High Levels Of Cytotoxicity Research Articles

CD52 Is Expressed on Human Mast Cells and Is a Therapeutic Target for the Anti-CD52 Monoclonal Antibody Campath-1H in Waldenstrom's Macroglobulinemia and Mast Cell Disorders.

Campath-1H is a monoclonal antibody used in the treatment of CD52 expressing B-cell malignancies. Recently, we and others have observed that Campath-1H shows unusually high activity in patients with relapsed/refractory Waldenstrom's macroglobulinemia (WM), a B-cell malignancy with excess bone marrow mast cells (BMMC). Importantly, as we have recently shown, BMMC appear to provide direct support for WM tumor cell growth (Tournilhac et al, JCO 2004 22:571S), and are therefore a potential therapeutic target in WM. We therefore examined BMMC from patients with WM and other mast cell disorders for cell surface expression of CD52, and targeting by Campath-1H in preclinical studies. Multicolor flow cytometric analysis demonstrated CD52 expression on BMMC (FceRI+, CD117+) from 13/15 WM; 2/2 systemic mastocytosis (SM) patients; 2/4 healthy donors; as well as on the LAD and HMC MC lines. Moreover, RT-PCR analysis confirmed CD52 expression in sorted BMMC from 6/7 WM, along with 6/6 healthy donors. Importantly, Campath-1H induced high levels of antibody dependent cell mediated cytotoxicity (ADCC) actvity against LAD mast cells using activated NK effector cells. No direct cytotoxicity or antiproliferative activity by Campath-1H on LAD cells was observed. These studies demonstrate that CD52 is widely expressed on human MC and provide support for the use of Campath-1H in the treatment of WM and other systemic mast cell disorders.

Interleukin-15 enhances cytotoxicity, receptor expression, and expansion of neonatal natural killer cells in long-term culture.

Newborn infants have a higher susceptibility to various pathogens due to developmental defects in their host defense system, including deficient natural killer (NK) cell function. In this study, the effects of interleukin-15 (IL-15) on neonatal NK cells was examined for up to 12 weeks in culture. The cytotoxicity of fresh neonatal mononuclear cells (MNC) as assayed by K562 cell killing is initially much less than that of adult MNC but increases more than eightfold after 2 weeks of culture with IL-15 to a level equivalent to that of adult cells. This high level of cytotoxicity was maintained for up to 12 weeks. In antibody-dependent cellular cytotoxicity (ADCC) assays using CEM cells coated with human immunodeficiency virus gp120 antigen, IL-15 greatly increased ADCC lysis by MNC from cord blood. IL-15 increased expression of the CD16+ CD56+ NK markers of cord MNC fivefold after 5 weeks of incubation. Cultures of neonatal MNC with IL-15 for up to 10 weeks resulted in a unique population of CD3- CD8+ CD56+ cells (more than 60%), which are not present in fresh cord MNC. These results show that IL-15 can stimulate neonatal NK cells and sustain their function for several weeks, which has implications for the clinical use of IL-15.

Attenuated poxviruses generate clinically relevant frequencies of CMV-specific T cells

AbstractImmunotherapeutic approaches to limit cytomegalovirus (CMV) morbidity and mortality after hematopoietic stem cell transplants (HSCTs) are currently under investigation as alternatives to antiviral drugs. In this context, we have inserted full-length and ubiquitin-modified CMV phosphoprotein 65 (pp65), phosphoprotein 150 (pp150), and immediate early protein 1 (IE1) immunodominant antigens into the virulent Western Reserve strain of vaccinia virus (VV) and the highly attenuated strain, modified vaccinia Ankara (MVA). Recombinant (r) VV or rMVA stimulated vigorous expansion of CMV-specific CD8+ T cells in CMV-positive donor peripheral blood mononuclear cells (PBMCs), which showed minimal alloreactivity and high levels of HLA tetramer binding, cytokine production, and cytotoxicity. Ubiquitinated antigens had a profound effect when expressed in VV. Single antigen rMVA expressing pp65 or IE1, either ubiquitin-modified or native, stimulated both cytotoxic T lymphocyte (CTL) populations to be expanded up to 500-fold in a 60-mL blood draw from the same donor. This result demonstrates the clinical feasibility of simultaneously amplifying multiple CMV-CTL populations. Transgenic HLA A2.1 (HHD II) mice, immunized with the same rMVA as used with human PBMCs, produced a robust cytotoxic response to both CMV pp65 and IE1. The specificity of the vigorous immunologic response to rMVA, both in vitro and in vivo, makes them candidates for clinical evaluation in the context of adoptive immunotherapy for hematopoietic stem cell transplant (HSCT) recipients or donor vaccination.

