Effects of recombinant human insulin-like growth factor I (IGF-I) therapy on the growth hormone-IGF system of a patient with a partial IGF-I gene deletion.

We have previously reported a 17.2-yr-old boy with severe growth retardation and undetectable serum levels of insulin-like growth factor I (IGF-I) due to a partial deletion of the IGF-I gene. The aim of this study was to investigate the effects of recombinant human IGF-I (rhIGF-I) therapy on the GH-IGF system of this patient to gain further insights into its growth-promoting and metabolic actions. To assess the changes in GH, IGFs, IGF-binding proteins (IGFBPs), acid-labile subunit (ALS), and insulin levels, blood samples were obtained before therapy and during the first year of treatment. Hormones were analyzed by specific RIAs. Overnight GH profiles were performed before and at 1, 6, and 12 months of therapy. Fasting ALS, IGF-II, IGFBP-3, IGFBP-2, IGFBP-1, and insulin levels before rhIGF-I treatment were 46.3 mg/L, 1044 microg/L, 5.8 mg/L, 73 ng/mL, 4.7 ng/mL, and 27.3 mU/L, respectively. IGF-II, ALS, and insulin levels were elevated, whereas IGFBP-1 and IGFBP-2 levels were decreased compared to reference values. Twenty-four hours after a single s.c. injection of rhIGF-I (40 microg/kg), the concentrations were 46 mg/L, 888 microg/L, 6.9 mg/L, 112 ng/mL, 5.0 ng/mL, and 21.0 mU/L, respectively. After a single s.c. injection of rhIGF-I of 40 or 80 microg/kg x day and modelling the data using a two-compartment model, the half-lives of elimination were 15.7 and 14.3 h, with a maximum increase in IGF-I levels to 341 and 794 microg/L around 7 h, respectively. An increase in IGFBP-3 levels was observed with both doses of rhIGF-I, with a peak values of 9 mg/L. GH profiles showed a decrease in peak amplitude from 342 to 84 mU/L at 1 month, to 67 mU/L at 6 months, and to 40 mU/L at 1 yr of therapy, with no significant changes in peak number. A significant increase in IGFBP-1 levels was observed during treatment with 80 microg/kg x day IGF-I, reflecting the inhibitory effect of rhIGF-I on insulin secretion. The clinical response to rhIGF-I therapy was an increased height velocity from 3.8 cm/yr before treatment to 6.6 cm/yr. Increased lean body mass correlated with changes in the doses of rhIGF-I and, in turn, with the biochemical changes in the GH-IGF axis. Similar to healthy individuals, this patient had normal IGFBP-3 and ALS levels, which are the major regulators of the pharmacokinetics of rhIGF-I. In summary, rhIGF-I treatment has improved linear growth and insulin sensitivity in this patient by restoring IGF-I levels and by normalizing circulating GH, IGFBP, and insulin levels.

Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli.

A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz. Cys182-Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (Tv18-19) and the corresponding tryptic peptide from rhGH (T18+19) were identical. Electrospray mass spectrometry (ES MS) of Tv18+19 isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T18+19 from rhGH. A synthetic Tv18+19 peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragment in ES/MS compared to Tv18+19 isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cys182-SSS-Cys189) bridge not found in the corresponding T18+19 disulfide peptide. Furthermore, the synthetic Tv18+19 co-eluted in RP-HPLC with Tv18+19 isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic T18+19 and Tv18+19 peptides were performed. Using these data all protons were assigned. The major chemical shift changes (delta delta > 0.05 ppm) observed were for the beta-protons of Cys182 and Cys189 in Tv18+19 as compared to T18+19. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data rhGH-HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties.

Number of interleukin-4- and interferon-gamma-secreting human T cells reactive with tetanus toxoid and the mycobacterial antigen PPD or phytohemagglutinin: distinct response profiles depending on the type of antigen used for activation.

