Cell Surface Reactivity Research Articles

Carbohydrate characterization of quail primordial germ cells during migration and gonadal differentiation

A selection of lectins were used to study changes in the distribution of sugar residues in primordial germ cells (PGCs) during gonadal colonization and differentiation. Although the cytochemical characteristics of PGCs have been described in the chick, little is known about such characteristics in other avian species of interest to experimental biology. Therefore, we studied embryos of Japanese quail (Coturnix coturnix japonica) by light and laser confocal scanning microscopy, using the QH1 antibody as a tool to identify PGCs in both sexes and at all stages. LEA, WGA and RCA-I bound to PGCs in a similar way to QH1. LEA was the best marker for all stages. The presence of acid phosphatase and the intense reaction of LEA, WGA, RCA-I and WFA at the cell surface were shown to be a useful tool in the study of the migratory PGCs of the quail. Quails were sexed histologically in younger embryos than in chick; results were confirmed by PCR. The lectin-binding pattern changed drastically in differentiated gonads. Cell surface reactivity was almost entirely lost. Quail differentiating oogonia showed a characteristic binding pattern to QH1 and to the lectins WGA, RCA-I and WFA. Binding was observed in intense spots in the Golgi complex, and there was a specific PNA reaction. These results suggest that some selective sugar binding sites on the PGCs play a significant role in their migration, colonization and maturation.

Overexpression of the Cytokine Osteopontin Identifies Aggressive Laryngeal Squamous Cell Carcinomas and Enhances Carcinoma Cell Proliferation and Invasiveness

Osteopontin is a secreted cytokine that binds to the cell surface CD44v6 receptor. We studied osteopontin and CD44v6 expression in laryngeal squamous cell carcinomas and correlated osteopontin expression levels with clinicopathologic tumor features. We used immunohistochemistry, immunoblotting, and reverse transcription-PCR to study osteopontin expression in 58 laryngeal squamous cell carcinomas. Cultured squamous carcinoma cells were treated with exogenous osteopontin or with RNA interference to knockdown osteopontin expression. Osteopontin expression was higher in all the invasive carcinomas than in patient-matched normal mucosa. Its expression levels were significantly correlated with tumor stage and grade and with the presence of lymph node and distant metastases. Osteopontin positivity was negatively correlated with overall survival (P = 0.03). Osteopontin expression was paralleled by intense cell surface reactivity for CD44v6. Treatment of squamous carcinoma cells with recombinant osteopontin sharply increased proliferation and Matrigel invasion in comparison with the untreated cells parallel to activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase signaling cascade. Osteopontin knockdown by RNA interference, anti-CD44 antibodies, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition prevented these effects. These results identify osteopontin as a marker and a potential therapeutic target in cases of aggressive laryngeal squamous cell carcinomas.

Antibodies against Tumor Cell Glycolipids and Proteins, but Not Mucins, Mediate Complement-Dependent Cytotoxicity

One of several effector mechanisms thought to contribute to Ab efficacy against cancer is complement-dependent cytotoxicity (CDC). Serological analysis of a series of clinical trials conducted over a 10-year period suggested that six vaccines containing different glycolipids induced Abs mediating CDC whereas four vaccines containing carbohydrate or peptide epitopes carried almost exclusively by mucin molecules induced Abs that did not mediate CDC. To explore this further, we have now compared cell surface reactivity using flow cytometry assays (FACS), complement-fixing ability, and CDC activity of a panel of mAbs and immune sera from these trials on the same two tumor cell lines. Abs against glycolipids GM2, globo H and Lewis Y, protein KSA (epithelial cell adhesion molecule, also known as EpCAM) and mucin Ags Tn, sialylated Tn, Thomsen Friedenreich (TF), and MUC1 all reacted comparably by FACS with tumor cells expressing these Ags. Compared with the strong complement binding and CDC with Abs against glycolipids and KSA, complement binding was diminished with Abs against mucin Ags and no CDC was detected. A major difference between these two groups of Ags is proximity to the cell membrane. Glycolipids and globular glycoproteins extend less than 100 A from the cell membrane while mucins extend up to 5000 A. Although complement activation at sites remote from the cell membrane has long been known as a mechanism for resistance from complement lysis in bacteria, it is identified here for the first time as a factor which may contribute to resistance from CDC against cancer cells.