Green fluorescent protein expression in dendritic cells enhances their immunogenicity and elicits specific cytotoxic T-cell responses in humans

Objective Green fluorescent protein (GFP) has been used to monitor and select cells transduced with vectors encoding other transgenes of interest. We investigated the immunogenic nature of GFP in humans and further explored whether this xenoprotein could be used as a functional adjuvant to enhance T-cell immunity to the melanoma tumor antigen MART1. Methods Peripheral blood lymphocytes from healthy donors were stimulated by autologous dendritic cells expressing GFP, then cloned by limiting dilution and tested for antigen specificity following coculture with GFP-expressing or GFP-negative targets. In a parallel experiment, lymphocytes from HLA A 0201 + healthy donors were stimulated with four different Melan-A/MART1 27-35 peptide-pulsed stimulators: 1) MART1 peptide-pulsed DCs, 2) MART1 peptide-pulsed DCs loaded with GFP protein, 3) MART1 peptide-pulsed GFP adenovirus–transduced DCs, and 4) MART1 peptide-pulsed null adenovirus–transduced DCs. The percentage of CD3 +/CD8 + MART1 peptide-specific T cells was determined by intracellular cytokine staining for γ-IFN. Results Multiple CD4 + and CD8 + T cell clones were expanded which secreted γ-IFN and demonstrated high levels of cytotoxicity to GFP-expressing targets as assessed by ELISA and Cr 51 release respectively. We next investigated the impact of GFP expression on DCs used to stimulate cytotoxic T cells specific for a tumor-associated peptide. The percentage of MART1-specific CD8 + T cells that were generated was higher when MART1-pulsed GFP adenovirus–transduced DCs were used as stimulators (28%) compared to MART1-pulsed DCs alone (11%, p = 0.01), MART1-pulsed null adenovirus–transduced DCs (11.7%, p = 0.02), or MART1-pulsed DCs loaded with GFP protein (12.2%). Conclusions These findings further support GFP's immunogenicity and suggest this xenoprotein might further be used to enhance the expansion of tumor-specific T cells.

DNA damage produced in HaCaT cells by combined fluoranthene exposure and ultraviolet A irradiation.

Fluoranthene is a polycyclic aromatic hydrocarbon (PAH) and a principal constituent of PAH-contaminated aquatic systems. In the present study, fluorescein diacetate uptake and the Comet assay were used to assess the cytotoxicity and genotoxicity of fluoranthene in HaCaT (human adult low calcium high temperature) cells in the presence or absence of ultraviolet A (UVA) irradiation. Exposure of cells to 0.1, 0.25, 0.75, 2, and 5 microM fluoranthene alone for 30 min or to 6.1 +/- 0.07 J/cm2 UVA alone did not cause cytotoxicity or cellular DNA damage. However, concomitant exposure to both caused a nonlinear dose-response in cytotoxicity to HaCat cells. The same exposure conditions also resulted in a dose-responsive DNA damage in HaCaT cells. Because DNA damage mainly was detected at relatively high levels of cytotoxicity, we cannot rule out the possibility that it occurred as a consequence of cellular toxicity mechanisms.