The enzyme-linked immunospot (ELISPOT) assay has been proven to be an efficient and sensitive method for the enumeration of single cells secreting antibodies or cytokines. Here we have used this method to determine the number of interleukin-4 (IL-4)- and interferon-gamma (IFN-gamma)-producing cells in vitro secondary responses to tetanus toxoid (TT) and the mycobacterial antigen (purified protein derivative; PPD) or the mitogen phytohemagglutinin (PHA). PHA-induced IL-4 and IFN-gamma secretion was well correlated suggesting polyclonal activation of cells. This was not the case with the specific antigens, where PPD preferentially induced IFN-gamma- and very few IL-4-producing cells, while TT-induced both IL-4 and IFN-gamma. These differences are probably a reflection of the types of immunity the two antigens induce, mycobacteria preferentially inducing a cell-mediated T helper type 1 (Th 1) type of immunity, while immunity to tetanus is an antibody-dependent, Th 2 type of response. In individuals recently boosted with TT, a significant increase in both IL-4- and IFN-gamma-producing cells in response to TT was seen at day 7 after boost, followed by decline. This was in contrast to what was seen in response to PPD where an increase of IFN-gamma-producing cells after the TT boost at day 7 persisted for at least 14 days. These results suggest that after an in vivo boost both antigen-specific and nonspecific T cells are activated and that antigen-specific cells home to other organs and therefore may be difficult to demonstrate in the circulation. Our data show that the ELISPOT assay is a powerful tool for determining the frequency of cells secreting cytokines. The assay has several advantages over other assays since it is sensitive, measures the number of actually secreting cells, and avoids the problems of binding of cytokines to their cell-bound or soluble receptors.

Radioimmunolocalisation of bladder tumors xenotransplanted in nude mice.

We have previously reported on the derivation of mouse monoclonal antibodies (Mabs), identifying several cell surface antigens selectively associated with cancer of the urinary bladder (TCC) (1-4). Three of these Mabs (4E8, SK4H and 8F4) have now been assessed for their ability to localise TCC-tumor xenografts in nude mice. The biodistribution of 125I-labeled intact Mabs as well as the corresponding Fab and F(ab')2 fragments from two of them were investigated in animals carrying TCC tumors or antigen negative control tumors. Using direct measurements of excised tissues, all three antibodies were seen to accumulate specifically in the TCC tumors, giving tumor to normal tissue ratios of between 3 and 20 depending on the Mab used and the time after injection. Antibody fragments were generally more efficient in their localisation, mainly due to a dramatic reduction in the blood background as compared to intact Ig. One of the antibodies, 4E8, was also employed for external imaging with gamma camera scintigraphy using 111In or 131I as tracers. Excellent visualisation of the tumor sites could be obtained both with Fab fragments and intact antibody within 12-24 hours after injection. As expected, background radioactivity was significantly lower with fragments than with whole molecules. 111In labeled antibodies appeared in all instances to be superior to the corresponding 131I conjugates. In conclusion, the present study indicates that the three anti TCC antibodies may become useful for the in vivo diagnosis of bladder cancer in man.

MHC nonrestricted cytotoxic T cell clones with selective specificity from patients with transitional cell carcinoma (TCC) of the urinary bladder.

Lymphocytes from patients with transitional cell carcinoma (TCC) of the urinary bladder are more cytotoxic to bladder tumor cells than to a variety of control cells. This disease-related cytotoxicity has previously been shown to involve several mechanisms and different types of effector cells. To analyze further the nature of the effector cells operative in this system, peripheral blood lymphocytes from eight TCC patients were stimulated in vitro with TCC extract and cultured in the presence of interleukin 2 and allogeneic feeder cells. When tested for cytotoxicity in vitro on a target cell panel including both adherent and nonadherent cell lines, the lymphocytes killed a broad spectrum of targets in a major histocompatibility complex (MHC)-unrestricted fashion. When cloned by limiting dilution, clones were obtained which displayed a more restricted pattern of target cell killing. Some of the clones were highly but not exclusively selective for TCC-derived target cells. Phenotypically, these cells resembled mature T cells of CTL-type (CD8+/CD4-). They also expressed the CD3/5 T cell antigen receptor complex but target cell killing was not MHC-restricted. The results of various inhibition experiments suggested that the CD3/TCR complex was involved in the cytotoxicity exhibited by these effector cells. However, its precise role in target cell recognition and the identification of the tumor cell structures recognised by the effector cells require further studies.

