T-cell-receptor engagement and tumor ICAM-1 up-regulation are required to by-pass low susceptibility of melanoma cells to autologous CTL-mediated lysis

Tumor-specific and non-specific CD3⁺, TcRαβ⁺, CD8⁺ cytotoxic T-cell (CTL) clones, isolated from tumor-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) of a melanoma patient and allogeneic LAK cells, were used to investigate the requirements for bypassing the low lysability of some melanoma clones derived from an S.C. metastasis from which highly lysable clones were also obtained. Cytofluorimetric analysis showed that all melanoma clones expressed ICAM-1, although to different extents, reaching a 10-fold difference in fluorescence units, while HLA class-1 antigens were similarly expressed. The differences in expression of ICAM-1 among tumor clones correlated with differences in lysability, by both specific and non-specific CTL, but were not large enough to affect lymphocyte-tumor conjugate formation. Cytokine- or gene-transfer-mediated up-regulation of ICAM-1 did not induce de novo lysis of ICAM-1^low tumor cells; however, it markedly enhanced a low level of killing of the same cells by tumor-specific, TcR-dependent and HLA-restricted CTL clones but not by non-specific, TcR-independent effectors. In addition, lysis of melanoma clones by any effector was similarly inhibited by anti-ICAM-1 and anti-LFA-1 antibodies. This indicates that by-pass of low lysability of ICAM-1^low melanoma clones by CTL clones, after ICAM-1 up-regulation, is possible only if simultaneous LFA-1 and TcR engagement takes place. In addition, these results suggest that the constitutive high level of expression of ICAM-1 on the subset of ICAM-1^high melanoma cells must be only one of the factors contributing to the high lysability of these cells by any effector.     

CIITA‐driven MHC‐II positive tumor cells: Preventive vaccines and superior generators of antitumor CD4+ T lymphocytes for immunotherapy

In our study, we have investigated whether tumors of distinct histological origin can be rejected if expressing CIITA‐driven MHC class II molecules. Moreover, we assessed whether antitumor lymphocytes generated by this approach could be used as an immunotherapeutic tool for established cancers. Stable CIITA‐transfectants of C51colon adenocarcinoma, RENCA renal adenocarcinoma, WEHI‐164 sarcoma as well as TS/A mammary adenocarcinoma were generated. Tumor cells transfectants were injected in vivo, and their growth kinetics and recipient's immune response were analyzed. Tumor rejection and/or retardation of growth was found for the first 3 CIITA‐transfected tumor cell lines and confirmed for TS/A‐CIITA. Animals rejecting CIITA‐transfected tumors acquired specific immunological memory as demonstrated by resistance to challenge with parental tumors. Adoptive cell transfer experiments demonstrated that tumor immunity correlates with the efficient priming of CD4⁺ T helper cells and the consequent activation of CD8⁺ T lymphocytes. T cells from TS/A‐vaccinated mice were used in an adoptive immunotherapy model of established tumors. The results showed the cure at early stages and significantly prolonged survival at later stages of tumor progression. Importantly, CD4⁺ T cells were clearly superior to CD8⁺ T cells in antitumor protective function. Interestingly, the protective phenotype was associated to both a Th1 and Th2 polarization of the immune effectors. These results establish the general application of our tumor vaccine model and disclose the additional application of this strategy for producing better lymphocyte effectors for adoptive antitumor immunotherapy.     

Lu-ECAM-1-mediated adhesion of melanoma cells to endothelium under conditions of flow