The Characterization of Cationic Fusogenic Liposomes Mediated Antisense Oligonucleotides into HeLa Cells

Antisense oligonucleotides (ODNs) are potential therapeutic agents, but their development is still limited due to poor cellular uptake and high degradation rate in biological media. To resolve these problems, we propose to attach the Sendai virus to cationic liposomes. Cationic–fusogenic liposomes (CFLs) were prepared by reverse‐phase evaporation and fused with the Sendai virus. The mean diameter was about 186 nm, determined by photon correlation laser light scattering method. The cytotoxicity of CFLs and the ODN loading efficiency depended on the +/− charge ratio. The fluorescence intensity in cytoplasm was enhanced with the increasing of DC‐Chol content and +/− charge ratio. We also investigated the mechanism of cellular uptake using temperature shifts and lysosomotropic agent. The results indicated that the vector was introduced into the cells, not via endocytosis but membrane fusion. The preliminary experiment showed that CFLs are a promising formulation for ODN delivery with high levels of transfection and minimal cytotoxicity.

Ontogeny of Xenopus NK cells in the absence of MHC class I antigens

This paper explores the ontogeny of NK cells in control and early-thymectomized (Tx) Xenopus laevis through phenotypic analysis of cells expressing the NK cell antigen 1F8 and by performing in vitro cytotoxic assays. Dual color flow cytometry reveals that a few 1F8positive splenocytes first emerge in late larval life, at ∼7-weeks post-fertilization. This is about 2-weeks after the time when surface MHC class Ia expression can first be detected. The proportion of splenocytes expressing 1F8 remains very low in 3–4 month-old froglets, but by 1 year there is a sizeable 1F8positive population, which is proportionally elevated in Tx frogs. The ontogeny of NK cell function is monitored by a 5 h DNA fragmentation (JAM) assay. Control and Tx larval splenocytes (from either 5- or 7-week-old tadpoles) fail to kill MHC-deficient thymus-derived tumor cell targets. Such in vitro killing is still relatively poor in 3–4 month froglets, compared with high levels of tumor cell cytotoxicity mediated by splenocytes from older frogs. Immunoprecipitation studies identify that the major ligand for the 1F8 mAb is a 55 kDa polypeptide. Finally, further evidence is provided that 1F8positive lymphocytes are indeed bona fide NK cells, distinct from T cells, since purified 1F8positive splenocytes from Tx Xenopus fail to express fully rearranged TCRβ V region transcripts. We conclude that NK cells fail to develop prior to MHC class I protein expression and, therefore, do not contribute to the larval immune system, whereas they do provide an important backup for T cells in the adult frog by contributing to anti-tumor immunity.

Abstracts of the 8th Meeting of the Italian Peripheral Nerve Study Group: 52

Gangliosides (Gs) have been implicated as potential antigens in autoimmune neuropathies and anti‐tumoral immune response, but their role in paraneoplastic neuropathy has been poorly investigated. We describe one patient affected by lung adenocarcinoma and subacute motor axonal neuropathy with high titres of IgG1 and IgG3 to several Gs. Expansion (3% of total T cells) of peripheral g/d T lymphocytes was found, as compared to healthy controls (<0.5%) and to patients with lung neoplasia without neuropathy (1.1%). At three months after neoplasia removal, we observed stabilization of the neuropathy, decline of anti‐Gs IgG and of g/d T cells, which dropped to normal values. Expression of GM1, GD1a, GM2 and GM3 was found by immunohistochemistry on patient's neoplastic cells, but not in control lung neoplasia. Absorption experiments of anti‐Gs IgG revealed that only GD1a and GM1, among all Gs tested, significantly decreased anti‐Gs IgG reactivity. Indirect immunohistochemistry with biotinylated IgG purified from the patient showed positive signals on motoneurons, dorsal root ganglia, axonal structures and Schwann cells and his own neoplastic cells. At autopsy, IgG deposits were found on the same neural targets. Purified IgG from the patient exerted high levels of cytotoxicity on both Schwann and neuroblastoma cells, as compared to healthy controls. The association of neoplasia, peripheral neuropathy and autoimmune responses to Gs observed in our patient proposes a pathogenetic role for Gs as putative autoantigens in paraneoplastic neuropathy.

