revealed an overall limited variation, and that our antibodies constitute a novel set. Based on produced and analysed chimeric mouse-human antibodies, extensive chain shuffling experiments were performed in order to analyse influences of the respective H and L chains on the specificity of the antibodies, and to generate modified antibodies with improved properties. One chIgG1 out of the shuffled antibodies revealed improved specificity and markedly enhanced functional affinity to Lewis Y compared to the parental chIgG1 antibodies. Therefore, the combinatorial approach of chain shuffling provides a platform to improve specificity and/or affinity of anti-carbohydrate antibodies.

A new method for the preparation of rat liver lysosomes. Separation of cell organelles of rat liver by carrier-free continuous electrophoresis.

A combination of differential centrifugation and carrier-free continuous electrophoresis is introduced as a new method for the isolation of animal cell organelles. Various buffers were systematically checked in order to find the system which preserves the organelles and gives as well a good separation in the free-flow electrophoresis apparatus. Triethanolamine-acetate buffer (10 mM), pH 7.4 was used. The isolated lysosomes were pure according to marker enzymes and electron micrographs. A heterogeneity of the lysosomes in electrophoretic mobility was demonstrated with respect to the marker enzymes arylsulfatase and beta-glucuronidase. The lysosomes with higher mobility showed a maximum enrichment of 240-fold with respect to arylsulfatase. The lysosomes with lower electrophoretic mobility showed a 65-fold enrichment with respect to beta-glucuronidase. The ratio of beta-glucuronidase to arylsulfatase varied from 2:1 to 1:2 in lysosomes of different mobility. The yield amounted to approximately 1 mg of lysosomal protein per gram of liver protein. 5-8 mg of lysosomes can be obtained in one experiment. The electrophoretic separation proves to be an effective tool in obtaining pure and well preserved lysosomes.

The surface charge of rat liver mitochondria and their membranes. Clarification of some controversies concerning mitochondrial structure.

The surface charge of intact mitochondria and submitochondrial particles was examined by the technique of preparative free flow electrophoresis. When submitochondrial preparations obtained by a swelling-contraction procedure were examined with this technique, two fractions were observed. One of these fractions exhibited the same electrophoretic properties as intact mitochondria, which indicated that it was derived from the outer limiting membrane of the mitochondrion. This fraction was found to contain the enzymes monoamine oxidase and rotenone-insensitive NADH-cytochrome c reductase which have been reported to be localized in the outer mitochondrial membrane. The other fraction exhibited an electrophoretic mobility which was different from that of intact mitochondria, and this fraction contained enzymes characteristic of the inner membrane-matrix fraction such as soluble and particulate enzymes of the Krebs cycle. Microsomes exhibited an electrophoretic mobility which was almost identical with that of the outer mitochondrial membrane. In addition to resolving the localization of enzymes in mitochondrial membranes, these data indicate that the outer limiting membrane of the mitochondrion is the sole determinant of the surface charge of mitochondria.

Physical properties and pharmacological activity in vitro and in vivo of optimised liposomes prepared from a new cancerostatic alkylphospholipid.

Liposomes from octadecyl-(1,1-dimethyl-4-piperidino-4-yl)-phosphate (OPP), a new alkylphospholipid derivative with an improved cancerostatic activity, were prepared for the first time and the activity in vitro and in vivo was characterised. The formation of liposomes (MLV, SUV and LUVET) differing in cholesterol content, charge, and sterical stabilisation is possible without serious problems, despite the lysolipid-like structure of the OPP. Liposomes with a low amount of cholesterol and with PEG2000DSPE-coating were the most stable OPP liposomes, both in buffer and in serum. The cytotoxicity of micellar or liposomal OPP against breast cancer cell lines in vitro was in the range of 20-60 microM. The cytotoxicity of the liposomal formulation was inversely related to the content of cholesterol, whereas the sterical stabilisation and/or the incorporation of a positive charge had only a very moderate modulating effect on the inhibition of cell proliferation. The strongest antitumour effect on the xenotransplanted breast cancer MT-3 in vivo was obtained with sterically stabilised OPP liposomes with low CH content. The beneficial therapeutic effect of these liposomes was accompanied by better tolerance and a significant inhibition of haemolysis compared to micellar OPP.