A rapid method for detection of cellular proliferation using carboxyfluorescein. Assay of growth factors (IL-2, IL-1) and growth inhibiting antibodies.

The uptake of carboxyfluorescein diacetate (CFDA) into live cells was used as the basis for a simple, rapid and fully automated micromethod for determination of cell growth. The aim of the investigation was to adapt the CFDA method for detection of cell growth factors from cell culture supernatants. Thus, the biological activities of the growth factors IL-2 or IL-1 could be detected and quantitated at the same level of sensitivity as with the conventional [3H]thymidine incorporation. Similarly, the method was well suited to assay inhibition of IL-2-dependent lymphocyte growth by the monoclonal antibody anti-Tac, binding to the human lymphocyte membrane receptor for IL-2. The CFDA method proved to be rapid, reliable and well suited for several applications involving limiting numbers of cells and biological reagents.

Monoclonal antibodies against carcinoma cells of the human urinary bladder: immunohistochemical staining of tissues.

We have previously described the generation of mouse monoclonal antibodies (Mabs) directed to cell surface antigens of human bladder carcinoma. Based on experiments with cultured cells and a limited number of freshly isolated tissues, four distinct antigens were identified as being associated with this disease. In the present investigation, comprising the immunostaining of tissues of normal, malignant, and fetal origin, we have confirmed and extended the close association of these antigens with bladder cancer. Antibodies to all four antigens could clearly discriminate between malignant and normal uroepithelium. Two of the antibodies, SK4H-12 and 4E8, showed no additional reaction when tested with various adult tissues of normal or malignant origin. Antibodies to the remaining two antigens gave a positive staining of a few other tissue types.

Specificities and binding properties of 2 monoclonal antibodies against carcinoma cells of the human urinary bladder.

Mice were immunized with cultured cells derived from transitional cell carcinoma of the human urinary bladder (TCC). Spleen cells were fused with mouse myeloma cell line Sp2/0-Ag14 and the hybridomas obtained screened for antibody production against a panel of human cells. Two hybridomas were selected for further studies. The antibodies from one of these hybridomas (P7A5-4) could clearly discriminate between malignant and normal cells from the bladder, both when tested with cultured cells and fresh tissue. The P7A5-4 antibodies, however, also reacted with some non-TCC cultured carcinoma and melanoma cells but to a lesser extent. This difference in reactivity was even more pronounced in the fresh tumours tested, thus indicating a quantitative difference in antigen expression between TCC and other cells. From extracts of TCC cells, P7A5-4 bound three polypeptides of mol. wts 92Kd (ConA+), 23 and 17Kd (ConA-). The antibody derived from hybridoma SK4H-12 bound a ConA reactive glycopeptide of 100Kd mol. wt, the expression of which was almost entirely restricted to urothelial cell lines and tissue of TCC origin, as shown by immunocytochemical studies. The finding in this study of new antigens associated with urinary bladder carcinoma, extend the results obtained previously in our laboratory (Koho et al., 1984; Paulie et al., 1984) and further delineate the heterogeneity of tumour-associated antigens in this human tumour system.

Production of antibodies to cellular antigens by EBV-transformed lymphocytes from patients with urinary bladder carcinoma.