Lu-ECAM-1 is a lung-derived, venular endothelial cell adhesion molecule. It promotes the selective adhesion of lung-metastatic B16-F10 melanoma cells to endothelium under static conditions and mediates colonization of the lungs by the same tumor cells. To test whether Lu-ECAM-1 by itself is sufficient to cause vascular arrest of B16-F10 cells, we measured here under conditions of flow tumor cell adhesion to endothelia that express different amounts of Lu-ECAM-1 on their surfaces. At physiological shear stresses, adhesion of B16-F10 melanoma cells to endothelia correlates positively with the amount of Lu-ECAM-1 expression on the endothelial cell surface and inversely with the level of the applied shear stress. Tumor cell trajectories are biphasic; i.e., B16-F10 melanoma cells initially move along the endothelial surface with a velocity similar to the theoretical velocity, then arrest within a fraction of a second. Arrest is permanent for most B16-F10 melanoma cells at all shear stresses tested. Tumor cells never engaged in a rolling motion prior to arrest. Masking of the Lu-ECAM-1 ligand on the surface of B16-F10 melanoma cells with soluble Lu-ECAM-1 impedes arrest of tumor cells on the surface of the test endothelium. Purified Lu-ECAM-1 also mediates B16-F10 arrest, but arrest is mostly transient at shear stresses of 0.59 dynes/cm² and higher, implying adhesion by single receptor/ligand bonds. Our data suggest that Lu-ECAM-1 plays a critical role in the recognition and initial arrest of murine melanoma cells in lung venules. © 1996 Wiley-Liss, Inc.     

Tumor antigen extracts stimulate cytotoxicity against unrelated target cells

A new cytotoxicity assay for the study of tumor antigens is described. Splenocytes from normal and mammary-tumor-bearing BALB/c mice were depleted of glass-adherent cells, exposed to various concentrations of tumor extracts and mixed with ⁵¹Cr-labelled chick red blood cells. Cytolysis evidenced by ⁵¹Cr release was determined 20 h after incubation at 37°C. Lymphocytes from tumor bearers exerted no cytotoxic action per se, but became cytolytic after confrontation with immunogenic tumor extracts. Although this cytotoxicity is expressed against targets other than the cells containing the sensitizing antigen, i.e., a non-specific activity, its induction is strictly specific as evidenced by the following: (1) extracts of D1-DMBA-3 tumors exerted an inductive effect only in lymphocytes from mice bearing this tumor and not in lymphocytes from normal mice; and (2) extracts of normal tissues or of a non-immunogenic mammary tumor, D1-DMBA-2, did not induce cytotoxicity in lymphocytes from normal or tumor-bearing mice. This approach may provide a useful method for in vitro determination of cell-mediated immunity in which specific reactions can be induced by tumor extracts as opposed to intact tumor cells.     

Colony stimulating factor 1 receptor blockade improves the efficacy of chemotherapy against human neuroblastoma in the absence of T lymphocytes

Tumor‐associated macrophages can promote growth of cancers. In neuroblastoma, tumor‐associated macrophages have greater frequency in metastatic versus loco‐regional tumors, and higher expression of genes associated with macrophages helps to predict poor prognosis in the 60% of high‐risk patients who have MYCN‐non‐amplified disease. The contribution of cytotoxic T‐lymphocytes to anti‐neuroblastoma immune responses may be limited by low MHC class I expression and low exonic mutation frequency. Therefore, we modelled human neuroblastoma in T‐cell deficient mice to examine whether depletion of monocytes/macrophages from the neuroblastoma microenvironment by blockade of CSF‐1R can improve the response to chemotherapy. In vitro, CSF‐1 was released by neuroblastoma cells, and topotecan increased this release. In vivo, neuroblastomas formed by subcutaneous co‐injection of human neuroblastoma cells and human monocytes into immunodeficient NOD/SCID mice had fewer human CD14⁺ and CD163⁺ cells and mouse F4/80⁺ cells after CSF‐1R blockade. In subcutaneous or intra‐renal models in immunodeficient NSG or NOD/SCID mice, CSF‐1R blockade alone did not affect tumor growth or mouse survival. However, when combined with cyclophosphamide plus topotecan, the CSF‐1R inhibitor BLZ945, either without or with anti‐human and anti‐mouse CSF‐1 mAbs, inhibited neuroblastoma growth and synergistically improved mouse survival. These findings indicate that depletion of tumor‐associated macrophages from neuroblastomas can be associated with increased chemotherapeutic efficacy without requiring a contribution from T‐lymphocytes, suggesting the possibility that combination of CSF‐1R blockade with chemotherapy might be effective in patients who have limited anti‐tumor T‐cell responses.     

