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.

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 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.

Sialyl Lewis(x)-liposomes as vehicles for site-directed, E-selectin-mediated drug transfer into activated endothelial cells.

E-selectin, exclusively expressed on activated endothelial cells, is a potential target for site-directed delivery of agents. We and others have shown that sialyl LewisX-liposomes (sLe(x)-liposomes) are recognized by E-selectin. We now report an approach employing sLe(x)-liposomes to deliver antisense oligonucleotides (AS-ODNs) directed against the adhesion molecule ICAM-1 to activated vascular endothelial cells. ICAM-1 expression was analyzed at the protein level by immunofluorescence and a cell surface ELISA, and at the RNA level by RT-PCR. We have investigated two different AS-ODNs complementary to the 3' untranslated region and the AUG translation initiation codon of ICAM-1 mRNA. Both inhibited protein expression, but did not influence the mRNA level, pointing to a hybridization of AS-ODNs with the mRNA in the cytoplasm. Our results demonstrate the feasibility of a novel approach for the delivery of agents to activated endothelial cells by glycoliposomes targeted to E-selectin.

Cancerostatic octadecylpiperidinoylphosphate liposomes: effect of composition on uptake by and toxicity to J774 mouse macrophage cells and MT1 breast cancer cells in vitro.

Liposomes prepared from the cancerostatic octadecyl-(N,N-dimethylpiperidino-4-yl)-phosphate (OPP) were investigated in order to characterize the influence of composition on cytotoxicity and to minimize side effects on the immune system. Differently composed liposomes with respect to charge, cholesterol content and steric stabilization were used. Fluorescence measurements and the MTT assay were applied to investigate the effect of uptake and cytotoxicity, respectively, on J774 mouse macrophages and MT1 human breast cancer cells in vitro. Because of their endocytotic capability, uptake was generally higher for macrophages compared with tumour cells. OPP liposomes, which are negatively charged, cholesterol-poor and sterically stabilized, showed the lowest total and internal uptake by both cell lines. On the other hand, these liposomes were also the most cytotoxic ones for both cell lines investigated, with an inhibitory concentration of between 50 and 80 microM. Cytotoxicity does not correlate with cellular uptake and is most likely caused by other mechanisms. The results demonstrate that cancerostatic liposomes have composition-dependent toxic effects on macrophages which have to be seriously considered. For therapeutic experiments in vivo liposomes should be negatively charged and sterically stabilized and composed of OPP and cholesterol in a molar ratio of approximately 1.

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.

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.

Functional characterization of atherosclerosis-associated Ser128Arg and Leu554Phe E-selectin mutations.

The cellular adhesion molecule E-selectin is expressed on activated endothelial cells, and is involved in the process of adherence of blood cells to vessel endothelium in inflammatory events such as atherosclerosis. In a recent study we found a Ser128Arg mutation in the EGF domain as well as a Leu554Phe mutation in the membrane domain of E-selectin. We also established increased frequencies of both mutations among young patients with severe coronary atherosclerosis. In the present study we investigated the influence of these mutations on cell adhesion and on the release of soluble E-selectin. Mutants were created by site-directed mutagenesis and COS cells were transfected with E-selectin, either wild-type or mutant. Antibody-binding studies and cell-adhesion assays were performed on transfected COS cells and on interleukin-1 beta-stimulated HUVECs. Soluble E-selectin in supernatants of wild type and Leu554Phe mutant-transfected COS cells was measured by ELISA. We discovered significant differences in the strength of HL-60 cell adhesion for the Ser128Arg mutant: in comparison with the wild type, the strength of adhesion to the mutant was reduced on transfected COS cells (P < 0.01) as well as on stimulated HUVECs (P < 0.01). Significantly diminished release of soluble E-selectin was detected for the Leu554Phe membrane domain mutant, in comparison with the wild type. In summary, the mutations studied here influence the E-selectin function in vitro and may be considered as one of the risk factors involved in the complex pathogenesis of atherosclerosis.

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.