Investigation of the biological activities of Siphonochilus aethiopicus and the effect of seasonal senescence

Siphonochilus aethiopicus (Schweinf.) B.L. Burtt (Zingiberaceae), commonly known as wild ginger, is one of the most important and threatened medicinal plants in South Africa. A study of the pharmacological properties of S. aethiopicus and the effect of seasonal senescence on antibacterial and anti-inflammatory properties was undertaken. Water, ethanol and ethyl acetate extracts were prepared from the leaves, rhizomes and roots of S. aethiopicus plants. The extracts were tested in a variety of pharmacological assays. Results for the general screening showed antibacterial and anti-inflammatory activity. Some cytotoxicity was observed with the aqueous extracts of the rhizome. However, no significant activity against the herpes simplex virus types 1 and 2, the influenza A virus, and in the anthelmintic, antischistosomal and biochemical induction assays were observed. In the microdilution antibacterial assay, no inhibitory activity against the test bacteria was detected with the aqueous extracts. The ethanol and ethyl acetate extracts tested showed greater antibacterial activity at minimal inhibitory concentrations ranging from 0.78 to 3.13 mg ml − 1 against the Gram-positive bacteria ( Bacillus subtilis , Staphylococcus aureus ) than the Gram-negative bacteria ( Escherichia coli , Klebsiella pneumoniae ). Little difference was observed between the ethanol and ethyl acetate extracts, or between the different plant parts. Ethanol extracts were prepared from the different plant parts before and after seasonal senescence, and tested for antibacterial and anti-inflammatory activity. There appeared to be a loss of antibacterial activity in the leaves with senescence, concomitant with an increase of activity in the alpha-roots. In the cyclooxygenase-1 assay, the aqueous extracts showed no significant prostaglandin synthesis inhibition. For the ethanol and ethyl acetate extracts, the leaves showed the highest levels of activity at a concentration of 250 ng ml −1 per test solution, in both the cyclooxygenase-1 and −2 assays. Some differences in the levels of anti-inflammatory activity in the roots following senescence were also observed. There was a slight loss of activity as a result of drying the rhizome material prior to extraction. This suggests that fresh rhizome material may be more effective for medicinal use, although it should be noted that the aqueous rhizome extracts displayed moderately high levels of cytotoxicity, and may require further investigation.

Genetic disruption of host interferon-gamma drastically enhances the metastasis of pancreatic adenocarcinoma through impaired expression of inducible nitric oxide synthase.

Synergistic induction of the inducible nitric oxide synthase (NOS II) gene requires a combination of interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). In this study, we determined whether the induction of IFN-gamma was required for NOS II-mediated antitumor activity in vivo. Highly metastatic H7 murine pancreatic adenocarcinoma cells were implanted into the subcutis, footpad, and pancreas of syngeneic IFN-gamma(+/+) and IFN-gamma(-/-) mice. These cells grew and produced metastases and ascites in IFN-gamma(+/+) mice. In sharp contrast, the same tumor cells grew much more aggressively, metastasized more extensively, and produced a larger amount of malignant ascites in IFN-gamma(-/-) mice. Also, induction of IFN-gamma correlated with NOS II gene expression and NO production in IFN-gamma(+/+) injected with the tumor cells but not in IFN-gamma(-/-) mice or IFN-gamma(+/+) mice without tumor challenge. In vitro, only LPS plus IFN-gamma induced a high level of NO production and cytotoxicity against H7 cells. These data suggested that the tumor cells stimulated IFN-gamma secretion from host cells, which in turn stimulated NO production by host cells and suppressed tumor growth and metastasis.