Calcium ions as efficient cofactor of polycation-mediated gene transfer.

We investigated the effect of calcium on the transfection of non-viral DNA transfer systems. Cationic proteins such as the nuclear protein H1, the polycation polylysine and a number of commercial transfection agents exhibited high transfection rates in the presence of Ca2+. Without Ca2+ H1 and HMG1 were inactive in transfection of the human permanent endothelial cell line ECV 304 while cationic liposomes such as Lipofectin and Lipofectamine did not show any Ca2+ dependence. More detailed experiments showed that Ca2+ was replaceable by the lysosomotropic agent chloroquine. Furthermore, it was possible to separate the transfection-enhancing role of Ca2+ from the actual transfection process by adding Ca2+ to the cells after the transfection period and still to obtain a significant transgene expression. This makes it possible to distinguish between cellular uptake of H1 (or mediator)-DNA complexes and endocytotic release. We also replaced soluble Ca2+ by Ca-phosphate precipitates not containing DNA and obtained similar transfection results. This allowed us to suggest that the addition of free Ca2+ to the transfection medium resulted in nascent Ca-phosphate microprecipitates. The known fusogenic and membranolytic activity of such microprecipitates could facilitate the transport through and the release of the transfecting complexes from the endosomal/lysosomal compartment.

Diabetes and its complications among selected tribes in North Dakota, South Dakota, and Nebraska.

OBJECTIVE: To determine the prevalence and incidence rates of diabetes and two specific complications for selected American-Indian tribes in North Dakota, South Dakota, and Nebraska. RESEARCH DESIGN AND METHODS: A descriptive epidemiological study was conducted using ambulatory care data during 1987 for prevalence and diabetes registries and complication case reporting during 1988 from IHS facilities on reservations in these states. RESULTS: The Winnebago and Omaha tribes had the highest age-adjusted diabetes rates, with prevalence 8.8 times and incidence 7.7 times the respective U.S. rates. The diabetes prevalence rate of combined data for the Sioux was 3.7 times the U.S. rate. Among Sioux Indians, the age-adjusted incidence rate for ESRD was 4.8 times the American-Indian/Alaska-Native rate and 13.4 times the rate for U.S. whites. The proportion of new diabetes-related ESRD (86%) was almost 3 times greater than the general U.S. population rate (30%). Also, among the Sioux, the age-adjusted incidence rate for LEA (86.7/10,000 diabetic population) was 1.5 times higher than the U.S. rate; the proportion of diabetes-related LEA (84%) was 1.8 times higher than the general U.S. population rate (45%). CONCLUSIONS: The age-adjusted rates of diabetes and certain complications among these Northern Plains tribes are greater than the U.S. rates. Improved health services to detect and monitor diabetes and its complications and community-based prevention activities directed at the epidemic of diabetes among the various Indian tribes are urgently needed.

The prevalence of diabetic retinopathy and associated risk factors among Sioux Indians.

OBJECTIVE: To estimate the prevalence of and risk factors for diabetic retinopathy among Sioux Indians of South Dakota. RESEARCH DESIGN AND METHODS: Strong Heart Study (SHS) participants with diabetes who are members of the Cheyenne River Sioux Tribe and the Oglala Sioux Tribe were invited to have ophthalmological examinations in 1991. A total of 417 people had eye examinations out of the 488 diabetic SHS participants of the two tribes (85% participation rate). Fundus photographs were obtained of each eye and graded for severity of retinopathy using the modified Airlie House Classification Scheme. Risk factors for retinopathy were determined from the SHS database. RESULTS: The prevalence of diabetic retinopathy among participants from these tribes was 45.3%. Risk factors associated with severity of retinopathy include mean fasting glucose, level. HbA1c, systolic blood pressure, urinary albumin-to-creatinine ratio, renal dialysis, and duration of diabetes. CONCLUSIONS: The prevalence of diabetic retinopathy among diabetic Sioux Indians is similar to or higher than the prevalence in other diabetic Indian and non-Indian populations. Aggressive glycemic and blood pressure control is urgently needed to reduce this high rate, and annual eye examinations to detect and treat diabetic retinopathy should be emphasized.