Local but no systemic immunomodulation by intraperitoneal treatment of advanced ovarian cancer with autologous T lymphocytes re-targeted by a bi-specific monoclonal antibody

We have reported a 27% overall anti-tumor response using i.p. immunotherapy of advanced ovarian carcinoma with autologous, ex vivo expanded, T lymphocytes re-targeted with bi-specific monoclonal antibody OC/TR, combined with soluble OC/TR and low-dose recombinant interleukin-2 (IL-2). This treatment had no effect on extraperitoneal disease. Therefore we studied in 13 patients whether this immunotherapeutic protocol resulted only in local or also in systemic immunomodulation. The phenotype of the ex vivo expanded lymphocytes was mainly CD3⁺, 4⁻, 8⁺, 16⁻, 56⁻. Their OC/TR-re-targeted cytolytic activity against Igrov-1 ovarian-carcinoma cells was approximately as high in responders as in non-responders. Following most therapeutic cycles, the immunophenotype of lymphocytes recovered from the peritoneal fluid was similar to that of the infused T cells (i.e., mainly CD3⁺, 4⁻, 8⁺) and they were coated with OC/TR. However, cytolytic activity of the recovered lymphocytes against Igrov-1 cells was low in direct assays, and only slightly increased after additional in vitro re-targeting with OC/TR. Systemically, the i.p. immunotherapy resulted in a transient lymphopenia lasting for about 7 days, low (i.e., 5 to 13 ng/ml) serum concentrations of free, functional OC/TR, and very weak coating of circulating T lymphocytes with OC/TR. These peripheral-blood T lymphocytes did not exert OC/TR-re-targeted cytolytic activity. Thus, locoregional OC/TR-re-targeted cellular immunotherapy resulted in substantial local immunomodulation and anti-tumor effects but virtually no systemic immunomodulation. Int. J. Cancer 73:211–219, 1997. © 1997 Wiley-Liss, Inc.     

Inhibition of vascular adhesion protein-1 enhances the anti-tumor effects of immune checkpoint inhibitors

Modulation of the immunosuppressive tumor microenvironment (TME) is essential for enhancing the anti-tumor effects of immune checkpoint inhibitors (ICIs). Adhesion molecules and enzymes such as vascular adhesion protein-1 (VAP-1), which are expressed in some cancers and tumor vascular endothelial cells, may be involved in the generation of an immunosuppressive TME. In this study, the role of VAP-1 in TME was investigated in 2 murine colon cancer models and human cancer cells. Intraperitoneal administration of the VAP-1-specific inhibitor U-V296 inhibited murine tumor growth by enhancing IFN-γ-producing tumor antigen-specific CD8⁺ T cells. U-V296 exhibited significant synergistic anti-tumor effects with ICIs. In the TME of mice treated with U-V296, the expression of genes associated with M2-like macrophages, Th2 cells (Il4, Retnla, and Irf4), angiogenesis (Pecam1), and fibrosis (Acta2, Loxl2) were significantly decreased, and the Th1/Th2 balance was increased. H₂O₂, an enzymatic product of VAP-1, which promoted the production of IL-4 by mouse Th2 and inhibited IFN-γ by mouse Th1 and human tumor-infiltrating lymphocytes, was decreased in tumors and CD31⁺ tumor vascular endothelial cells in the TMEs of mice treated with VAP-1 inhibitor. TCGA database analysis showed that VAP-1 expression was a negative prognostic factor in human cancers, exhibiting a significant positive correlation with IL-4, IL4R, and IL-13 expression and a negative correlation with IFN-γ expression. These results indicated that VAP-1 is involved in the immunosuppressive TMEs through H₂O₂-associated Th2/M2 conditions and may be an attractive target for the development of combination cancer immunotherapy with ICIs.     

