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J Hansson

Publications and source records attributed to J Hansson.

At least 55 records · Page 3Linked to original sources
PubMed1997

Functional characterization of the interaction between the superantigen staphylococcal enterotoxin A and the TCR.

In this report, we show that despite an overall amino acid residue identity of more than 80% between the staphylococcal enterotoxins (SE) A and E, these proteins markedly differ in their absolute requirement for the MHC class II during T cell activation. The superantigens were produced as C215Fab-SE fusion proteins and analyzed for their ability to activate T cells in a MHC class II-independent manner, using C215 Ag expressing cell lines as pseudo super-APCs. C215Fab-SEA, but not C215Fab-SEE, induced T cell cytotoxicity and proliferation in these MHC class II-independent systems. Introduction of a region from SEA, comprising amino acids 20-27, to SEE transferred the ability to engage T cells in the absence of MHC class II. Analysis of the Vbeta specificity of the chimeric SEA/SEE molecules and a panel of SEA mutants demonstrated that the site for TCR interaction covers the edge surrounding the shallow cavity on top of the SEA molecule.

Amino Acid Sequence↗
PubMed1997

Genetically engineered superantigens as tolerable antitumor agents.

Superantigens (SAg) are a family of bacterial and viral proteins with strong immunostimulatory properties. SAg bound to major histocompatibility complex (MHC) class II molecules activate a high frequency of T cells and represent the most potent known activators of T cells to date. To explore the use of SAg for T cell-based tumor therapy we have created a tumor-reactive SAg by engineering a fusion protein composed of a tumor-reactive mAb (C215Fab) and the bacterial SAg staphylococcal enterotoxin A (SEA). A point mutation D227A was introduced at the major MHC class II binding site in SEA to reduce systemic toxicity. Treatment of tumor bearing mice with the Fab-SEA D227A fusion protein resulted in profound antitumor effects with a markedly reduced toxicity as compared with the wild-type Fab-SEA fusion protein. The reduced toxicity was probably due to a weak distribution of the SEA D227A fusion protein in tissues with a high MHC class II expression and low systemic cytokine levels as exhibited in mice and rabbits. The data presented demonstrate the efficacy of immunoconjugates containing a mutated SAg in directing a T cell attack against tumor cells with minimal systemic immune activation.

Animals↗
PubMed1997

Allogeneic heart transplantation activates alloreactive NK cells.

The ability of natural killer (NK) cells to recognize and reject transplants has so far been shown in hematopoietic grafts only. This study was designed to ascertain whether NK cells may also be involved in the rejection of transplanted organs. In most rat strain combinations, immunization with allogeneic cells induces a T cell response with cytotoxic T lymphocyte (CTL) activation. We have previously found one exception to this. In contrast to Wistar Furth rats (WF, RT1u), which manifest allospecific CTL activation in response to immunization with Brown Norway (BN, RT1n) cells, BN rats immunized with repeated intraperitoneal (i.p.) injections of allogeneic WF spleen cells manifest activation of alloreactive NK effector cells. The alloreactive NK cells were of the TCR-, CD3-, CD8+, and NKR-P1 intermediate phenotype and killed target cells with alloselectivity. In this study we used a heart transplantation model to study the rejection response of BN rats receiving WF grafts. NK cell infiltration was greater in WF hearts transplanted to BN recipients than in BN hearts transplanted to WF recipients. Furthermore, the extent of T cell infiltration was less in BN recipients. In WF rats transplanted with allogeneic BN hearts, CTL were activated in response to i.p. challenge with allogeneic BN cells, whereas BN rats transplanted with allogeneic WF hearts and i.p. challenged with allogeneic WF cells, manifested activation of alloreactive NK cells but no measurable activation of classic CTL. The alloreactive NK cells killed their allogeneic targets with specificity and with potency comparable to that of CTL. Furthermore, WF grafts were rejected in BN recipients as efficiently as were BN grafts in WF recipients. These results not only show cardiac allografts to be able to activate alloreactive NK cells, but also suggest that NK cells may be involved in the rejection of solid organ transplants and function as classic CTL in certain donor-recipient combinations.