Assessment of diabetes care by medical record review. The Indian Health Service model.

OBJECTIVE: To evaluate the adherence to minimum standards for diabetes care in multiple primary-care facilities using a uniform system of medical record review. RESEARCH DESIGN AND METHODS: In 1986, the Indian Health Service (IHS) developed diabetes care standards and an assessment process to evaluate adherence to those standards using medical record review. We review our assessment method and results for 1992. Charts were selected in a systematic random fashion from 138 participating facilities. Trained professional staff reviewed patient charts, using a uniform set of definitions. A weighted rate of adherence was constructed for each item. RESULTS: Medical record reviews were conducted on 6,959 charts selected from 40,118 diabetic patients. High rates of adherence (> 70%) were noted for blood pressure and weight measurements at each visit, blood sugar determinations at each visit, annual laboratory screening tests, electrocardiogram at baseline, and adult immunizations. Lower rates of adherence (< or = 50%) were noted for annual eye, foot, and dental examinations. CONCLUSIONS: IHS rates of adherence are similar to rates obtained from medical record reviews and computerized billing data, but are less than rates obtained by provider self-report. Medical record review, using uniform definitions and inexpensive software for data entry and reports, can easily be implemented in multiple primary-care settings. Uniformity of data definition and collection facilitates the aggregation of the data and comparison over time and among facilities. This medical record review system, although labor intensive, can be easily adopted in a variety of primary-care settings for quality improvement activities, program planning, and evaluation.

Human primary T-cell lines in lectin-free media.

A method of establishing human T-cell lines in tissue culture media containing TCGF but not PHA is described. PBL, initially stimulated by PHA to produce TCGF, continue production for at least 48 hrs after the lectin has been washed off. The TCGF-conditioned medium initiates blast formation of T-cells from freshly isolated PBL and supports indefinite growth of the T-cell blasts from the primary PBL cultures and also from blast-cells obtained from clonally derived T-cell colonies grown in soft agar culture. The established cell lines are identified by cytochemical and biological means as belonging to the T-cell compartment and express spontaneous cytotoxicity against HeLa-cells. The continuously growing T-cells are unable to produce TCGF and depend strictly on external supply of the growth factor. PHA by itself does not support T-cell growth in spite of its ability to elicit a mitogenic response in PBL-cultures. Thus two types of cells must be involved in the mitogenic event: (i) a TCGF-producer and (ii) a TCGF-responder. PHA elicits in the former TCGF production and then TCGF in the later the mitogenic T-cell response. Therefore, not PHA but TCGF might be considered to be the T-cell mitogen.

A comitogenic serum glycoprotein in T lymphocyte activation.

The production of Interleukin-2 is induced in helper cells, probably of T-cell origin, after stimulation by Interleukin-1. PHA is known to induce production of Interleukin-1 and Interleukin-2 in human PBL. We observed that PHA-induced mitogenesis in PBL requires the presence of a 90-KD-serum glycoprotein, which we were able to distinguish from other known serum proteins of similar molecular weight. Its biological activity can be destroyed by removal of its sialic acid residues. Evidence presented in this paper indicates that the function of this protein is related to the induction of synthesis and/or release of Interleukin-2. The presence of PHA does not seem to be essential at the level of Interleukin-2 production; the glycoprotein is able to induce Interleukin-2 in cooperation with a soluble mediator, which is produced by adherent cells. This mediator causes T-cell mitogenesis in PBL, provided serum or the 90-KD glycoprotein is present in the culture. It is not able to support the growth of a CTL line. We suggest the name PHILIP (Plasmatic Human Interleukin-2-Inducing Protein) for the 90-KD molecule.