Lymphocytes from patients with transitional-cell carcinoma (TCC) of the urinary bladder were transformed by infection with Epstein-Barr virus. To obtain B cells secreting antibodies reactive with TCC cells, the transformed cells were either adhered to irradiated monolayers of cultured allogeneic TCC cells or subcultured at limiting dilution. Supernatants from these cultures were tested in a modified enzyme-linked immunosorbent assay against fixed cells, isolated plasma membranes, or lipid antigens or were tested by antibody-dependent cellular cytotoxicity (ADCC). Reactions with antigens derived from the serum source were excluded by proper controls. By this approach a majority of the patients tested (7/12) gave rise to cultures producing antibodies recognizing various cellular antigens. The antibody-containing supernatants from these cultures were usually of high titres and the reactive antibodies of IgM isotype. One culture, which had been selected by repeated adherence to TCC cells, produced antibodies reactive with such cells in ADCC. None of the antibodies investigated detected antigens exclusively present on TCC cells.

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder. I. Determination of the selectivity of six antibodies by cell ELISA and immunofluorescence.

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.

Monoclonal antibodies to antigens associated with transitional cell carcinoma of the human urinary bladder. II. Identification of the cellular target structures by immunoprecipitation and SDS-PAGE analysis.

The cellular target structures for six monoclonal antibodies raised against cultured human bladder carcinoma cells (TCC) were investigated. The specificities of these antibodies when tested against a large panel of cells have been described in the companion paper. Radiolabeled cell lysates were precipitated with the different monoclonal antibodies bound to protein A (Staphylococcus aureus) on a matrix of Sepharose beads. The precipitates were separated by sodium dodecyl sulfate- gel electrophoresis (SDS-PAGE) and analyzed by autoradiography. The antibodies 4B5, 7E9, and 14B11 have previously been found to react in a similar way with TCC-targets and some non-TCC tumor cells, but not with normal urothelial cells or cells of hematopoietic origin. When tested with lysates of a TCC-cell line (TCCSuP) a strong 92K band and a weak 23K band were precipitated with any one of these antibodies. These polypeptides were expressed on the cell surface and were not linked by disulfide bonds. Depletion experiments confirmed that the three antibodies recognized the same antigens. Another antibody (4E8) probably directed to a differentiation antigen present on both urothelial and melanoma cells detected two high molecular polypeptides, 190K and 170K. Antibodies from the S2C6 hybridoma, which displayed a distinct dual specificity for TCC- targets and for malignant or transformed cells of B cell origin, precipitated a 50K component from extracts of either TCC- or B cell-derived cell lines. Antibodies produced by the S2A9 hybridoma were shown to bind to a framework epitope of the HLA-A, B, C heavy chain.

Lectins as probes for identification of tumor-associated antigens on urothelial and colonic carcinoma cell lines.

In the search for tumor-associated antigens, seven lectins were used to investigate the cellular distribution of membrane-associated glycoproteins on a panel of human cells derived from tumor or normal tissues. Surface labelled lysates of the different cells were precipitated with the lectins and the precipitates were separated on SDS-PAGE. Comparison of the autoradiographic patterns revealed that a La-reactive 115K glycopeptide (gp 115) was present on transitional-cell carcinoma cells of the urinary bladder, on two spontaneously transformed urothelial cell lines and on a melanoma cell line. Gp 115 was absent from a non-transformed urothelial cell line, a squamous bladder carcinoma line and five unrelated cell lines of miscellaneous tissue origin. When precipitation was performed with a rabbit antiserum raised against the La-reactive components of a TCC cell line the same distribution of gp 115 was observed. From Helix pomatia hemagglutinin (HP) precipitates a 150K glycopolypeptide co-migrating with a previously described HP-reactive differentiation antigen associated with human T cells was present on one of the urothelial cell lines and on a colon carcinoma cell line. When different extracts depleted of ConA binding glycopeptides were compared, a group of three antigens (32K, 35K and 40K) were identified in the extracts of the colon carcinoma cell line, HT29. These antigens were shared by two other colon carcinoma cell lines but were absent from the unrelated cells of our panel. Furthermore, an extensively absorbed rabbit anti-HT29 serum specifically precipitated one of these antigens (35K) from the three colon cell lines.