Bioactive compounds from coral reef invertebrates

Abstract Manzamine A (1) known as a cytotoxin exhibited potent antimalarial activity with 40% recovery 60 days after single intraperitoneal (i.p.) injection against mice infected with the malaria parasite Plasmodium berghei. Pachastrisamine (6), a simple derivative of a sphingosine, isolated from a sponge, Pachastrissa sp. showed a high level of cytotoxicity. New sesquiterpene carbonimidic dichlorides and related aldehydes (8­12) have been isolated as cytotoxic constituents of the sponge Stylotella aurantium and shown to have moderate cytotoxicity. Cyclic undecapeptides, barangamides A­D (13­16), have been isolated from the sponge Theonella swinhoei. Their structures were established by spectroscopic analysis and chemical reaction.

Intensified regression of colon cancer liver metastases in mice treated with irinotecan and the immunomodulator JBT 3002.

The authors recently reported that tumoricidal activation of macrophages by a new synthetic bacterial lipopeptide, JBT 3002, can augment chemotherapy-mediated tumor-cell killing. The aim of this study was to identify the mechanism responsible for the destruction of metastatic cells. Three daily oral doses of JBT 3002 before once-weekly intraperitoneal injections of 100 mg/kg irinotecan for 3 weeks significantly increased the eradication of established CT-26 murine colon cancer liver metastases compared with treatment with irinotecan alone. Immunohistochemical analyses revealed that the hepatic metastases in mice given combination therapy contained infiltrating CD8+ lymphocytes and a dense infiltrate of macrophages expressing both inducible nitric oxide synthase (iNOS) and interleukin-15. In vitro treatment of peritoneal macrophages with JBT 3002 plus interferon-gamma induced the expression of iNOS and the production of nitric oxide. In the presence of a low (subtoxic) dose of irinotecan, these activated macrophages produced significant lysis of CT-26 cells. The high level of cytotoxicity was inhibited by the specific inducible nitric oxide synthase inhibitor, NG-methyl-L-arginine. In contrast, irinotecan-mediated lysis of normal intestinal epithelial IEC-6 cells was not increased by activated macrophages. Scanning electron microscopy revealed that activated macrophages bound to CT-26 tumor cells but not to normal IEC-6 cells, confirming that nitric oxide-mediated cytotoxicity is specific for tumor cells. Collectively, the results suggest that augmentation of the therapeutic efficacy of irinotecan against colon cancer liver metastases by immunomodulation with JBT 3002 may be associated with elevated inducible nitric oxide synthase and endogenous interleukin-15 in tumor-infiltrating macrophages.

Expression of cytokines and factors modulating apoptosis by human sinusoidal leucocytes

Background/Aims: Liver sinusoids contain a large population of spontaneously cytotoxic cells (NK cells), CD8+ T cells and macrophages. The physiological role of these leucocytes remains unclear. They may participate in immune surveillance and peripheral tolerance by deleting tumour cells, virus-infected cells and activated T cells as they traffic through the liver. In order to gain further information about the function of these leucocytes within the hepatic sinusoids, we examined their production of immunomodulatory cytokines and apoptosis-related molecules.Methods: Semi-quantitative polymerase chain reaction and immunohistochemistry were used to determine the spontaneous production of cytokines and apoptosis-related molecules by sinusoidal leucocytes isolated from donor liver preservation solution.Results: In comparison with matched peripheral blood mononuclear cells, sinusoidal leucocytes produced more mRNA for IL-10, IL-15, TNF-α, IL-18, IFN-γ, FasL, perforin and granzyme. IL-4 and IL-12 were not detected and IL-2 was only faintly detected in the liver-derived CD4+ population. Less bcl-2 was expressed in liver-derived CD4+ and CD8+ cells in comparison with matched peripheral blood cell populations.Conclusions: The cytokines produced spontaneously by sinusoidal leucocytes are consistent with their high level of activation and spontaneous cytotoxicity. Their strong expression of apoptosis-mediating molecules (FasL, perforin, granzyme and TNF-α) support a role for these cells in immune surveillance and peripheral tolerance induction.