Relationship of binding of immunoglobulin G to Plasmodium falciparum-infected erythrocytes with parasite endemicity and antibody responses to conserved antigen in immune individuals.

To investigate the potential for use of a well-established strain of Plasmodium falciparum as a reference strain for infected red blood cell (IRBC) surface reactivity, we monitored the binding of specific immunoglobulin G (IgG) from immune individuals to the reference Knob-positive FCR3 strain by flow cytometry. To permit interassay comparison for 162 plasma samples drawn after the rainy season, a labeling index (LI) was defined as the percentage of labeled parasites multiplied by the mean peak intensity. An LI ratio (LIR) was then calculated as the LI of the sample divided by the LI of the control. LIRs were calculated for individuals living in Dielmo and Ndiop, two Senegalese villages where P. falciparum is transmitted holoendemically and mesoendemically, respectively. The incidence (persons with an LIR of >3) observed in Dielmo was lower than that observed in Ndiop. Significantly higher LIRs were observed (i) for samples from Ndiop than for samples from Dielmo (P < 0.01) and (ii) in Ndiop, in subjects with hemoglobin AS (HbAS) than in those with hemoglobin AA (P = 0.03). No correlation with the cumulative age-associated immune status of the villagers was evidenced, contrary to antibody (Ab) responses against conserved IRBC-associated antigen (Ag) measured by enzyme-linked immunosorbent assay. These results are consistent with the notions that protection in HbAS individuals may relate to an increased IgG response to IRBC membrane Ags and that cell surface reactivity parallels IgG responses even though it is in itself a distinct indicator of the anti-P. falciparum Ab response. Measures of IgG binding to live IRBC are thus relevant for the functional screening of conserved IRBC-associated Ags that contribute to parasite destruction in vivo, as these Ags might be included in a multitarget vaccine.

Characterization and implications of the cell surface reactivity of Calothrix sp. strain KC97.

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK(a)] between 5.0 and 6.2) and amine groups (mean pK(a) of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK(a) spectra similarly dominated by carboxyls (pK(a) of 4.6 to 6.1) and amines (pK(a) of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK(a) values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 x 10(-5) and 12.3 x 10(-5) mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.

Effect of immunological adjuvant combinations on the antibody and T-cell response to vaccination with MUC1–KLH and GD3–KLH conjugates

A year ago we described a comparison of 19 immunological adjuvants for their ability to augment antibody and T-cell responses against vaccines containing two cancer antigens, GD3 ganglioside and MUC1 peptide, covalently attached to keyhole limpet hemocyanin (KLH). As in our previous experience, the saponin fraction QS-21 was the most potent single adjuvant but several other adjuvants also had potent adjuvant activity. Induction of an immune response against cancer antigens is generally difficult because these antigens are autoantigens. To get maximal benefit from the adjuvant component of cancer vaccines we have now tested whether combinations of the optimal adjuvants induced an improved immune response compared to QS-21 alone. Since over the intervening year a new semi-synthetic saponin adjuvant (GPI-0100) containing the dodecylamide derivative of hydrolyzed naturally-occurring saponins had become available, this was tested as well. Twelve different adjuvant combinations and GPI-0100 were compared for their ability to augment (1) antibody responses against GD3 and MUC1 and (2) T-cell responses against GD3, MUC1 and KLH. GPI-0100 and five adjuvant combinations were superior to QS-21 alone for induction of IgM and IgG antibodies against MUC1 and/or GD3: QS-21 plus bacterial nucleotide CpG, QS-21 plus monophosphoryl lipid A (MPL), QS-21 plus non-ionic block copolymer CRL-1005, QS-21 plus Titermax and Titermax plus CpG. Antibody responses were documented both by ELISA against purified antigens and by FACS for cell surface reactivity. There was no evidence for T-cell immunity against GD3 or MUC1. The antibody responses against GD3 and MUC1 were, however, strongly correlated with IFN-γ release and DTH against KLH. These results demonstrate that combinations of immunological adjuvants are able to augment antibody and T-cell responses to these conjugates beyond that attainable with QS-21 alone, and again confirm the absolute necessity of potent adjuvants or adjuvant combinations for optimal immunogenicity with conjugate vaccines.