Diverse effect of cytokine treatment of tumor cells on specific versus non-specific cytotoxicity

(First submitted 15 Nov 1991;accepted 11 December 1991) The effect of IFN-γ and TNF-α treatment of an ovarian carcinoma line on the sensitivity to lysis by specific CTL clones and non-specific Tumor Associated Lymphocytes (TAL), isolated from the ascites fluid, was analyzed. Thein vitro established TAL line displayed a non-specific lytic activity against the autologous tumor as well as against several allogeneic tumor lines. Pretreatment with IFN-γ alone, or in combination with TNF-α, rendered the carcinoma line less susceptible to lysis by the autologous TAL line. Conversely, susceptibility to lysis by tumor specific T cell clones, isolated from the TAL line, was increased as a result of cytokine pretreatment. Several TCR-α/β⁺, CD8⁺ T-cell clones showing a more specific pattern of lysis against the autologous tumor were isolated. Lysis of the autologous tumor by these clones involved the TCR-α/β via a MHC-class I restricted mechanism dependent on the adhesion molecules ICAM-1 and LFA-3, as inferred from antibody blocking studies. The enhanced sensitivity to specific CTL clones seen after cytokine treatment may be related to theenhanced expression of ICAM-1 molecules on the ovarian carcinoma. These results have implications for cytokine based immunotherapy, where IFN-γ may enhance the effects of tumor associated specific CTL while decreasing that of non-specific effector cells.     

Studies on bovine lymphosarcoma: formation of syncytia and detection of virus particles in mixed-cell cultures

This paper reports studies on mixed-cell cultures of human diploid embryonic lung (WI-38) cells, embryonic bovine tracheal (EBTr) cells, and bovine embryonic kidney (BEK) cells with thoracic duct lymphocytes and buffy coat cells from lymphosarcoma and normal cattle, and lymphocytes from lymphosarcoma cattle that have been established as cell lines (NBC) in suspension culture. Peripolesis and emperipolesis were very pronounced during the first hours after initiation of mixed cultures with ?lymphosarcoma”? lymphocytes, whereas little or no peripolesis or emperipolesis characterized mixed cultures with ?normal”? lymphocytes. Syncytial formation, cytoplasmic vacuolation, and nuclear abnormalities occurred in all the mixed cultures containing cells from lymphosarcoma animals. Cells from normal animals did not provoke the characteristic cytopathic effect, with the exception of buffy coat cells from one animal. Interferon was demonstrated in some of the syncytia-positive cultures. The data suggest that the cultures were infected with a latent syncytial virus. Immunological data indicate that this virus was not infectious bovine rhinotracheitis, bovine parainfluenza 3, or bovine viral diarrhea, and preliminary serological data suggest that the lymphosarcoma animals may develop neutralizing antibodies against it. In two cell lines derived from mixed cultures of WI-38 and EBTr with NBC-17, numerous virus particles were observed by electron microscopy both extracellularly and, in many instances, budding from the plasma membranes or from cytoplasmic vacuole membranes. The possible relationship of the virus to bovine lymphosarcoma remains to be elucidated.     

HIV protease inhibitors differentially inhibit adhesion of Candida albicans to acrylic surfaces

Highly active antiretroviral therapy (HAART), using HIV protease inhibitors, is commonly used in the management of HIV infection. HIV protease inhibitors also have a direct effect on a key virulence factor of Candida albicans, its secreted aspartyl proteinase (Sap). Although protease inhibitors can attenuate Candida adhesion to human epithelial cells, their effects on adhesion to acrylic substances, which is a common component of oral appliances, is unknown. This study investigated whether protease inhibitors affect C. albicans adhesion to acrylic substances. C. albicans suspensions were pretreated with different concentrations of saquinavir, ritonavir or indinavir for 1 h and allowed to adhere on acrylic strips, which had been pretreated with pooled human saliva for 30 min, for another hour in the presence of each drug. The test groups showed a significantly lower degree of adhesion than the controls. Adhesion was reduced by 50% at drug concentrations of 100, 100 and 20 μmol l^?1 for saquinavir, ritonavir and indinavir respectively. In conclusion, protease inhibitors attenuated C. albicans adhesion to an acrylic surface in vitro in a dose‐dependent manner, and different protease inhibitors exhibited different degrees of inhibition.     

