Characterization of CD4+ CD25+ T cells in solid organ transplant recipients

Although important progresses have been achieved in the therapeutic management of transplant recipients, acute and chronic rejections remain the leading causes of premature graft loss after solid organ transplantation. This, together with the undesirable side effects of immunosuppressive drugs, has significant implications for the long-term outcome of transplant recipients. Thus, a better understanding of the immunological events occurring after transplantation is essential. The immune system plays an ambivalent role in the outcome of a graft. On one hand, some T lymphocytes with effector functions (called alloreactive) can mediate a cascade of events eventually resulting in the rejection, either acute or chronic, of the grafted organ ; on the other hand, a small subset of T lymphocytes, called regulatory T cells, has been shown to be implicated in the control of these harmful rejection responses, among other things. Thus, we focused our interest on the study of the balance between circulating effectors (alloreactive) and regulatory T lymphocytes, which seems to play an important role in the outcome of allografts, in the context of kidney transplantation. The results were correlated with various variables such as the clinical status of the patients, the immunosuppressive drugs used as induction or maintenance agents, and past or current episodes of rejection. We observed that the percentage of the alloreactive T lymphocyte population was correlated with the clinical status of the kidney transplant recipients. Indeed, the highest percentage was found in patients suffering from chronic humoral rejection, whilst patients on no or only minimal immunosuppressive treatment or on sirolimus-based immunosuppression displayed a percentage comparable to healthy non-transplanted individuals. During the first year after renal transplantation, the balance between effectors and regulatory T lymphocytes was tipped towards the detrimental effector immune response, with the two induction agents studied (thymoglobulin and basiliximab). Overall, these results indicate that monitoring these immunological parameters may be very useful for the clinical follow-up of transplant recipients ; these tests may contribute to identify patients who are more likely to develop rejection or, on the contrary, who tolerate well their graft, in order to adapt the immunosuppressive treatment on an individual basis.

Toxic effects of brake wear particles on epithelial lung cells in vitro

Background: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. Results: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. Conclusion: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress. [Authors]

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

CD62L(high) Treg cells with superior immunosuppressive properties accumulate within the CNS during remissions of EAE.

The role of regulatory T cell populations within the CNS in the regulation of CNS-autoimmunity is controversial. We show that during recovery from relapsing remitting experimental autoimmune encephalomyelitis, regulatory T cells accumulate within the CNS that express high levels of CD62L. These CD62L(high) Treg cells express increased amounts of CTLA-4, ICOS and TGF-β and are more potent than CD62L(low) Treg cells in suppressing proliferation and inducing apoptosis in effector T cells. CD62L(high) Treg cells thus represent a population of Treg cells that display superior immunosuppressive properties and accumulate in the CNS during recovery from CNS-autoimmunity.

Characterization of a specific 20- to 25-kD interleukin-1 inhibitor from cultured human lung macrophages.

Alveolar macrophages have the ability to downregulate immune processes in vitro. We have recently suggested the presence of interleukin-1 (IL-1) inhibitors in the supernatants of human bronchoalveolar lavage cells from patients with idiopathic pulmonary fibrosis or sarcoidosis. In the present study, we further analyze the cellular origin and the biologic properties of a 20- to 25-kD IL-1 inhibitor spontaneously produced by cultured human alveolar macrophages (AM). The inhibitor blocks IL-1-induced prostaglandin E2 production by human fibroblasts and the IL-1-related increase of phytohemagglutinin-induced murine thymocyte proliferation. After rigorous IL-1 alpha and IL-1 beta depletion, supernatants of lung macrophages specifically block the binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1 in a dose-dependent manner. These results indicate that AM from both normal donors and patients produce a specific IL-1 inhibitor that may be of importance in protecting the alveolar environment from the deleterious effects of excessive IL-1 production.

Coculture of bladder urothelial and smooth muscle cells on small intestinal submucosa: potential applications for tissue engineering technology.

