Sialylated glycolipid antigens on human leukemic cell lines

Many granulocyte-specific mouse monoclonal antibodies recognize the carbohydrate sequence 3-fucosyllactosamine, Ga 1 beta 1-4[Fuc alpha 1-3]GlcNAc, which occurs in cell-surface glycolipids and glycoproteins. In general, these antibodies bind to blast cells from most patients with acute myeloblastic leukemia, but not to those with acute lymphocytic leukemia. Neuraminidase treatment, however, increases exposure of this antigen on both myeloid and lymphoid cells. In the present study, the glycolipids from 13 lymphoid and nonlymphoid human cell lines were examined for the presence of unsialylated and sialylated 3-fucosyllactosamine sequences using a thin-layer chromatography immunostaining method. Nine of the cell lines were also tested by indirect immunofluorescence both before and after neuraminidase treatment. None of the six B-cell and T-cell lines had detectable neutral or sialylated glycolipid antigen. In contrast, six out of seven and five out of seven nonlymphoid cell lines had neutral and sialylated glycolipid antigens, respectively. These results agreed, in general, with those found by indirect immunofluorescence. They also represent the first direct demonstration of these sialylated glycolipids on human leukemic cells. Thus, in some cases increased antibody binding to neuraminidase-treated cells can be explained by the presence of sialylated glycolipid antigen.     

Expression of CD11, CDw15, and transferrin receptor antigens on human hematopoietic progenitor cells

The expression of transferrin receptor-associated antigens and of CD11 and CDw15 antigens was investigated on myeloid committed progenitor cells (CFU-GM day 10, CFU-GM day 7, and cluster-forming cells [CFC] day 4), on erythroid committed progenitor cells (BFU-E and CFU-E), and on multilineage progenitor cells (CFU-GEMM). Both complement-dependent cytotoxicity and fluorescence-activated cell-sorting assays were performed. Complement-dependent cytotoxicity appeared to be the more sensitive assay. Transferrin receptor-associated antigens appeared to be clearly present on all myeloid and erythroid committed progenitor cells, but were found to be only weakly expressed on CFU-GEMM. CD11 antigens appeared to be strongly expressed only on mature granulocytes, monocytes, and certain lymphocytes, but not significantly on myeloid committed precursor cells. Surprisingly, CD11 antigens were weakly, but significantly, present on CFU-E. CDw15 antigens appeared to be restricted to myeloid differentiation and were increasingly expressed from CFU-GM day 10 to CFC day 4. Thus, antitransferrin receptor, CD11, and CDw15 antibodies can be used to separate hematopoietic progenitor cells and may be useful tools in the study of hematopoietic differentiation.     

Regulation of glycolipid synthesis in HL-60 cells by antisense oligodeoxynucleotides to glycosyltransferase sequences: effect on cellular differentiation.

Treatment of the human promyelocytic leukemia cell line HL-60 with antisense oligodeoxynucleotides to UDP-N-acetylgalactosamine:beta-1,4-N-acetylgalactosaminyl-transferase (GM2-synthase; EC 2.4.1.92) and CMP-sialic acid:alpha-2,8-sialyltransferase (GD3-synthase; EC 2.4.99.8) sequences effectively down-regulated the synthesis of more complex gangliosides in the ganglioside synthetic pathways after GM3, resulting in a remarkable increase in endogenous GM3 with concomitant decreases in more complex gangliosides. The treated cells underwent monocytic differentiation as judged by morphological changes, adherent ability, and nitroblue tetrazolium staining. These data provide evidence that the increased endogenous ganglioside GM3 may play an important role in regulating cellular differentiation and that the antisense DNA technique proves to be a powerful tool in manipulating glycolipid synthesis in the cell.     

Generation of human monoclonal antibodies reactive with cellular antigens.

Human lymphocytes from lymph node, peripheral blood, spleen, and tumor specimens have been fused with the LICR-LON-HMy2 (LICR-2) or SKO-007 human cell lines or the NS-1 mouse myeloma line. Over 75 fusions with the three myeloma-lymphoblastoid lines have been performed. Several factors appeared to improve the fusion outcome, including maintenance of the myeloma-lymphoblastoid lines in logarithmic phase growth at greater than or equal to 95% viability, a delay of 24 hr in the introduction of aminopterin to the fused cells, and preselection of the fetal calf serum used in the medium. For a given number of lymphocytes, fusions with NS-1 produced 5-20 times more clones than fusions with LICR-2 or SKO-007, and LICR-2 produced 4 times as many clones as SKO-007. The percentage of clones secreting human immunoglobulin, the range of immunoglobulin production, and the proportion of IgM, IgA, and IgG secretors were comparable for clones derived from the three myeloma-lymphoblastoid lines. Stable Ig-secreting clones were isolated with approximately equal frequency from LICR-2 and NS-1 fusions. A number of stable clones producing human monoclonal antibodies reacting with cell-surface, cytoplasmic, or nuclear antigens have been isolated from tumor-bearing patients and normal individuals. A surface antigenic system present on normal and malignant cells has been defined with a human monoclonal antibody derived from a patient with breast cancer. Techniques for producing human monoclonal antibody now appear to be sufficiently advanced to initiate a serological dissection of the humoral immune response to cancer.     