Role of chicken IL-2 on γδ T-cells and Eimeriaacervulina-induced changes in intestinal IL-2 mRNA expression and γδ T-cells

Continuous culture of concanavalin A (Con A)-activated spleen cells in the presence of chicken recombinant IL-2 (rIL-2) promoted preferential growth of γδ T-cells. These cells displayed a high level of spontaneous cytotoxicity against LSCC-RP9 tumor cells, an avian NK cell target. Stimulation of IL-2-dependent γδ T-cells with Con A induced IFN-γ and IL-2 mRNA transcripts, whereas stimulation with rIL-2 induced only IFN-γ mRNA. Subcutaneous injection of 3-week-old chickens with IL-2 DNA increased splenic cells, expressing the CD8 and γδ TCR antigens. To investigate the role of IL-2 and γδ T-cells in parasitic infection, chickens were orally infected with Eimeria acervulina and the expression of IL-2 mRNA transcripts in the spleen and duodenum and the percentage of γδ T-cells in the duodenum were examined. Following both, the primary and secondary infections, a significant enhancement of IL-2 mRNA transcripts in the spleen and intestine and increased percentage of intraepithelial γδ T-cells in the duodenum were observed. These results indicate that host immune responses to E. acervulina involve an up-regulation of IL-2 secretion and an increased duodenum γδ T-cells.

Human biological relevance and the use of threshold-arguments in regulatory genotoxicity assessment: experience with pharmaceuticals

Issues of biological relevance and thresholds for genotoxicity are discussed here based upon the background of experience with the submissions for the approval of new pharmaceuticals to the German regulatory authority over the period between 1990 and 1997. This experience shows that out of the genotoxicity test systems which are required according to existing guidelines in the European Union (EU), the in vitro tests for chromosomal aberrations (CA) and the mouse lymphoma tk assays (MLA) yield a rate of positives that is about four-fold higher than that of other genotoxicity tests. A detailed analysis of chemical and pharmacological classes of compounds and their effects in these systems reveals that in addition to direct DNA reactivity several mechanisms of indirect genotoxicity such as nucleoside analogue incorporation into DNA, interaction with microtubule assembly, topoisomerase inhibition and high levels of cytotoxicity are relevant. New pharmaceuticals, for which the latter mechanisms apply, often display threshold-like characteristics in their genotoxic effects in vitro or even in vivo in experimental animals. This casts doubt upon the relevance of positive in vitro test results for such compounds. However, the discussion of examples shows that it may not be easy to demonstrate the exact thresholded mechanism of genotoxicity in a given case. In particular, the demonstration of a coincidence of genotoxicity and high levels of cytotoxicity, which seems to be a major factor for biologically non-relevant in vitro positive new pharmaceuticals, usually requires quite extensive testing. Hence, for new pharmaceuticals it is practice to provide in addition to in vitro results that may be thresholded a wealth of information from in vivo studies on genotoxicity, carcinogenicity, metabolism, pharmacokinetics, etc. the results of which help in assessing the biological relevance of in vitro positives. The regulatory acknowledgement of biologically non-relevant, thresholded mechanisms of (in vitro) genotoxicity in addition to those that are considered relevant for human risk ensures a better understanding of test results and is needed for the credibility of genotoxicity testing practice in general.

Proteosome delivery of a protective 9B-antigen against Schistosoma mansoni

We have previously characterized a stage specific, partially protective protein denoted 9B-antigen. This antigen is of 450 kDa in its native form but upon SDS–PAGE in reducing conditions it exhibits two subunits of 30 kDa and 45 kDa. The 9B-antigen is localized at the surface of schistosomula and persists at the surface of lung schistosomula. The 9B-antigen is also localized in internal organs of a vital function in the parasite such as flame cells and cytoplasmic tubes. Infected individuals or mice vaccinated with irradiated cercariae recognize the 9B-antigen. We have previously shown that when injected with complete Freunds adjuvant, the 9B-antigen can induce 40% protection against challenge infection. In this study we have used a more effective delivery system for this antigen. The 9B-antigen was coupled to proteosomes derived from meningoccocal outer membrane proteins. Vaccination of mice with this complex increased the protection level to 60%. Sera from these vaccinated mice induced high levels of complement mediated cytotoxicity of the parasite. Since the proteosomes are approved for human use, these results are promising towards the development of a vaccine against schistosomiasis.