Animals↗
PubMed1997

Systemic therapy of malignant melanoma.

The present status of medical treatment of malignant melanoma is briefly reviewed, both with regard to adjuvant therapy for individuals with high-risk melanoma and a high probability of harbouring subclinical micrometastases, as well as to therapy for established disseminated (macrometastatic) disease. At present, disseminated, macrometastatic melanoma is incurable in the majority of cases. Single agent chemotherapy has modest effects and results in disease remission in a minority of patients, usually of short duration, Combination chemotherapy, or the combination of chemotherapeutic drugs and cytokines, results in increased response rates and occasionally remissions of prolonged duration. So far, no regimen has demonstrated improved survival compared to single agent therapy in disseminated melanoma. New insights into the mechanisms of resistance to chemotherapeutic drugs may lead to development of predictive tests that can identify individuals with tumors sensitive to a specific agent, as well as to the development of strategies to circumvent drug resistance. It has recently been shown that adjuvant therapy of high-risk melanoma with large doses of interferon-alpha 2b significantly prolongs relapse-free and overall survival, at the price of considerable toxicity. Ongoing studies aim to define the optimum dose and duration of adjuvant interferon therapy. Recent advances in molecular biology and immunology may lead to the development of new treatment modalities, such as improved vaccines and other biologic therapies, which may benefit patients with malignant melanoma.

Clinical Trials as Topic↗
PubMed1997

Immunohistochemical examination of the expression of O6-methylguanine-DNA methyltransferase in human melanoma metastases.

In tumour cell lines, an inverse relationship has been shown between susceptibility to the cytotoxic effects of the O6-alkylating agents and the expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). One of the most effective single agents in chemotherapy of metastatic melanoma is the O6-alkylating drug, dacarbazine. We therefore examined the distribution of MGMT in 37 skin and lymph node melanoma metastases using rabbit antihuman MGMT antiserum. MGMT expression was undetectable in tumours from 2 out of 34 patients and low in 4 further patients. When present, staining was mainly nuclear and showed a marked variation both among tumour cells within the same metastases, between separate metastases in the same patient and between tumours in different patients. MGMT expression determined by immunohistochemistry showed a relation to MGMT activity measurements, but was not related to the number of proliferating cells, as identified by staining with MIB-1 antibody. Tumour cells with moderate to strong immunostaining with MGMT antiserum were significantly more abundant in metastases excised after dacarbazine-based chemotherapy (n = 8) than in those excised before treatment (n = 29).

Adult↗
PubMed1997

Role of O6-methylguanine-DNA methyltransferase, glutathione transferase M3-3 and glutathione in resistance to carmustine in a human non-small cell lung cancer cell line.

O6-methylguanine-DNA methyltransferase (MGMT), glutathione transferase (GST) M3-3 and glutathione (GSH) have all been implicated in the resistance of cells to the cytostatic drug carmustine. U1810, a human non-small cell lung cancer cell line, expresses all of these putative resistance factors. The U1810 cells show a 4.4-fold lower sensitivity to carmustine compared with the U1690 cell line, a human small cell lung cancer cell line lacking detectable levels of both MGMT and GST M3-3. We investigated the effect of the MGMT inhibitor O6-benzylguanine, the GST inhibitor ethacrynic acid and the GSH synthesis inhibitor D,L-buthionine-S,R-sulfoximine (BSO) on the cytotoxicity of carmustine to U1810 cells. No potentiation to carmustine was observed after treatment with ethacrynic acid, while a 2-fold potentiation was found after exposure to O6-benzylguanine. Depletion of GSH with BSO showed a similar sensitising effect as that obtained with O6-benzylguanine. Thus, MGMT and GSH are the predominant resistance factors to carmustine in the U1810 cell line, whereas it is unclear whether GST M3-3 plays any role.

Antineoplastic Agents, Alkylating↗
PubMed1997

Efficient harvest of in vivo IL-2-activated CD3+ lymphocytes for adoptive immunotherapy by selective leukapheresis (lymphocytapheresis).