Experimental Research on the TrkA Gene Inhibition of Angiogenesis in Human Neuroblastoma

Objective This study was designed to investigate the feasibility of gene therapy for human neuroblastoma (NB) with the TrkA gene inhibition of tumor angiogenesis, growth and metastasis. Methods Three groups of cells including SY5Y, SY5Y-TrkA and SY5Y-Vec NB cells, were cultured by routine methods. Comparison of oncogenicity was performed among the three groups of cells. Tumor volume and angiogenesis in nude mice were also compared with VEGFmRNA expression (by RT-PCR analysis), immunohistochemistry and microvessel counting. Results The TrkA-transfected SY5Y NB cells showed significantly reduced oncogenicity and tumor angiogenesis. Tumor volumes were statistically different among the control, Empty-Vec and the experimental group, namely 1.736±0.485cm³, 1.803±0.751cm³ and 0.395±0.015cm³, respectively (P< 0.01). The difference of vascular endothelial growth factor (VEGF) expression between the experimental group and the control group was significant (P<0.01 ). Microvessel density (MVD) of the control, Empty-Vec and the experimental group were 27.21±14.58, 27.76±14.15 and 4.08±4.72 respectively, with statistical differences from the experimental group (P< 0.001). Conclusion The tumor angiogenesis and growth of NB were significantly inhibited by the TrkA gene. These studies provide a theoretical basis for application of NB antiangiogenesis gene therapy. This work was supported by National Nature Science Foundation (No. 39470739).     

Changes in peripheral lymphocyte subsets in patients after partial microwave ablation of the spleen for secondary splenomegaly and hypersplenism: A preliminary study

Purpose: Microwave ablation therapy for secondary splenomegaly and hypersplenism has been shown to be effective from pre-clinical animal models and clinical investigations. This study was performed to determine its effects on the status of peripheral lymphocyte subsets in patients receiving microwave ablation of the spleen.

Materials and methods: Ten patients with secondary splenomegaly and hypersplenism received microwave ablation of the spleen during laparoscopy or percutaneously under ultrasound guidance. The percentage peripheral blood T cells, B lymphocytes and NK cells were measured using flow cytometry before and on days 1, 3 and 7 after therapy, as well as 1 and 3 months afterwards.

Results: Percentages of CD3⁺ and CD4⁺ cells increased rapidly 1 month after therapy. There was no significant change in CD8⁺, CD4⁺/CD8⁺ or NK cells of the pre- and post-therapy levels and B lymphocytes increased significantly after therapy. In patients with an ablation volume (AV) less than 20% (group A), T cells increased 1 month after ablation but decreased 3 months after ablation. B lymphocytes increased significantly after surgery. Levels of NK cells were lower than that before therapy on each testing. In patients with 20–40% AV (group B), levels of T cells, B lymphocytes and NK cells showed an increase. Levels of CD4⁺ cells were significantly higher in group B than in group A, 3 months after therapy.

Conclusions: Microwave ablation therapy for splenomegaly and hypersplenism appears to have a favourable effect on peripheral lymphocyte subsets. A relationship may exist between the ablation volume and the level of peripheral lymphocyte subsets.     

Quantitation of proliferative and cytotoxic precursor cells directed against human tumours: Limiting dilution analysis in peripheral blood and at the tumour site

Blood and tumour-infiltrating lymphocytes (TIL) from 16 cancer patients have been examined under limiting dilution conditions to determine the frequency of cells responding in mixed tumour-lymphocyte cultures (MLTC) to autologous tumour and Interleukin-2 (IL-2). Tumour-derived lymphocytes showed a high spontaneous response to IL-2 alone 1/1,900 in TIL; 1/6,000 in PBL suggesting the presence of “activated” T cells in situ. Proliferative frequencies were increased in MLTC in both blood (1/3,779) and TIL (1/1,084). Phenotypic analyses showed that total T-cell contents of the responder populations were comparable but TIL were enriched for the OKT₈⁺ subset with a corresponding reduction in OKT₄⁺. TIL showed increased numbers of OKMI⁺ and Tac⁺ lymphocytes. The major cytotoxic precursor expanding under these conditions was reactive against autologous tumour. K562 (NK) were present at a lesser frequency -particularly in TIL The data show a concentration and activation of reactive lymphocytes at the tumour site and establish conditions for the clonal expansion of specifically cytotoxck T cells.     