The role of type II phospholipase in membrane polishing in the human placenta: A review

During periods of cellular quiescence, the cell membrane bilayer is characterized by an asymmetric phospholipid distribution. The exoplasmic monolayer is principally composed of neutral, choline-containing phospholipids and presents a relatively inert surface to the environmental milieu. Aminophospholipids (e.g., phosphatidylserine) are almost exclusively confined to the cytoplasmic monolayer. Upon cellular activation and increases in cytosolic calcium concentrations, phospholipid asymmetry is rapidly relaxed. The appearance of aminophospholipids on the cell surface dramatically affect many aspects of cellular function, including: cell surface protein binding; membrane fusion events; terminal differentiation; and recognition by phagocytes. Secreted, extracellularly active PLA 2 s that display a substrate preference for aminophospholipids may regulate their expression in the exoplasmic monolayer of the cell membrane and thereby alter cellular function and reactivity. One such secreted PLA 2 isozyme (Type II) is abundantly expressed in and released from human placenta. Although this isozyme contributes to gestational tissue phospholipid metabolism, its role in regulating the expression of aminophopholipids on the cell surface and cell reactivity has yet to be established.

The Amino Terminus of Human CCR5 Is Required for Its Function as a Receptor for Diverse Human and Simian Immunodeficiency Virus Envelope Glycoproteins

The chemokine receptor CCR5 plays a key role in the CD4-dependent entry of human and simian immunodeficiency viruses into target cells. We have mapped the interaction sites on CCR5 for a number of novel anti-CCR5 monoclonal antibodies and have used these to study the role of the CCR5 N-terminal ectodomain in viral entry and to demonstrate differential CCR5 epitope expression on different cell types. Deletions of the CCR5 amino terminal domain or substitution with equivalent regions from other chemokine receptors did not affect cell surface expression or reactivity with loop-specific antibodies, suggesting that the loop regions remained conformationally intact. Exchanges of the amino terminal segment of CCR5 with the equivalent domains of CCR1, CCR2, and CXCR4 did not significantly affect infection with virus pseudotyped with envelope glycoproteins (Envs) from HIV-2 and SIV, but substitution with the CXCR4 sequence abrogated entry mediated by Env from HIV-1. In contrast, deletion of the amino terminus abrogated CCR5 receptor activity for all viral Envs examined. These data indicate that the amino terminus of CCR5 has an essential role in entry mediated by diverse viral Envs but that the sequence requirements are more relaxed for the HIV-2 and SIV Envs compared to the HIV-1 Env examined. This suggests that different viral Envs make distinct and specific interactions with the amino terminus of CCR5. Viral Env utilization of CCR5 expressed on 293-T cells does not always correlate with the cellular tropism of the virus, and one possible explanation is that Env-accessible interaction sites on CCR5 differ on different cell types. We therefore analyzed binding of several anti-CCR5 monoclonal antibodies to cell lines and primary cells that express this chemokine receptor and found that whereas all antibodies bound to CCR5-transfected 293T cells, several did not bind to PBMC. The results suggest that CCR5 undergoes cell type specific structural modifications which may affect interaction with different HIV and SIV envelope glycoproteins.

Monoclonal antibody against adult marrow-derived mesenchymal stem cells recognizes developing vasculature in embryonic human skin.

We have described previously a monoclonal antibody (SH2) that specifically recognizes undifferentiated mesenchymal progenitor cells isolated from adult human bone marrow. These cells, which we operationally refer to as mesenchymal stem cells, have the capacity to differentiate and form distinct mesenchymal tissues such as bone and cartilage when the isolated cells are placed in the appropriate in vivo or in vitro environment. We report here the partial biochemical characterization of the antigen recognized by the SH2 antibody. Metabolically radiolabelled adult marrow-derived mesenchymal stem cells in culture were extracted and immunoprecipitated with the SH2 antibody. The purified antigen migrated as a single band of 90 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed under reducing conditions. The SH2-immunoprecipitated protein exhibited a molecular weight band shift after removal of N-linked oligosaccharides. We investigated the expression of the SH2 antigen, along with the endothelial markers factor VIII-related antigen and Ulex europaeus I (UEA-I) lectin during specific developmental periods in human dermal embryogenesis and in the postnatal period through aged adults. Frozen sections of human embryonic, fetal, or postnatal skin ranging from 8 weeks estimated gestational age (EGA) through 84 years of age were immunostained or double immunolabelled with antibodies SH2, UEA-I, or factor VIII-related antigen followed by second antibodies with fluorescent markers. Positive cell surface reactivity with the SH2 antibody was seen in cells in the vascular plane in the earliest specimens (day 55 EGA) corresponding to the late cellular dermis period. During the period of the cellular to fibrous transition, in which the initiation of appendage development occurs, most SH2-reactive cells colocalized with vasculature markers UEA-I and factor VIII-related antigen, although there was a subset of cells recognized by SH2 antibody that did not colocalize with the endothelial markers. In contrast to the endothelial markers UEA-I and factor VIII-related antigen, in which the number of immunopositive cells became more prominent with age and maturation of the dermis, the frequency of cells that contained the SH2-reactive antigen diminished with age. The SH2 reactivity evident in embryonic, fetal, and early postnatal periods was not observed in human skin specimens taken from adults greater than 30 years old. These observations support the hypothesis that the SH2 antigen is a cell surface marker of developing microvasculature and may play a role in dermal embryogenesis and angiogenesis.