Ligand-histone H1 conjugates: increased solubility of DNA complexes, but no enhanced transfection activity.

We introduced galactose and a short RGD sequence as ligands into H1 histone to target the asialoglycoprotein receptor or integrins on cells expressing these receptors. The efficiency of the gene transfer mediated by galactosylated H1 histone was strongly affected by the transfection conditions. Galactosylation of H1 led to an increase of the basic H1-mediated gene transfer activity only, when H1 itself did not develop its optimal transfection activity. Under other conditions any specific gene transfer mediated by the asialoglycoprotein receptor was covered by the high transfection efficiency of H1 itself. Similar results of a marginal increase in the transfection efficiency were obtained by conjugates of a short RGD sequence and H1. This unexpected failure in the receptor specificity of both conjugates could be due to the unspecific cell-binding capacity of the H1 moiety and to increasing solubility of the complexes as shown by gel shift and solubility measurements.

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.

Multivalent sialyl Lewis x ligands of definite structures as inhibitors of E-selectin mediated cell adhesion.

We report on the efficiencies of structurally different but well defined multivalent sLex-ligands (di- and trivalent sLex-peptides and sLexbearing liposomes) to block receptor mediated HepG2-cell binding. Using three types of binding assays with distinct receptor accommodations (soluble anti-sLexmonoclonal antibody CSLEX1, immobilized E-selectin, activated HUVECs), we quantified considerable differences of the inhibition efficiencies for the same multivalent sLex-ligands. Compared to the monovalent sLexthe inhibition powers of both (sLex)2-peptides and (sLex)3-peptides were enhanced up to 50-fold for cell binding to the soluble antibody, and that of sLex-liposomes by 7 orders of magnitude. Directed to immobilized E-selectin the inhibition activity was enhanced only 3-fold for (sLex)2-peptides, 10-fold for (sLex)3-peptides but 5 orders of magnitude for sLex-liposomes, respectively. Further decrease of the inhibition efficiencies of glycoligands prepared was observed for cell binding to activated HUVECs. Compared to monovalent sLexwe measured relative efficiencies of 1 for (sLex)2-peptides, of 2 for (sLex)3-peptides but about 20,000 for sLex-liposomes. We concluded that the multivalency of the sLex-ligands prepared is an essential but not sufficient precondition for a high inhibition potency. Additionally, structural properties of the inhibitors determine their binding behavior, which must be considered for the design of potential therapeutic probes.

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.

Acanthosis Nigricans among Native Americans: an indicator of high diabetes risk.

Prevalence of the skin lesion acanthosis nigricans was determined in two tribal communities in Texas and Nebraska. Thirty-eight percent of the Alabama-Coushatta tribe of Texas had acanthosis nigricans. Nineteen percent of Omaha and Winnebago tribal children had the skin lesion; the youngest children had the least acanthosis nigricans. Among weight-matched Alabama-Coushatta, fasting insulin concentrations were twofold higher in subjects with the lesion. It was concluded that acanthosis nigricans is highly prevalent among Native Americans and that its presence suggests insulin resistance. Thus, it may identify those with the highest risk for non-insulin-dependent diabetes mellitus in this population.

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.

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.

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.

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.

Steroid economising effects of a calf thymus extract in three patients with juvenile chronic arthritis.

Recent therapeutic trials in rheumatology using different immunomodulating agents have given encouraging results. In this study an aqueous calf thymus extract (CTE) was administered to three patients, two with systemic juvenile chronic arthritis (JCA), Still's disease, who could not be weaned from steroids during 2 years of conventional therapy, and one girl with a chronic juvenile monarthritis who had responded unsatisfactorily to nonsteroidal antirheumatics for 19 months. A striking clinical improvement was observed in all three patients. Prednisone (PRED) was discontinued in one case with systemic (JCA) and 0.25 mg/kg body weight/day is presently being given to the other patient. The girl is doing well on 4 mg chloroquin kg body weight/day; indomethacin (IND) was discontinued. Laboratory data including cellular immunoreactivity normalized in all three patients.