Effects of nerve growth factor administration of N-ethyl-N-nitrosourea carcinogenesis.

We attempted to determine whether the sympathetic nervous system in rodents is susceptible to co-carcinogenesis, employing murine salivary nerve growth factor (NGF) as a co-carcinogenic agent. NGF had no co-carcinogenic effect with either methylcholanthrene or ethylnitrosourea (ENU) on the sympathetic nervous system of the mouse, whether administered transplacentally, postnatally, or both transplacentally and postnatally. At a dose of ENU of 30 mug/g body weight, NGF did not shorten the latent period for tumor induction of BD-IX rats. In contrast, a 25% reduction in latent period was brough about by NGF for tumor appearance in BD-IX rats receiving 90 mug/g ENU. In both cases the frequency of urogenital tumors in rats was increased as a result of NGF administration, at the apparent expense of neural tumors.

Binding of tumor antigen mucin (MUC1) derived peptides to the heat shock protein DnaK.

The human epithelial mucin encoded by the gene MUC1 is a tumor-associated antigen expressed on breast, pancreatic, colon and ovarian cancer cells recognized by cytotoxic T-cells and antibodies. Underglycosylated as well as glycosylated mucin-peptide epitopes are promising targets for vaccination against cancer. Heat shock proteins of 70 kDa (HSP70), also highly expressed in tumor cells, can function as chaperones for peptides and proteins and are involved in antigen processing. The involvement of HSP70 molecules in mucin antigen binding, processing and presentation has not yet been examined. Here we present first results concerning the relative binding affinities of various mucin-derived peptides to the bacterial 70 kDa heat shock protein DnaK. Interestingly, longer mucin peptides reveal a higher affinity to DnaK than short peptides. The non-glycosylated mucin-derived peptides of 5-8 amino acids length were able to compete with a high affinity (unrelated) reference peptide at millimolar concentrations. Glycosylation of the investigated short peptides lowers their binding affinity to DnaK, depending on the position of the carbohydrate. The binding affinity is not influenced by free charges at unprotected ends. The peptide (MUC1)5 consisting of five repeating units has an affinity enhanced by a factor of three as compared to the peptide with only one repeating unit. Mucin-peptide-HSP-complexes could be the basis of developing new kinds of tumor vaccines.

Aggregation of influenza virus haemagglutinin in the presence of detergents.

In the haemagglutinin (HA) detergent mixtures coexist various protein complexes. Using 125I-HA tracer, gel chromatography, and centrifugation techniques we found protein aggregates comprising up to eight HA trimers. Formation of these structures seemed to be a function of the protein storage medium only. By contrast, special properties of the detergent as well as physicochemical conditions during the protein/detergent interaction had nearly no influence.

Quantitative reconstitution of isolated influenza haemagglutinin into liposomes by the detergent method and the immunogenicity of haemagglutinin liposomes.

The reconstitution of influenza virus haemagglutinin into liposomes from lipid/protein/detergent mixtures by detergent removal provides vesicles that are similar in structure to viral particles. The dissociation properties of haemagglutinin aggregates and the molar ratio of lipid to protein in the starting mixture are the key factors for the individual and total yield of protein incorporation into liposomes. Structural properties of the detergent used as well as special reconstitution conditions are of minor importance for the formation of haemagglutinin liposomes. As determined by radial immunodiffusion-, haemolysis- and fusion experiments, specific properties of haemagglutinin were maintained to a large extent on liposomal incorporation, but its immunogenicity is increased, if the antigen is incorporated into the lipid bilayer of liposomes.

A novel series of anti-human glycophorin A (CD235a) antibodies defining five extra- and intracellular epitopes.

Glycophorin A (GPA, CD235a) is a major membrane glycoprotein and marker of cells of the erythroid lineage. It is also the target of Plasmodium falciparum and of influenza virus. We describe a novel series of 10 antibodies towards GPA, recognizing four extra- and intracellular peptide epitopes of this molecule (defined by epitope mapping) and one mixed peptide/carbohydrate epitope. All antibodies bind better to the desialylated than to the fully sialylated molecule, including those specific for the intracellular epitope. For some of the antibodies (representing all five epitopes) functional binding constants were determined by Surface Plasmon Resonance. The new panel complements the already known anti-glycophorin antibodies and offers several potential applications for, e.g., differential diagnosis of erythroleukemias, lineage analyses of erythroid cells, isolation of senescent erythrocytes, or a highly sensitive neuraminidase assay.