Autologous activated lymphocytes are an alternative to tumor-infiltrating lymphocytes for adoptive immunotherapy. We developed a method of selective lymphocytapheresis that harvests large numbers of interleukin-2 (IL-2)-activated autologous T lymphocytes. Five patients with metastatic malignant melanoma received 2.4 x 10(6) IU/m2 IL-2 sc. once a day 5 days a week for 3 weeks before lymphocytapheresis. Four patients went through lymphocytapheresis without IL-2 pretreatment. After IL-2 pretreatment, activated memory T cells increased significantly. Increasing CD3+ cells paralleled the significant enhancement of the cytotoxic activity against an HLA-A2-matched allogeneic melanoma cell line during the 3 weeks of IL-2 pretreatment. Lymphocytapheresis was performed 72 h after the last IL-2 injection to obtain the maximum recovery of activated lymphocytes at the peak of the rebound phenomenon. IL-2 pretreatment resulted in many more lymphocytes in the harvest than without pretreatment. The percentage, number, and lytic units of CD3+ cells harvested by the differential apheresis were significantly higher than were present in peripheral blood before lymphocytapheresis. These results show that pretreatment of melanoma patients with low-dose IL-2 before lymphocytapheresis allows the selective harvest of large numbers of activated T lymphocytes for adoptive immunotherapy.

Adult↗
PubMed1997

Tamoxifen-induced DNA adducts in leucocytes of breast cancer patients.

Tamoxifen-induced DNA adducts were searched in leucocyte DNA from breast cancer patients. Total white blood cell DNA from tamoxifen-treated and control patients was analysed by 32P-postlabelling using HPLC-radioactivity detection. Rat liver DNA was used as a positive standard. In blinded analysis four of the six treated patients showed DNA adducts; none of the five controls were positive. The identity of fraction as a tamoxifen adduct was confirmed by using different chromatographic systems, each with spiked rat liver samples. The level of adducts in the treated patients was 5.5 adducts/10(9) nucleotides as compared to an apparent level of 1.9/10(9) in the controls.

Adult↗
PubMed1997

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro.

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.

Areca↗
PubMed1997

A mutation of F47 to A in staphylococcus enterotoxin A activates the T-cell receptor Vbeta repertoire in vivo.

The bacterial superantigen staphylococcal enterotoxin A (SEA) binds with high affinity to major histocompatibility complex (MHC) class II molecules and subsequently activates T cells bearing particular T-cell receptor (TCR) Vbeta chains. Structural and mutational studies have defined two distinct MHC class II binding sites located in the N-terminal and C-terminal domains of SEA. The N-terminal F47 amino acid is critically involved in a low-affinity interaction to the MHC class II alpha-chain, while the C-terminal residues H187, H225, and D227 coordinate a Zn2+ ion and bind with moderate affinity to the beta-chain. In order to analyze whether the SEA-MHC class II alpha-chain interaction plays a role in dictating the in vivo repertoire of T-cell subsets, we studied distinct Vbeta populations after stimulation with wild-type SEA [SEA(wt)] and SEA with an F47A mutation [SEA(F47A)]. Injections of SEA(wt) in C57BL/6 mice induced cytokine release in serum, strong cytotoxic T-lymphocyte activity, expansion of T-cell subsets, and modulated expression of the T-cell activation antigens CD25, CD11a, CD44, CD62L, and CD69. SEA-reactive TCR Vbeta3+ and Vbeta11+ T cells were activated, while TCR Vbeta8+ T cells remained unaffected. The SEA(F47A) mutant protein induced a weaker T-cell response and failed to induce substantial interleukin-6 production compared to SEA(wt). Notably, SEA(F47A) failed to activate TCR Vbeta11+ T cells, whereas in vivo expansion and modulation of T-cell activation markers on TCR Vbeta3+ T cells were similar to those for SEA(wt). A similar response to SEA(F47A) was seen among CD4+ and CD8+ T cells. Activation of TCR Vbeta3+ and TCR Vbeta11+ T-cell hybridomas confirmed that SEA(F47A) activates TCR Vbeta3+ but not TCR Vbeta11+ T cells. The data support the view that the SEA-N-terminal MHC class II alpha-chain interaction defines a topology that is required for engagement of certain TCR Vbeta chains in vivo.

