Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40

The Ag CDw40 (p50, Bp50) is a phosphoprotein expressed on the surface of both B lymphocytes and on certain malignant cell types of nonhemopoietic origin. Antibodies to this Ag have been shown to act as a potent co-mitogen for B cells. In order to elucidate the function of this Ag, we have now investigated some of its biochemical characteristics as well as the relationship of B cell derived CDw40 to that derived from urinary bladder carcinoma (transitional cell carcinoma, TCC) cells. CDw40 from normal B cells or from the Burkitt lymphoma line Raji showed a characteristic pattern of three bands when analyzed by SDS-PAGE and Western blotting: a main band of 47 kDa, a degradation product of 43 kDa, and a dimer of 85 kDa. The dimer was disrupted by reduction with 2-ME but was reformed spontaneously from the purified monomers under nonreducing conditions. CDw40 from two bladder cancer cell lines gave a similar pattern but formed little or no dimer. Thirty amino acids of the amino terminal end of CDw40 from Raji and 22 amino acids of that from TCC cells (HU549) were sequenced. The sequences were unusually rich in cysteines and differed only in that the cysteine in position 6 in Raji CDw40 had been replaced by glutamine in HU549. In addition there were two conservative changes in positions 15 and 19. Taken together these results show that CDw40 derived from B cells or from TCC cells are the same or closely related molecules. Comparisons of the amino acid sequence and biochemical characteristics of CDw40 with proteins having receptor functions indicated a close structural resemblance of CDw40 to the nerve growth factor-receptor.     

Tumor marker measurements of cells in a fine needle used for aspiration cytology

OBJECTIVE: To investigate whether the needle washing could yield sufficient cells for tumor marker (TM) measurements as an ancillary technique to ensure the accuracy of fine needle aspiration cytology (FNAC) of tumors. STUDY DESIGN: After obtaining preliminary data that aspirated tumor cells within a 22-gauge needle could be collected by washing it with distilled water for TM measurements, we studied tumor cell numbers and TM values obtained by washing a 22-gauge needle directly after tumor aspiration and another needle after FNAC. RESULTS: Using 8 resected hepatobiliary and pancreatic carcinomas, the used needles yielded 16.8+/-10.5 x 10(4) cells per milliliter. Used needles from 6 adenocarcinomas expelled 479.2+/-406.5 ng/mL of carcinoembryonic antigen, and 6,561.3+/-5,713.1 ng/mL of CA 19-9, while the needles from 2 hepatomas showed normal values of those markers. CONCLUSION: A needle used for FNAC contains sufficient cells for TM measurements, which can be ancillary to the differential diagnosis.     

Vertical migration, nitrate uptake and denitrification: survival mechanisms of foraminifers (Globobulimina turgida) under low oxygen conditions

(15)NO(3)(-) isotope labelling experiments were performed to investigate foraminiferal nitrate uptake strategies and the role of pseudopodial networks in nitrate uptake. Globobulimina turgida were placed below the nitrate penetration depth in homogenized sediment cores incubated in artificial seawater containing (15)NO(3)(-) . A nylon net prevented the vertical migration of foraminifera to strata containing nitrate and oxygen, but allowed potential access to such strata by extension of pseudopods. No (15)NO(3)(-) was found in G. turgida in these cores, suggesting that foraminifera cannot extend their pseudopods for nitrate uptake through several millimetres of sediment, but must physically migrate upwards closer to nitrate-containing strata. However, foraminiferal migration patterns in control cores with no nylon net were erratic, suggesting that individuals move in random orientations until they find favourable conditions (i.e. free nitrate or oxygen). A second experiment showed that foraminifera actively collect nitrate both in the presence and in the absence of oxygen, although uptake was initiated faster if oxygen was absent from the environment. However, no systematic influence of the size of the intracellular nitrate pool on nitrate uptake was observed, as specimens containing a large range of intracellular nitrate (636-19 992 pmol per cell) were measured to take up (15)NO(3)(-) at comparable rates.     

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

Summary We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.     