Invariant NK T cells: potential for immunotherapeutic targeting with glycolipid antigens

Invariant NK T (iNKT) cells are a subset of T lymphocytes that recognize glycolipid antigens bound with the antigen-presenting molecule CD1d. iNKT cells have potent immunoregulatory activities that can promote or suppress immune responses during different pathological conditions. These immunoregulatory properties can be harnessed for therapeutic purposes with cognate glycolipid antigens, such as the marine sponge-derived glycosphingolipid α-galactosylceramide. Preclinical studies have shown substantial promise for iNKT cell-based treatments of infections, cancer and autoimmune and inflammatory diseases. Translation of these preclinical studies to the clinic, while faced with some obstacles, has already had some initial success. In this article, we review the immunodulatory activities of iNKT cells and the potential for developing iNKT cell-based prophylactic and curative therapies of human disease.     

Age-related expression of the cellular prion protein in human peripheral blood leukocytes

BACKGROUND AND OBJECTIVE: Creutzfeldt-Jakob disease typically affects older patients, yet victims of new-variant Creutzfeldt-Jakob disease (nvCJD) are unusually young. Because the cellular prion protein PrP(C) is required for disease development, we investigated age-dependent variability in cell surface PrP(C) expression on various subclasses of human peripheral blood leukocytes (PBL) as a possible susceptibility factor. DESIGN AND METHODS: Three age groups of healthy individuals (mean ages: 6, 33 and 68) were studied by two-color FACS analysis of PBL with fluorescent monoclonal antibodies to PrP(C) and to the lineage markers CD3, CD19, CD4, and CD8. For each subclass marker, surface PrP(C) levels were expressed as mean fluorescence intensity ratios (MFIR) by dividing the geometric mean of the fluorescence of each test antibody by the geometric mean of its isotype-matched control antibody. PrP(C) expression levels in each age and lineage group were compared using appropriate non-parametric tests. RESULTS: We found significant age-related differences in PrP(C) expression on lymphocytes (p=0. 0004). The elderly expressed significantly higher levels than children (p=0.0006) and adults (p=0.0009). PrP(C) expression was also significantly higher in CD3(+) compared to CD19(+ )(p=0.0004) and in CD8(+) compared to CD4(+ )lymphocytes (p=0.0044). INTERPRETATION AND CONCLUSIONS: If PrP(C) expression on PBL were a significant susceptibility factor for nvCJD, young persons would display higher levels. Instead, the elderly expressed the highest amounts of PrP(C) on PBL. This argues against the hypothesis that variability in cell surface expression of PrP(C) in PBL contributes to the exquisite susceptibility of the young to nvCJD.     

Atomic resolution structural characterization of recognition of histo-blood group antigens by Norwalk virus

Members of Norovirus, a genus in the family Caliciviridae, are causative agents of epidemic diarrhea in humans. Susceptibility to several noroviruses is linked to human histo-blood type, and its determinant histo-blood group antigens (HBGAs) are regarded as receptors for these viruses. Specificity for these carbohydrates is strain-dependent. Norwalk virus (NV) is the prototype genogroup I norovirus that specifically recognizes A- and H-type HBGA, in contrast to genogroup II noroviruses that exhibit a more diverse HBGA binding pattern. To understand the structural basis for how HBGAs interact with the NV capsid protein, and how the specificity is achieved, we carried out x-ray crystallographic analysis of the capsid protein domain by itself and in complex with A- and H-type HBGA at a resolution of approximately 1.4 A. Despite differences in their carbohydrate sequence and linkage, both HBGAs bind to the same surface-exposed site in the capsid protein and project outward from the capsid surface, substantiating their possible role in initiating cell attachment. Precisely juxtaposed polar side chains that engage the sugar hydroxyls in a cooperative hydrogen bonding and a His/Trp pair involved in a cation-pi interaction contribute to selective and specific recognition of A- and H-type HBGAs. This unique binding epitope, confirmed by mutational analysis, is highly conserved, but only in the genogroup I noroviruses, suggesting that a mechanism by which noroviruses infect broader human populations is by evolving different sites with altered HBGA specificities.     