Monoclonal antibody against adult marrow‐derived mesenchymal stem cells recognizes developing vasculature in embryonic human skin

We have described previously a monoclonal antibody (SH2) that specifically recognizes undifferentiated mesenchymal progenitor cells isolated from adult human bone marrow. These cells, which we operationally refer to as mesenchymal stem cells, have the capacity to differentiate and form distinct mesenchymal tissues such as bone and cartilage when the isolated cells are placed in the appropriate in vivo or in vitro environment. We report here the partial biochemical characterization of the antigen recognized by the SH2 antibody. Metabolically radiolabelled adult marrow-derived mesenchymal stem cells in culture were extracted and immunoprecipitated with the SH2 antibody. The purified antigen migrated as a single band of 90 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed under reducing conditions. The SH2-immunoprecipitated protein exhibited a molecular weight band shift after removal of N-linked oligosaccharides. We investigated the expression of the SH2 antigen, along with the endothelial markers factor VIII–related antigen and Ulex europaeus I (UEA-I) lectin during specific developmental periods in human dermal embryogenesis and in the postnatal period through aged adults. Frozen sections of human embryonic, fetal, or postnatal skin ranging from 8 weeks estimated gestational age (EGA) through 84 years of age were immunostained or double immunolabelled with antibodies SH2, UEA-I, or factor VIII–related antigen followed by second antibodies with fluorescent markers. Positive cell surface reactivity with the SH2 antibody was seen in cells in the vascular plane in the earliest specimens (day 55 EGA) corresponding to the late cellular dermis period. During the period of the cellular to fibrous transition, in which the initiation of appendage development occurs, most SH2-reactive cells colocalized with vasculature markers UEA-I and factor VIII–related antigen, although there was a subset of cells recognized by SH2 antibody that did not colocalize with the endothelial markers. In contrast to the endothelial markers UEA-I and factor VIII–related antigen, in which the number of immunopositive cells became more prominent with age and maturation of the dermis, the frequency of cells that contained the SH2-reactive antigen diminished with age. The SH2 reactivity evident in embryonic, fetal, and early postnatal periods was not observed in human skin specimens taken from adults greater than 30 years old. These observations support the hypothesis that the SH2 antigen is a cell surface marker of developing microvasculature and may play a role in dermal embryogenesis and angiogenesis. Dev. Dyn. 1998;212:119–132. © 1998 Wiley-Liss, Inc.

Efficient selection of scFv antibody phage by adsorption to in situ expressed antigens in tissue sections

The present report describes the development and application of an efficient method for the direct adsorption/selection of antibody phage using antigens expressed in situ in cryostat tissue sections. In a model system, scFv phage directed towards an epitope on the GA733-2 epithelial glycoprotein expressed in colorectal carcinoma tissue could be specifically enriched up to 1500 fold in single-pass experiments and a million fold after three rounds of selection. Enrichment efficacy was directly proportional to the fraction of antigen positive area over the total area. Sufficient enrichment was achieved at an area fraction of less than four percent, thereby permitting the selection of antibodies to sub-populations of cells or to tissue sub-structures. The general usefulness of the method was demonstrated when a combinatorial scFv antibody phage library derived from melanoma immunized non-human primates was selected in tissue sections of metastatic melanoma. Individual scFv antibodies from enriched phage populations demonstrated different binding specificities, reflected in extracellular and cellular tissue staining patterns which included tumor cell surface reactivity. This method should be particularly useful for the identification of antigens which are only expressed during specific in vivo conditions, and overcomes a major limitation of currently used selection protocols.