Distribution of Adoptively Transferred,Tumor-sensitized Lymphocytes in the Glioma-bearing Rat

For adoptively transferred lymphocytes to exert anti-tumor effects in vivo, they must traffic or initiate the migration of endogenous immune cells to the site of tumor. Using a rat model, we examined the trafficking of tumor-sensitized lymphocytes to an intracerebral glioma. By labeling the cells with ¹¹¹Indium oxine (¹¹¹In) prior to intravenous injection, we were able to quantify the relative number of lymphocytes that traveled to the tumor site. There was no difference in lymphocytic influx between the tumor-bearing and non-tumor-bearing cerebral hemispheres in 3-day rat glioma models. However, in 7-day models, significantly greater numbers of ¹¹¹In-labeled lymphocytes resided in the tumor-bearing hemisphere at 12 h post-administration. This number increased more than two-fold by 24 h post-adoptive transfer. Using fluorescent-labeled lymphocytes and microscopy, we confirmed that the detection of radioactivity within the brain was truly due to tumor infiltrating ¹¹¹In-labeled lymphocytes. Adoptively transferred cells were found in perivascular and peritumoral locations. These data demonstrate that tumor-sensitized lymphocytes traffic to an intracerebral target site where they can exert an effect, further supporting adoptive immunotherapy as a treatment for glioma.     

Generation of CD4+ cytotoxic T lymphocytes stimulated by immobilized anti-CD3 monoclonal antibody and interleukin-2 in cancer patients

The proliferation of autologous tumor-reactive cytotoxic T lymphocytes (CTL), induced by autologous mixed lymphocyte tumor-cell culture, was remarkably enhanced by activation with immobilized anti-CD3 monoclonal antibody (MAb) and interleukin-2 (IL-2), as compared with IL-2 alone. The activated CTL exhibited high cytotoxicity against autologous tumor cells. Cytotoxicity against autologous tumor cells was inhibited by anti-HLA-DR MAb. In negative selection with immunomagnetic beads, cytotoxicity against autologous tumor cells was inhibited by the elimination of CD4⁺ cells. The major cell-surface antigens of the activated CTL were CD3⁺, CD4⁺, CD25⁺, CD45RO⁺and CD45RA^-, suggesting helper T cells, and the activated CTL produced IL-2. It is concluded that the CTL activated by immobilized anti-CD3 MAb and IL-2 were CD4 cells that had both killer and helper functions. Our findings indicate that adoptive immunotherapy using these activated CTL would be effective in cancer patients. © 1995 Wiley-Liss, Inc.     

Schannoma cells induce a tumor-cell-specific cytotoxic- T-cell response upon transplantation into syngeneic rats but escape elimination through the secretion of immunosuppressive factors

Tumor cells often express antigens that can be recognized by the immune system. Despite induction of an immune response, the tumor cells escape their elimination. We have studied the mechanisms and factors which mediate these events in a syngeneic tumor model. NV2Cd rat schwannoma cells were transplanted into BDIX rats. After injection of 10⁷ to 2 × 10⁷ cells, tumors grew very slowly for 10 to 12 days. After that time, rapid growth was observed. The tumors consisted of compact areas of spindle-shaped cells with small cysts, many blood vessels and central necrotic areas. During tumor growth, the number of spleen cells and T lymphocytes increased, and cytotoxic T cells with specificity for the NV2Cd tumor cells were detected. The strong specific cellular immune response did not prevent the increase in tumor volume. We studied the biological activity of the fluid present in the cysts of the tumor. At a concentration of 1 ng to 10 μg protein per ml, the cyst fluid inhibited the proliferation of splenic T lymphocytes and B lymphocytes and of lymphoma cells, but enhanced the proliferation of NV2Cd tumor cells. The cyst fluids contain the immunosuppressive transforming growth factors (TGF)-β1, -β2 and -β3, also the vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF). Antibodies directed against TGF-β relieved the suppression of T-cell growth by cyst fluid, but did not influence the proliferation of NV2Cd cells. The growth-modulating factors present in the tumor cyst fluid were also detected in conditioned medium from NV2Cd cells cultured in vitro. Our data suggest that tumors can escape the cellular immune response by the production of factors that inhibit lymphocytes. They also enhance their own growth environment by secreted factors. Int. J. Cancer 70:542–550. © 1997 Wiley-Liss Inc.     