Animals↗
PubMed1997

Tumor therapy with an antibody-targeted superantigen generates a dichotomy between local and systemic immune responses.

Repeated injections of a fusion protein containing the superantigen staphylococcal enterotoxin A (SEA) combined with a Fab fragment of a tumor-specific antibody is a highly efficient immunotherapy for mice expressing lung melanoma micrometastasis. In the present study, the systemic and local immune responses generated by this therapy were analyzed at a cellular level. Two distinct but coupled immune reactions occurred after repeated therapy. Tumor necrosis factor and macrophage inflammatory protein-1 alpha and -1 beta were immediately synthesized, in the absence of T lymphocytes, at the local tumor site in the lung. This was followed by the induction of VCAM-1 adhesion molecule expression on pulmonary vascular endothelial cells. Concurrently, the early response in the spleen was characterized by the induction of selective T cells producing interleukin (IL)-2. The primed and expanded SEA-reactive V beta 3- and V beta 11-expressing T lymphocytes accumulated to the tumor area only after Fab-SEA therapy and were not present in the lung when SEA, Fab fragment, or recombinant IL-2 was injected. The tumor-infiltrating T cells produced large amounts of interferon-gamma, but no IL-2 or Th2 type of lymphokines were detected at the tumor site in the Fab-SEA-targeted antitumor immune response. These results emphasize the necessity to investigate several sites of antigen presentation to elucidate the effects of immunotherapy.

Animals↗
PubMed1996

Immune response during tumor therapy with antibody-superantigen fusion proteins.

To engineer superantigens (SAg) to express tumor reactivity, we genetically fused the Fab-part of the tumor-reactive MAb C215 and the bacterial SAg staphylococcal enterotoxin A (SEA). Treatment of mice carrying established lung micrometastases of the C215-transfected syngeneic B16 melanoma with 3-4 daily injections of C215Fab-SEA resulted in strong antitumor effects, while only moderate effects were seen when treatment was given every 4th day (intermittent treatment). High serum levels of IL-2, TNF-alpha, IFN-gamma and strong induction of CTLs (cytotoxic T lymphocytes) were noted after priming with the fusion protein. T cells responded well to 3 daily injections of C215Fab-SEA and then gradually entered a hyporesponsive state, characterized by a reduced ability to produce IL-2, TNF-alpha and IFN-gamma and failure to mediate cytotoxicity in vitro. Intermittent treatment was characterized by increased levels of IL-10, concomitant with accentuated loss of IL-2, TNF-alpha and IFN-gamma production. A 10-fold increase in SEA-reactive TCR V(beta)3+ CD4+ cells was observed in the spleen, while a loss of TCR V(beta)3+ CD8+ and V(beta)11+ CD8+ cells was noted. This is in striking contrast to injections of native SEA which induced a marked deletion of TCR V(beta)3+ CD4+ T cells, but not of CD8+ cells. Recovery of the TH1 cytokine profile occurred within 1-2 weeks, while restoration of cytotoxicity required several months and correlated with recovery of TCR V(beta)3+ CD8+ and TCR V(beta)11+ CD8+ T cells. These results show that the temporal relationship of SAg stimulations dictates the cytokine profile. Moreover, different mechanisms appear to regulate hyporesponsiveness in CD4+ and CD8+ T cells.

Animals↗
PubMed1996

Different mechanisms are responsible for c-jun mRNA induction by cisplatin and ultraviolet light.