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder

Summary Spleen cells from BALB/c mice immunized with cells derived from transitional cell carcinomas (TCC) of the human urinary bladder were fused with mouse myeloma Sp 2/0 Ag14 cells. Monoclonal antibodies from six established hybridomas were investigated for specificity in a cell ELISA and in indirect immunofluorescence against a large panel of fixed intact cells. Three of the antibodies reacted with half or more of the eight bladder tumors and with a few unrelated tumors. They did not react at all with malignant or normal cells of hematopoietic origin. A fourth antibody reacted with seven of eight bladder tumors. It also reacted weakly with a prostatic carcinoma, with five of six malignant or transformed B cell lines, and with a subpopulation of normal lymphocytes, but not with any of the other cells on the test panel. These four antibodies did not react with cells derived from normal urothelium. The results suggest that these antibodies might recognize cell-type-restricted antigens associated with malignancy. Another antibody reacted with almost all urothelium-derived cells. It also reacted with three of three melanomas but not with any other cells on the panel. The sixth antibody reacted with 32 of the 37 cells tested. The spectrum of reactivities displayed by the antibody suggested that it recognizes HLA antigens. Abbreviations used in this paper: TCC, transitional cell carcinoma of the urinary bladder; TAA, tumor-associated antigens; ELISA, enzyme-linked immunosorbent assay; NBCS, newborn calf serum; PBS, phosphate-buffered saline, pH 7.2; ALP, alkaline phosphatase; GDA, glutardialdehyde; BSA, bovine serum albumin; IF, indirect immunofluorescence; LCL, lymphoblastoid cell lines: B-lymphocytes transformed in vitro with Epstein-Barr virus     

The human B lymphocyte and carcinoma antigen, CDw40, is a phosphoprotein involved in growth signal transduction

The human B lymphocyte and carcinoma-associated Ag, CDw40, (p50, Bp50) is a receptor candidate for normal growth regulation. Interaction of mAb with this pan-B Ag, together with preactivating agents such as 12-O-tetradecanoylphorbol-13-acetate or anti-mu, deliver strong growth-promoting signals to the cells. We here demonstrate that signaling through this Ag is dependent on its aggregation on the cell surface. Thus, monovalent antibody fragments were relatively inefficient in this respect but effectively blocked stimulation by intact antibody. By using affinity purified CDw40 protein we have also demonstrated that it is antigenically distinct from other B cell-associated Ag, including the six differentiation clusters CD19 to CD24. The mAb S2C6 and G28.5, prepared by immunizing mice with human bladder carcinoma cells or tonsillar B-cells, respectively, were the only antibodies giving detectable binding. Either of these antibodies could also completely block the binding of the other, suggesting an identity or structural proximity of the epitopes recognized. The CDw40 Ag was shown to be a phosphoprotein lacking intrinsic protein kinase activity. The results provide further evidence for CDw40 being an important B cell growth factor receptor which may also have growth regulatory functions in the development of certain human carcinomas.     

Regulation of the NK cell alloreactivity to bone marrow cells by the combination of the host NK gene complex and MHC haplotypes

Host NK cells can reject MHC-incompatible (allogeneic) bone marrow cells (BMCs), suggesting their effective role for graft-vs leukemia effects in the clinical setting of bone marrow transplantation. NK cell-mediated rejection of allogeneic BMCs is dependent on donor and recipient MHC alleles and other factors that are not yet fully characterized. Whereas the molecular mechanisms of allogeneic MHC recognition by NK receptors have been well studied in vitro, guidelines to understand NK cell allogeneic reactivity under the control of multiple genetic components in vivo remain less well understood. In this study, we use congenic mice to show that BMC rejection is regulated by haplotypes of the NK gene complex (NKC) that encodes multiple NK cell receptors. Most importantly, host MHC differences modulated the NKC effect. Moreover, the NKC allelic differences also affected the outcome of hybrid resistance whereby F1 hybrid mice reject parental BMCs. Therefore, these data indicate that NK cell alloreactivity in vivo is dependent on the combination of the host NKC and MHC haplotypes. These data suggest that the NK cell self-tolerance process dynamically modulates the NK cell alloreactivity in vivo.     