Pemphigus Vulgaris Antigen (Desmoglein 3) Is Localized in the Lower Epidermis, the Site of Blister Formation in Patients

In Patients with pemphigus vulgaris, autoantibodies against the desmosomal glycoprotein desmoglein 3 (Dsg3) cause blisters due to loss of keratinocyte cell-cell adhesion in the basal and immediate suprabasal layer of the deeper epidermis, leaving the superficial epidermis intact. Autoantibodies from these patients, however, usually bind to the cell surface of keratinocytes throughout the entire epidermis, as determined by indirect immunofluorescence. To explain this apparent paradox, we immunoadsorbed pemphigus vulgaris sera with the extracellular domains of Dsg3 and desmoglein 1 (Dsg1) produced by insect cells infected with recombinant baculovirus. When adsorbed with extracellular domains of both Dsg3 and Dsg1, these sera no longer stained epidermis, demonstrating that most, if not all, of their cell surface reactivity can be attributed to antibodies against the extracellular domains of these desmogleins. Adsorption with only the Dsg1 extracellular domain left antibodies that stained only the basal and immediate suprabasal layers of the epidermis and immunoprecipitated only Dsg3, not Dsg1, from extracts of cultured cells synthesizing these molecules. In contrast, adsorption with only the Dsg3 extracellular domain left antibodies that stained only the more superficial epidermis and immunoprecipitated only Dsg1. These data localize Dsg3 exactly to the area in the epidermis where blisters occur in pemphigus vulgaris.

Use of synthetic peptides to identify surface-exposed, linear B-cell epitopes within outer membrane protein F of Pseudomonas aeruginosa.

In a previous study (Hughes EE, Gilleland LB, Gilleland HE Jr. [1992] Infect Immun 60:3497-3503), ten synthetic peptides were used to test for surface-exposed antigenic regions located throughout the length of outer membrane protein F of Pseudomonas aeruginosa. An additional nine peptides of 11-21 amino acid residues in length were synthesized. Antisera collected from mice immunized with each of the 19 synthetic peptides conjugated to keyhole limpet hemocyanin were used to determine which of the peptides had elicited antibodies capable of reacting with the surface of whole cells of the various heterologous Fisher-Devlin immunotypes of P. aeruginosa. Cell surface reactivity was measured by an enzyme-linked immunosorbent assay (ELISA) with whole cells of the various immunotypes as the ELISA antigens and by opsonophagocytic uptake assays with the various peptide-directed antisera, immunotype 2 P. aeruginosa cells, and polymorphonuclear leukocytes of human and murine origin. Three peptides located in the carboxy-terminal portion of protein F elicited antibodies with the greatest cell-surface reactivity. Peptide 9 (TDAYNQKLSERRAN), peptide 10 (NATAEGRAINRRVE), and peptide 18 (NEYGVEGGRVNAVG) appear to have sufficient potential for further development as vaccine candidates for immunoprophylaxis against infections caused by P. aeruginosa. A topological model for the arrangement of protein F within the outer membrane of P. aeruginosa is presented.

Cytoplasmic expression of a CD24-related epitope in human PHA activated normal T lymphocytes.

In this study we have analysed by immunoperoxidase (IPx) and indirect immunofluorescence (IIF) the intracellular and cell surface reactivity of VIB-E3 mAb, previously clustered as anti-CD24 antigen, on resting and activated normal human T lymphocytes. By IPx assay VIB-E3 mAb did not show reactivity with normal resting T cells. In contrast, the analysis of 11 different samples of PHA activated normal mononuclear cells, showed an intracytoplasmic expression of CD24. Kinetic studies showed that CD24 appears 24 to 48 h after PHA stimulation. To our knowledge, this is the first evidence that a CD24-related epitope is expressed in normal activated T lymphocytes.

Analysis of the Lewisx epitope in human pancreas and pancreatic adenocarcinomas.