Effect of intraperitoneally administered recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) on the cytotoxic potential of murine peritoneal cells.

We studied the effect of recombinant murine granulocyte–macrophage colony-stimulating factor(rmGM-CSF) on the cytotoxic potential of murine peritoneal cells. Mice received rmGM-CSF intraperitoneally using different dosages and injection schemes. At different time points after the last injection, mice were sacrificed, peritoneal cells isolated and their tumour cytotoxicity was determined by a cytotoxicity assay using syngeneic [methyl-3H]thymidine-labelled colon carcinoma cells. Also, the cytotoxic response to a subsequent in vitro stimulation with lipopolysaccharide was determined. Upon daily injection of 6000–54 000 U rmGM-CSF over a 6-day period, the number of peritoneal cells increased over ten fold with the highest rmGM-CSF dose. Increases in cell numbers was mainly due to increases in macrophage numbers. Upon injection of three doses of 3000 U rmGM-CSF per day for 3 consecutive days, the number of macrophages remained elevated for minimally 6 days. Although the peritoneal cells from rmGM-CSF-treated mice were not activated to a tumoricidal state, they could be activated to high levels of cytotoxicity with an additional in vitro stimulation of lipopolysaccharide. Resident cells isolated from control mice could be activated only to low levels of tumour cytotoxicity with lipopolysaccharide. Tumour cytotoxicity strongly correlated with nitric oxide secretion. When inhibiting nitric oxide synthase, tumour cell lysis decreased. Thus, the expanded peritoneal cell population induced by multiple injections of rmGM-CSF has a strong tumour cytotoxic potential and might provide a favourable condition for immunotherapeutic treatment of peritoneal neoplasms. © 1999 Cancer Research Campaign

Induction of HLA-unrestricted and HLA-class-II-restricted cytotoxic T lymphocytes against MUC-1 from patients with colorectal carcinomas using recombinant MUC-1 vaccinia virus.

We recently reported that immunization with a recombinant MUC-1 vaccinia virus (rVMUC-1) protected C57BL/6 mice from challenge with DF3/MUC-1-positive syngeneic tumors. To elucidate whether anti-MUC-1 tumor immunity, especially MUC-1-specific cytotoxic T lymphocytes (CTI), can be induced in cancer patients by rVMUC-1, we stimulated the peripheral blood lymphocytes from patients with DF3/MUC-1+ or DF3/MUC-1 colon carcinomas using the autologous monocytes infected with rVMUC-1 (rVAMN). The stimulated T lymphocytes from two patients with DF3/MUC-1-positive colorectal carcinomas (rVPY+T and rVPW+T) demonstrated HLA-unrestricted cytotoxicity against MUC-1, whereas those from the patient with DF3/MUC-1-negative colon carcinoma (rVPA-T) did not. The HLA-unrestricted cytotoxicity was demonstrated by the CD8+ T cells possibly recognizing an epitope present on the tandem repeats. Adoptive immunotherapy who performed three times with patient PY, at 4-week intervals. The adoptive transfer of the first stimulated lymphocytes, demonstrating a high level of HLA-unrestricted cytotoxicity against MUC-1, resulted in the significant reduction of the liver metastasis of patient PY. However, HLA-unrestricted cytotoxicity against MUC-1 was extremely reduced at the second transfer and finally eliminated at the third, whereas the CD4+ T cells demonstrating HLA-class-II-restricted cytotoxicity against MUC-1 predominantly proliferated at the third adoptive immunotherapy treatment. The liver metastasis and the serum levels of tumor markers (carcinoembryonic antigen CA19-9) demonstrated a rapid and marked increment after the second transfer and especially after the third. These results suggest that the HLA-unrestricted cytotoxic CD8+ T cells against MUC-1, induced in patients with DF3/MUC-1+ colorectal carcinomas using rVMUC-1, correlate with the antitumor activity in vivo.