Ultraviolet light (UV) and different DNA-damaging agents are known to induce AP-l-transcription-factor activity. Whereas UV induction appears to be triggered by events at the cell membrane, the mechanism of AP-l activation by alkylating or platinating agents is not known. We have here examined the effect of cisplatin on AP-l activity in RPMI-8322 melanoma cells. Cisplatin was found to induce binding of nuclear proteins to TRE elements from the c-jun and collagenase-gene promoters, and was also found to induce activation of a c-jun-promoter reporter construct. Compared with stimulation by UV, cisplatin stimulation of c-jun-promoter activity was found to be less sensitive to a dominant negative mutant of Raf-I protein kinase. Furthermore, whereas UV treatment resulted in strong MAP-kinase activation, cisplatin treatment resulted only in a weak and transient increase. These data suggest that the Raf-MAPK pathway is of minor importance for the induction of c-jun-promoter activity by cisplatin. Finally, we report that cisplatin induction of c-jun in RPMI-8322 cells was blocked by herbimycin A, an inhibitor of Src-family tyrosine kinases. In contrast, UV induction of c-jun was not blocked by herbimycin A. In conclusion, our data strongly suggest that UV and cisplatin induction of c-jun mRNA in RPMI-8322 melanoma cells occur by distinct mechanisms.

Benzoquinones↗
PubMed1996

Hydrogen peroxide secreted by tumor-derived macrophages down-modulates signal-transducing zeta molecules and inhibits tumor-specific T cell-and natural killer cell-mediated cytotoxicity.

Although alterations in CD3-associated signal-transducing molecules in tumor-infiltrating T cells of patients with advanced cancer have been previously described, the mechanism behind these changes is not known. We demonstrate that macrophages isolated from metastatic lymph nodes of patients with malignant melanoma down-regulate levels of CD3 zeta in autologous peripheral blood T cells. Lipopolysaccharide (LPS)- or phorbol 12-myristate 13-acetate (PMA)-stimulated monocytes derived from peripheral blood of healthy donors also induced decreased expression of CD3 and CD16-associated zeta chains similar to that observed in T cells and natural killer (NK) cells of patients with advanced cancer. Co-culture with activated monocytes impaired Ca2+ mobilization in peripheral blood derived-T cells when stimulated with monoclonal antibodies to CD3 and also strongly inhibited melanoma-specific cytotoxic T lymphocyte (CTL) activity and NK activity. The presence of catalase, a scavenger of H2O2, during co-culture almost totally abrogated the inhibitory effect of activated monocytes on melanoma-specific CTL lines and on NK cells. Pre-treatment of CTL or NK cells with nontoxic concentrations (1 x 10(-5) M) of H2O2 also severely reduced their cytotoxic activity which could be prevented by catalase. The decrease in CD3 zeta and in CD16 zeta expression, induced by macrophages isolated from metastatic lymph nodes or by LPS-stimulated monocytes, was also prevented by catalase when maintained throughout the co-culture period. The possibility that monocyte/macrophage-derived reactive oxygen metabolites contribute directly to alterations in signal transducing molecules of T cells and NK cells and to the mechanism of immunosuppression in individuals with cancer should be considered.

CD3 Complex↗
PubMed1996

Analysis of O6-methylguanine-DNA methyltransferase mRNA in fine needle biopsies from human melanoma metastases by reverse transcription and polymerase chain reaction.

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein, which removes alkyl groups from the O6 atom of guanine residues. Tumour cells which lack MGMT are sensitive to cytostatic drugs such as dacarbazine (DTIC), whose active species bind to this site. To explore whether analyses of MGMT expression can be used as a predictive test for clinical sensitivity to DTIC in melanomas, we developed a method to assay MGMT mRNA levels in cells obtained by fine needle aspiration biopsies of metastases. cDNA was synthesised from mRNA prepared from biopsy material. Polymerase chain reaction was performed using primers complementary to MGMT cDNA and to beta-actin, which served as an internal control. Analyses of 44 biopsies from 35 patients showed a considerable variation in MGMT mRNA, with 15 samples (34%) lacking detectable mRNA. In 6 out of 8 patients in whom more than one tumour was analysed, separate metastases had different levels of MGMT mRNA. There was no correlation between MGTM activity studied by a biochemical assay and MGMT mRNA levels when these were compared in 10 surgical biopsies.

Adult↗