The Lewisx epitope (Gal beta 1-4[Fuc alpha 1-3]GlcNAc-R) can be detected in most ductal pancreatic carcinomas. However, few data pertain to its expression in normal exocrine pancreas and its biochemistry. We compared the expression of the Lewisx epitope in normal pancreas with its expression in pancreatic adenocarcinomas and tumor cell lines and to further characterize the nature of the antigens bearing this epitope with immunochemical methods. On frozen tissue sections of normal pancreas, the Lewisx epitope was detected focally in acinar cell granules; sialosyl-Lewisx was detected in centroacinar cells and the cytoplasm of intralobular ducts. Sixteen of 19 pancreatic carcinomas expressed the Lewisx antigen on tissue sections; however, there was no correlation with prognostic parameters, such as tumor grade or stage. Eighteen of 19 tumor specimens were positive for sialosyl-Lewisx. Analysis of pancreatic carcinoma cell lines (CAPAN-1, CAPAN-2, and DAN-G) revealed intracytoplasmic expression of sialosyl-Lewisx epitopes in these cells, and cell surface reactivity was detected on flow cytometry for sialosyl-Lewisx. Lectin-affinity chromatography of cytoplasmac preparations showed that Lewisx-positive antigens of normal human pancreas bind to SBA and UEA-I lectins but have little or no affinity to WGA, GS-I, or DBA lectins, indicating the presence of Fuc and Gal oligosaccharide residues. It was concluded that the Lewisx and sialosyl-Lewisx antigens are nonpolymorphic carbohydrate determinants that are present in secretory granules of acinar cells, ductules, and pancreatic secretions. This makes the Lewisx antigen suitable for the analysis of the secretory process in the exocrine pancreas and the study of secretory differentiation antigens in ductal pancreatic carcinoma.

Human monoclonal antibody with dual GM2/GD2 specificity derived from an immunized melanoma patient.

GM2 ganglioside is a common cell surface constituent of human melanoma and other tumors of neuroectodermal origin, and vaccination with GM2 ganglioside results in high levels of anti-GM2 antibodies in patients with melanoma. Lymphocytes from a GM2-vaccinated patient (VS) were transformed by Epstein-Barr virus and tested for production of antibodies with reactivity for GM2-positive tumor cells. A high percentage of antibody-producing B cells was detected, but antibody reactivity was generally lost during culture expansion. Two cultures, however, remained stable for antibody productivity and one was used to develop a stable hybrid line with mouse myeloma. The monoclonal antibody (designated 3-207) derived from patient VS has dual specificity for GM2 and GD2, despite the fact that only GM2 antibody could be detected in the patient's serum. Monoclonal antibody 3-207 shows high-titered reactivity with a range of melanoma, astrocytoma, neuroblastoma, and leukemia cell lines, cells with prominent cell surface expression of GM2 and GD2. The cell surface reactivity of monoclonal antibody 3-207 was not abolished by treatment of target cells with neuraminidase, as the enzyme converted GD2 to GM2, which was still detected by monoclonal antibody 3-207.

Recognition of galactosylgloboside by monoclonal antibodies derived from patients with primary lung cancer.

Lymph node lymphocytes from patients with primary lung cancer were immortalized with Epstein-Barr virus, and culture supernatants were screened for cell-surface reactivity against allogeneic cancer cell lines. The percentage of wells containing detectable antibodies in initial screening ranged from 1 to 17%, but the vast majority of the cultures lost antibody activity on subsequent expansion. Two antibody-secreting clones, J309 and D579, derived from separate individuals and reactive with anaplastic lung cancer cell lines, were successfully expanded and fused with the NS-1 mouse myeloma cell line. The antibodies produced by these clones exhibited identical restricted serologic reactivity against cultured cell lines and detected a carbohydrate antigen present in the neutral glycolipid fraction of MCF-7 breast cancer cells. Serologic, immunochemical, and chemical analyses revealed that the antigen recognized by antibodies J309 and D579 is galactosylgloboside [Gal(beta 1----3)GalNAc(beta 1----3)Gal(alpha 1----4)Gal(beta 1----4)- GlcCer]. Conclusions regarding the significance of these findings with respect to the biology of lung cancer await further information concerning the distribution of galactosylgloboside in normal and malignant tissues and the frequency of antibodies to this structure in normal and tumor-bearing individuals.