A ligand for the murine NK activation receptor Ly-49D: activation of tolerized NK cells from beta 2-microglobulin-deficient mice

Mouse NK cells express inhibitory NK receptors that recognize target cell MHC class I molecules and activation receptors that are less well defined. The Ly-49D activation receptor on C57BL/6 NK cells recognizes Chinese hamster ovary cells and triggers natural killing. In this study, we demonstrate that a Chinese hamster classical MHC class I molecule is the ligand for Ly-49D in a reporter gene assay system as well as in NK cell killing assays. Ly-49D recognizes the Chinese hamster class I molecule better when it is expressed with Chinese hamster beta(2)-microglobulin (beta(2)m) than murine beta(2)m. However, it is still controversial that Ly-49D recognizes H-2D(d), as we were unable to demonstrate the specificity previously reported. Using this one ligand-one receptor recognition system, function of an NK activation receptor was, for the first time, investigated in NK cells that are tolerized in beta(2)m-deficient mice. Surprisingly, Ly-49D-killing activity against ligand-expressing targets was observed with beta(2)m-deficient mouse NK cells, albeit reduced, even though "tolerized" function of Ly-49D was expected. These results indicate that Ly-49D specifically recognizes the Chinese hamster MHC class I molecule associated with Chinese hamster beta(2)m, and indicate that the Ly-49D NK cell activation receptor is not tolerized in beta(2)m deficiency.     

Protection from lethal infection by adoptive transfer of CD8 T cells genetically engineered to express virus-specific innate immune receptor

CMV infection is one of the most common complications in immunocompromised individuals, such as organ and bone marrow transplant patients. Both innate and adaptive immune responses are required for defense against CMV infection. In murine CMV (MCMV) infection, strains harboring the MCMV-specific NK cell activation receptor, Ly49H (Klra8), are resistant. In contrast, MCMV infection of mice lacking Ly49H gene causes early mortality due to uncontrolled viral replication. In this study, we report the successful protection of mice from lethal MCMV infection with gene-transferred polyclonal CD8 T cells. CD8 T cells expressing a chimeric receptor comprising Ly49H extracellular and CD3zeta cytoplasmic domains are capable of killing target cells expressing the MCMV protein, m157. CD8 T cells expressing the chimeric receptor protect mice in vivo from lethality in the acute phase of MCMV infection, leading to the establishment of long-term protection. These data provide proof-of-principle evidence that a novel strategy for harnessing CD8 cytolytic function through TCR-independent yet pathogen-specific receptor can result in effective protection of hosts from pathogens.     

Expression of MCT1, MCT2 and MCT4 in the rumen, small intestine and liver of reindeer (Rangifer tarandus tarandus L.)

The expression of monocarboxylate transporters MCT1, MCT2 and MCT4 in the rumen, small intestine and liver was examined in free-ranging and captive reindeer. In addition, expression of chaperone protein CD147, which is needed for the activity of MCT1 and MCT4, was studied in the rumen of suckling calves. Immunoblotting of cell membrane proteins showed the expression of MCT1 and MCT4, but not that of MCT2 in the rumen of reindeer. In free-ranging reindeer the amount of MCT1 was higher than in the captive ones (P<0.01). Developing rumen of suckling calves expressed MCT1 and MCT4 and positive correlation was found between MCT1 and CD147. Both MCT1 and CD147 correlated also with age in calves less than 10 days. In the small intestine all the isoforms studied were expressed, but the amounts were lower than in the rumen (P<0.05). In the liver MCT1 and MCT2 were found while MCT4 was nearly undetectable. The expression of MCT isoforms in the rumen and small intestine reflects the site of absorption and concentrations of short chain fatty acids (SCFA). In the liver the expression of high affinity transporters, MCT1 and MCT2, is in accordance with almost complete uptake of propionate from portal blood.