Pharmacology of intracellular cytosine-arabinoside-5′-triphosphate in malignant cells of pediatric patients with initial or relapsed leukemia and in normal lymphocytes

Purpose The prodrug cytosinearabinoside (ara-C) is widely used in the treatment of acute leukemias. The active drug is the intracellular metabolite cytosine-arabinoside-5′-triphosphate (ara-CTP). The purpose of the present study was to investigate the relation between sensitivity and pharmacokinetic parameters C _max, t _1/2 and AUC of ara-CTP. The obtained results were compared to previous studies. Experimental design C _max, t _1/2 and AUC of ara-CTP were assessed in leukemic cells of 17 pediatric patients with acute lymphoblastic leukemia (ALL) and in 6 lymphoblastic cell lines and compared with normal lymphocytes of 9 healthy donors by high pressure liquid chromatography (HPLC). The sensitivity of the cells against ara-C was determined by the MTT assay. Results The intracellular accumulation of ara-CTP was significantly lower in normal lymphocytes (C _max 47.7–60.9 pmol/10⁶ cells) compared to leukemic cell lines (C _max 11–1128 pmol/10⁶ cells) and leukemic cells of our patients (C _max 85.9–631 pmol/10⁶ cells). Similar results were found for the AUC. There was no significant difference between initial and relapsed leukemias in our small cohort. A correlation between sensitivity in terms of IC₅₀ values and the intracellular ara-CTP accumulation was observed in cell lines, but not in leukemic cells and normal lymphocytes from healthy donors. Conclusions Pharmacokinetic parameters varied tremendously in leukemic cells in contrast to normal lymphocytes without a difference in sensitivity. It is worthwhile to compare literature data to assess an optimal dosage of ara-C in pediatric patients.     

Adhesion of human uroepithelial cells to E-selectin: Possible involvement of sialosyl LewisA-ganglioside

In a previous study we showed that tumorigenic and invasive human uroepithelial cell lines are characterized by the presence of sialosyl Le^a (sLe^a) ganglioside. Our data suggested that expression of this glycolipid correlated with acquisition of the malignant phenotype by human urothelial cells. To evaluate the postulated adhesion function of sLe^a antigen, we studied the adherence of 6 human urothelial cell lines with different expressions of this carbohydrate structure to E-selectin-expressing CHO cells. The only cell line that bound specifically to E-selectin was Hu 1703He, which expressed the highest level of sLe^a antigen. The involvement of carbohydrate-E-selectin interaction in the adhesion of Hu 1703He cells was indicated by the following facts: (i) anti-E-selectin monoclonal antibody (MAb) completely abolished binding to E-selectin-expressing CHO cells; (ii) removal of sialic acid from Hu 1703He cells highly decreased the adhesion. Adhesion correlated with the presence of several sLe^a-carrying glycoproteins, which was shown by immunoblotting of Hu 1703He cell lysate with anti-sLe^a MAb 19-9. The binding of antibody was abolished when cell lysate was treated with O-sialoglycoprotein endopeptidase, suggesting that sLe^a is present on O-linked oligosaccharides. However, incubation of Hu 1703He cells with O-sialoglycoprotease had no effect on adhesion to E-selectin or on binding of 19-9 MAb to the cell surface. Our data suggest that (i) protein-bound sLe^a oligosaccharides represent only a minor portion of whole sLe^a antigen produced by uroepithelial cells; (ii) effective binding to E-selectin occurs when sLe^a oligosaccharide present on cell-surface glycosphingolipids is expressed in high density since the cell lines with moderate expression of sLe^a ganglioside did not bind to E-selectin-transfected CHO cells. © 1996 Wiley-Liss, Inc.