Effects of site-specific mutation on structure and activity of influenza virus B/Lee/40 neuraminidase

A cDNA copy of the neuraminidase (NA) gene of a high-growing reassortant influenza virus which has the hemagglutinin of B/Hong Kong/8/73 and the NA of B/Lee/40 was cloned into plasmid pUC 9 and subcloned into a late-replacement SV40 vector so that the NA gene could be expressed in eukaryotic cells. The expressed protein was antigenically and enzymatically active. To study structure-function relationships in the B/Lee NA, particularly in comparison to the known structure of influenza A (N2) NA, specific mutations were introduced using synthetic oligonucleotides. Mutation of an apparently unpaired cysteine residue to serine at position 251 had no effect on protein transport or folding as judged by cell-surface reactivity with monoclonal antibodies, and the NA retained enzyme activity, confirming that this Cys is not essential for correct folding of the polypeptide. Mutation of Trp 364 to Leu abolished detectable enzyme activity, while mutation of Thr 368 to Val reduced enzyme activity to less than 25% of wild-type levels. Neither mutation affected antigenic properties. Therefore it is likely that both these side chains extend into the NA active site pocket. The results of these experiments are in accord with similarities in the structure of the B/Lee NA compared with that of influenza A (N2) NA. Although there is about 70% amino acid sequence difference between influenza A and B NAs, residues in the active site are highly conserved. Our in vitro mutagenesis experiments help to confirm the tentative alignment of sequences and have identified conserved amino acid side chains involved in enzyme activity.

Localisation on human chromosome 19 of three genes for cell surface antigens defined by monoclonal antibodies

Expression of three distinct human cell surface antigens defined by monoclonal antibodies (mAbs) was examined in a series of rodent-human somatic cell hybrids retaining different subsets of human chromosomes. Cell surface reactivity with mAbs F8 and G253, detecting a 95 kilodalton (kD) glycoprotein (gp95); with mAbs F10 and A103, detecting a 50 kD glycoprotein (gp50); and with mAb S7 was found to cosegregate with human chromosome 19. However, differential antigen expression was observed with hybrids containing fragments of the 19 and hybrids constructed with different human cell types. Comparison of results from the serological typing with the presence of a number of chromosome 19 DNA markers in hybrid cells and cytogenetic analysis suggests that MSK20, the gene coding for the F10/A103 antigen gp50, is located in chromosome region 19pter----19p13.2. The genes coding for the F8/G253 antigen, gp95 (gene symbol MSK19) and the S7 antigen (MSK37) are located in region 19p13.2----19q13.2. Thus, the cell surface antigens described in this study may be used as selectable markers for specific portions of human chromosome 19.

Phenotypic Analysis of Acute Lymphoblastic Leukemia (ALL) Cells Which Are Classified as Non-T Non-B and Negative for Common ALL Antigen

When phenotypic marker analysis of acute lymphoblastic leukemia (ALL) cells (102 cases) was performed, a group of ALL cells (15 cases) classified as non-T non-B, and negative for common-ALL antigen (CALLA) was characterized in a focused manner. "Non-T non-B" was defined as negative for T cell properties such as E-rosetting or reactivity with anti-human T-cell monoclonal antibodies (T101, WT1), and absence of any B-cell characteristics (cell surface and/or cytoplasmic immunoglobulin and reactivity with B1 monoclonal antibody). Despite their marked heterogeneity, CALLA(-) non-T non-B ALL cells revealed three different phenotypic patterns in terms of presence of terminal deoxynucleotidyl transferase (TdT) and of reactivity with antimyeloid (MCS1) or myelomonocyte (MCS2 and OKM1) monoclonal antibodies. Four of 15 cases reacted with some myeloid-specific antibodies, but were negative for TdT. Six cases had both MCS2 antigen and TdT. The remaining five cases expressed no myeloid antigens, but were positive for TdT with some exceptions. These findings showed that acute leukemias with myeloid antigens might be involved in CALLA(-) non-T non-B ALL having no relationship to the presence of TdT, and, furthermore, that the blasts with simultaneous expression of TdT and myeloid-specific antigen (MCS2) might represent an immature stage in hematopoietic differentiation closely corresponding with the bifurcation of the lymphocyte/myeloid pathway. Alternatively, only five cases remained "unclassified leukemia." We therefore think that the detailed examination of CALLA(-) non-T non-B ALL cells using myeloid specific antibodies is helpful in clarifying the characteristics of myeloid precursors and the common bipotential stem cell of lymphoid and myeloid progenitors.