Design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue

Capecitabine (N 4-pentyloxycarbonyl-5′-deoxy-5-fluorocytidine) is a novel oral fluoropyrimidine carbamate, which is converted to 5-fluorouracil (5-FU) selectively in tumours through a cascade of three enzymes. The present study investigated tissue localisation of the three enzymes in humans, which was helpful for us to design the compound. Carboxylesterase was almost exclusively located in the liver and hepatoma, but not in other tumours and normal tissue adjacent to the tumours. Cytidine (Cyd) deaminase was located in high concentrations in the liver and various types of solid tumours. Finally, thymidine phosphorylase (dThdPase) was also more concentrated in various types of tumour tissues than in normal tissues. These unique tissue localisation patterns enabled us to design capecitabine. Oral capecitabine would pass intact through the intestinal tract, but would be converted first by carboxylesterase to 5′-deoxy-5-fluorocytidine (5′-dFCyd) in the liver, then by Cyd deaminase to 5′-deoxy-5-fluorouridine (5′-dFUrd) in the liver and tumour tissues and finally by dThdPase to 5-FU in tumours. In cultures of human cancer cell lines, the highest level of cytotoxicity was shown by 5-FU itself, followed by 5′-dFUrd. Capecitabine and 5′-dFCyd had weak cytotoxic activity only at high concentrations. The cytotoxicity of the intermediate metabolites 5′-dFCyd and 5′-dFUrd was suppressed by inhibitors of Cyd deaminase and dThdPase, respectively, indicating that these metabolites become effective only after their conversion to 5-FU. Capecitabine, which is finally converted to 5-FU by dThdPase in tumours, should be much safer and more effective than 5-FU, and this was indeed the case in the HCT116 human colon cancer and the MX-1 breast cancer xenograft models.

Differential Cytotoxicity of Cord Blood and Bone Marrow–Derived Natural Killer Cells

Allogeneic cord blood is now being widely used as a source of stem cells for hematologic reconstitution after myeloablative therapy, with reported significantly lower levels of graft-versus-host disease (GVHD) compared with the use of allogeneic bone marrow (BM). This study was undertaken to investigate biologic aspects of natural killer (NK) cell activity, as recognized effector cells of the GVHD and graft-versus-leukemia (GVL) response, from cord blood and conventional BM. NK-cell activity levels of freshly isolated cells from cord blood and BM against K562 targets were comparable. Lymphokine activated killer (LAK) cells from both hematopoietic cell sources were compared for their ability to kill target cells by necrotic or apoptotic mechanisms using specific target cell lines. Cord blood cells had significantly higher necrosis-mediated cytotoxic activity against Daudi target cells compared with BM-derived cells. Cord blood LAK cells had relatively high levels of apoptotic-mediated cytotoxicity against YAC-1 target cells, whereas BM-derived LAK cells were unable to induce apoptosis in these cells. Interleukin-2 (IL-2) induced significant granzyme B activity in cord cells in contrast to BM cells, in which very little activity was measured. Western blotting confirmed these findings, with IL-2 inducing granzyme B protein expression in cord cells but not detectable levels in BM cells. BM cells had significantly lower cell surface expression of IL-2R and prolonged culture in IL-2 was only partially able to restore their deficient apoptotic cytotoxic activity. Thus, major differences exist between cord blood-derived and BM-derived mononuclear cells with respect to their NK-cell-associated cytotoxic behavior. This could have important implications for stem cell transplantation phenomena, because it suggests that cord blood may have increased potential for a GVL effect.