Isozymes of human phosphofructokinase: Identification and subunit structural characterization of a new system

The existence of a five-membered isozyme system for human phosphofructokinase (PFK; ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been demonstrated. These multimolecular forms result from the random polymerization of two distinct subunits, M (muscle type) and L (liver type), to form all possible tetrameters-i.e., M(4), M(3)L, M(2)L(2), ML(3), and L(4). Partially purified muscle and liver PFKs were hybridized by dissociation at low pH and then recombination at neutrality. Three hybrid species were generated in addition to the two parental isozymes, to yield an entire five-membered set. The various species could be consistently and reproducibly separated from one another by DEAE-Sephadex chromatography at pH 8.0 with a concave elution gradient of salt. Under similar experimental conditions, erythrocyte PFK from hemolysates was also resolved into five species chromatographically indistinguishable from those produced in the above experiment. Immunological and kinetic studies of the isozymes provided corroborative evidence to support the proposed subunit structures. Erythrocyte PFK was found to have kinetic properties intermediate between those of muscle and liver PFK and was neutralized only 50% by an antiserum against muscle PFK that completely neutralized muscle PFK. These data demonstrate that muscle and liver PFKs are distinct homotetramers-i.e., M(4) and L(4), respectively-whereas erythrocyte PFK is a heterogeneous mixture of all five isozymes. The structural heterogeneity of erythrocyte PFK provides a molecular genetic basis for the differential organ involvement observed in some inherited PFK deficiency states in which myopathy or hemolysis or both can occur.     

Partial purification and characterization of the high molecular weight latent collagenase from human leukocytes

Two latent collagenases whose apparent molecular weights were ca. 150 000 and 60 000 have been detected in human leukocytes and the partial purification of the high molecular weight component was accomplished by the following steps: acetone precipitation, ammonium sulphate fractionation, gel filtration on Sephacryl S-200, chromatography on DEAE-Sepharose, and a final gel filtration on Sephacryl S-200. After activation by an activator extracted from human rheumatoid synovial fluid, the enzyme was able to cleave collagen into the classical 1/4 and 3/4 fragments, and was inhibited by chelating agents and other typical collagenase inhibitors.     

Direct migration inhibition test with leukocytes as indicator of mycobacterial antigens in the human body.

In 32 patients with newly detected pulmonary tuberculosis positive on smear and culture, included in a controlled clinical trial, a significant correlation between diminishing inhibition of leukocyte migration (induced by PPD) and reduction of mycobacterial population in the sputum during the course of chemotherapy has been demonstrated. The changes of mycobacterial population in the host have been assessed in two ways: an indirect assessment was made from the time taken to achieve sputum smear and culture negativity; a direct assessment was based on repeated quantitative determinations of tubercle bacilli in the sputum. The direct migration inhibition test with peripheral buff-coated leukocytes, is an easy and reliable correlate of delayed hypersensitivity to mycobacterial antigens in the human body. It is suggested that its use in individual patients could indicate changes in mycobacterial population, particularly after microbiological negativity has been obtained, and thus could be helpful in estimating the optimum duration of chemotherapy.     

A comparison of cellular and humoral immune responses to trichuroid derived antigens in human trichuriasis

Individuals, residing in a region highly endemic for Trichuris trichiura, were examined for cytokine and proliferative responses to T. trichiura worm homogenate (TtAg), T. trichiura excretory/secretory products (TtES) and the equivalent antigenic preparations from the murine whipworm, Trichuris muris. Serum antibody levels against TtAg, T. muris worm homogenate and T. muris ES products were also studied. Measurable levels of immunoglobulin (Ig)G1, IgG4, IgA and IgE against T. muris antigens were detected, indicating a degree of conservation of epitopes between antigens derived from both species. Although levels of interleukin (IL)-4, IL-10, IL-13, tumour necrosis factor (TNF)-alpha and proliferative responses produced were comparable between homogenate antigens of either species and ES antigens of either species, a markedly different cellular response was observed in cultures stimulated with homogenate antigens compared to ES antigens. ES antigens preferentially induced IL-10 (P > 0.001) and TNF-alpha (P > 0.001) production, whereas levels of IL-4 (P > 0.001), IL-13 (P > 0.001) and proliferative responses (P > 0.001) were greater in cultures stimulated with whole worm extracts. Our findings suggest that T. muris preparations could be used as an alternative to T. trichiura proteins as a source of antigens in ex vivo cultures and that ES products stimulate a distinct immune response compared to somatic antigens.     

Identification and characterization of human B-cell epitopes in recombinant antigens of Leishmania aethiopica

Recombinant DNA fragments from Leishmania aethiopica that code for epitopes which react with human antibodies have been characterized by cross-hybridization studies and DNA sequence analysis. Twenty clones could be grouped into seven different groups (I–VII), probably representing seven different L. aethiopica antigens. The DNA sequences of representative clones from the seven groups have been obtained and the amino acid sequence of the respective recombinant antigens established. The recombinant antigens have been analysed by epitope scanning with patient sera, and octapeptides that contain potential B-cell epitopes have been identified in all seven recombinant antigens. These octapeptides have further been tested with additional patient sera and control sera, and three octapeptides (HAFCHEEG, YHSSVVHD and SYAPCSLK) were found to contain major epitopes recognizing specific antibodies in nine, seven and four, respectively, of the twenty sera tested. Fifteen of the twenty sera reacted with one or more of these three octapeptides.