A monoclonal antibody to Lewis Y/Lewis b revealing mimicry of the histone H1 to carbohydrate structures.

Antibodies to either peptide or carbohydrate tumour antigens are established tools for diagnostics and therapy. We here describe an antibody (A70-A/A9) recognizing a carbohydrate epitope common to the tumour-associated Lewis Y and Lewis b antigens (Fucalpha1-2Galbeta1-4/3[Fucalpha1-3/4]GlcNAcbeta-). Its specificity was established without doubt with a panel of 86 synthetic mono- and oligosaccharidic structures. This antibody was found to cross-react with the nuclear protein histone H1. Binding to H1 was specific, periodate-insensitive (non-carbohydrate) and saturable. Histone H1 was able to inhibit Lewis Y binding very effectively in a concentration-dependent manner. We conclude that it represents an example of natural peptide mimicry of a carbohydrate epitope. It may explain the observed occurrence of 'anti-histone autoantibodies' in cancer patients.

Cell adhesion inhibition by glycoliposomes: effects of vesicle diameter and ligand density.

The Thomsen-Friedenreich antigen (TF) is a pancarcinoma marker which is involved in the development of liver metastasis by binding tumour cells to the asialoglycoprotein receptor on hepatocytes. Blocking of this receptor prevents metastasis under certain circumstances. We report on conditions for an effective inhibition of the adhesion of KG-1 leukaemia cells expressing TF by lactosylated liposomes. In order to reach strong inhibition, carbohydrate blocking probes must be multivalent. Glycoliposomes are able to carry a large number of glycolipids accommodated in the lipid bilayer. They should be able to adapt their glycolipid pattern in order to achieve multiple binding. We found that, in addition to the number of carbohydrates on the liposome surface, their size, and probably the arrangement of neutral glycolipids in clustered domains, determine the inhibitory properties of glycoliposomes.

[T-lymphocyte activation. Studies on the function of mediator proteins (author's transl)]. / T-Lymphocyten-Aktivierung. Untersuchungen zur Funktion von Mediatorproteinen.

The functions of several mediator proteins involved in the T-cell blastogenesis have been investigated 1. Newly described is the Plasmatic Human IL-2 Inducing Protein (PHILIP), a glycoprotein from human serum with a molecular weight of about 85,000 D. Its presence is mandatory for the synthesis and/or secretion of Interleukin-2 (T-cell growth factor). The mediator protein can be distinguished from other known serum glycoproteins (e.g., transferrin and plasminogen) by affinity chromatography. Desialylation completely abolishes its biologic activity. 2. PMSF- and DFP-treatment of conditioned culture medium inhibit the blastogenesis in the peripheral mononuclear blood-cell fraction. However, the growth of a permanent T-cell line in the inhibitor-treated medium is not affected. This indicates the existence of a blastogenic factor with serine-protease activity.

Effect of oral aromatic retinoid (Ro 10-9359) on human papilloma virus-2-induced common warts.

Persisting human papilloma virus (HPV) 2 induced common warts of a chronic lymphatic leukemia patient were orally treated with aromatic retinoid Ro 10-9359 (Tigason). Clinically, the lesions improved rapidly. Virus-specific cytopathogenic effects (CPE), virus particles and viral DNA were no longer detectable. Therapy was discontinued because of the development of a liposarcoma, which led to a complete relapse of the cutaneous lesions. HPV-2 specific parameters, CPE and viral DNA, were restored. Comparison of the restriction enzyme cleavage patterns revealed that warts before and after therapy contained the same HPV-2 subtype. The implications of the observation on the effect of Tigason on virus-induced papillomas are discussed.