Humoral and cellular immune reactions against tumor cells in patients with urinary bladder carcinoma. Correlation between direct and antibody-dependent cell-mediated cytotoxicity.

Serum IgG fractions from a large and homogeneous group of patients with transitional cell carcinoma of the urinary bladder (TCC) were tested for their capacity to induce antibody-dependent cellular cytotoxicity (ADCC) with lymphocytes from healthy donors against a TCC-derived target cell and one derived from adenocarcinoma of the colon. Both targets have previously been shown to be of comparable susceptibility to cell-mediated lysis in vitro. Some of the IgG preparations showed strong and dose-dependent ADCC against either one or both targets, while others gave weak reactions or none at all. Similar results were obtained with IgG from a matched group of patients with prostatic carcinoma who were used as clinical controls (CC). In parallel experiments, lymphocytes taken from the two donor groups at the same time as the serum samples were tested for their direct cytotoxicity (CMC) against the two targets. CMC gave similar results to ADCC. The differences in cytotoxicity displayed by either IgG or lymphocytes from individual donors were analysed statistically, using nonparametric statistics. To avoid introducing bias due to arbitrary data selection, the entire set of results, comprising both high and low reactors, was included in the statistical assessment. ADCC of the TCC donors' IgG against the TCC target was significantly stronger than against the colon carcinoma and also significantly stronger than that of the control donors. Similarly, the TCC patients' lymphocytes displayed a significantly higher CMC against the TCC target than against the control targets. This was not seen when the lymphocytes from the patients with prostatic carcinoma were tested. When CMC and ADCC of individual donors were compared, a statistically significant correlation between these activities was seen in three of the four donor/target combinations. These results support earlier findings and suggest that a significant fraction of both the disease-related and the 'non-selective' CMC (NK) displayed by cancer patients lymphocytes against allogeneic tumor cells in vitro reflects antibody-dependent reactions.

Lymphocyte-mediated lysis of tumor cells in vitro (ADCC), induced by serum antibodies from patients with urinary bladder carcinoma or from controls.

IgG fractions from serum of patients with transitional-cell carcinoma of the urinary bladder (TCC), patients with carcinoma of the prostate (CC) and healthy donors (HD) were tested for their capacity to induce antibody-dependent lymphocyte-mediated cytotoxicity (ADCC) to tumor cells in vitro. Lymphocytes from healthy donors were selected for low natural cytotoxicity to the target cells from established cell lines of TCC or other origins. IgG was prepared by adsorption of serum to Sepharose-bound protein A from Staphylococcus aureus and subsequent acid elution. When tested against a panel of six different target cells, most individual IgG preparations from all three donor groups contained antibodies inducing ADCC to some of the target cells. When IgG preparations from II untreated TCC patients were studied for ADCC induction to the TCC target T24 and the colon carcinoma HT29, cytotoxicity to T24 was, on an average, significantly higher than that to HT29. For IgG preparations from 18 TCC patients, treated with radiotherapy, a similar difference was seen but was not statistically significant. IgG preparations from II patients with carcinoma of the prostate and from 12 healthy donors did not show this differences. Moreover, while individual IgG preparations from untreated TCC patients were, on the average, significantly more cytotoxic to T24 than those from either of the two control groups, no such differences were seen when HT29 was the target. On the contrary, IgG preparations from patients with prostatic carcinoma were significantly more cytotoxic to HT29 than those from healthy donors. The results suggest that TCC patients develop a disease-related humoral immune response, superimposed on a "natural" immunity to a variety of antigens on the target cells used. The nature of the antigens involved in these reactions remains to be established. However, the results are compatible with previous findings, in these patients, of a bladder-tumor-related cellular cytotoxicity, to a large extent caused by the patients' own antibodies.