PURPOSE: Small intestinal submucosa is a xenogenic, acellular, collagen rich membrane with inherent growth factors that has previously been shown to promote in vivo bladder regeneration. We evaluate in vitro use of small intestinal submucosa to support the individual and combined growth of bladder urothelial cells and smooth muscle cells for potential use in tissue engineering techniques, and in vitro study of the cellular mechanisms involved in bladder regeneration. MATERIALS AND METHODS: Primary cultures of human bladder urothelial cells and smooth muscle cells were established using standard enzymatic digestion or explant techniques. Cultured cells were then seeded on small intestinal submucosa at a density of 1 x 105 cells per cm.2, incubated and harvested at 3, 7, 14 and 28 days. The 5 separate culture methods evaluated were urothelial cells seeded alone on the mucosal surface of small intestinal submucosa, smooth muscle cells seeded alone on the mucosal surface, layered coculture of smooth muscle cells seeded on the mucosal surface followed by urothelial cells 1 hour later, sandwich coculture of smooth muscle cells seeded on the serosal surface followed by seeding of urothelial cells on the mucosal surface 24 hours later, and mixed coculture of urothelial cells and smooth muscle cells mixed and seeded together on the mucosal surface. Following harvesting at the designated time points small intestinal submucosa cell constructs were formalin fixed and processed for routine histology including Masson trichrome staining. Specific cell growth characteristics were studied with particular attention to cell morphology, cell proliferation and layering, cell sorting, presence of a pseudostratified urothelium and matrix penetrance. To aid in the identification of smooth muscle cells and urothelial cells in the coculture groups, immunohistochemical analysis was performed with antibodies to alpha-smooth muscle actin and cytokeratins AE1/AE3. RESULTS: Progressive 3-dimensional growth of urothelial cells and smooth muscle cells occurred in vitro on small intestinal submucosa. When seeded alone urothelial cells and smooth muscle cells grew in several layers with minimal to no matrix penetration. In contrast, layered, mixed and sandwich coculture methods demonstrated significant enhancement of smooth muscle cell penetration of the membrane. The layered and sandwich coculture techniques resulted in organized cell sorting, formation of a well-defined pseudostratified urothelium and multilayered smooth muscle cells with enhanced matrix penetration. With the mixed coculture technique there was no evidence of cell sorting although matrix penetrance by the smooth muscle cells was evident. Immunohistochemical studies demonstrated that urothelial cells and smooth muscle cells maintain the expression of the phenotypic markers of differentiation alpha-smooth muscle actin and cytokeratins AE1/AE3. CONCLUSIONS: Small intestinal submucosa supports the 3-dimensional growth of human bladder cells in vitro. Successful combined growth of bladder cells on small intestinal submucosa with different seeding techniques has important future clinical implications with respect to tissue engineering technology. The results of our study demonstrate that there are important smooth muscle cell-epithelial cell interactions involved in determining the type of in vitro cell growth that occurs on small intestinal submucosa. Small intestinal submucosa is a valuable tool for in vitro study of the cell-cell and cell-matrix interactions that are involved in regeneration and various disease processes of the bladder.

Induction of tolerogenic lung CD4+ T cells by local treatment with a pSTAT-3 and pSTAT-5 inhibitor ameliorated experimental allergic asthma.

Signal transducer and activator of transcription (STAT)-3 inhibitors play an important role in regulating immune responses. Galiellalactone (GL) is a fungal secondary metabolite known to interfere with the binding of phosphorylated signal transducer and activator of transcription (pSTAT)-3 as well of pSTAT-6 dimers to their target DNA in vitro. Intra nasal delivery of 50 μg GL into the lung of naive Balb/c mice induced FoxP3 expression locally and IL-10 production and IL-12p40 in RNA expression in the airways in vivo. In a murine model of allergic asthma, GL significantly suppressed the cardinal features of asthma, such as airway hyperresponsiveness, eosinophilia and mucus production, after sensitization and subsequent challenge with ovalbumin (OVA). These changes resulted in induction of IL-12p70 and IL-10 production by lung CD11c(+) dendritic cells (DCs) accompanied by an increase of IL-3 receptor α chain and indoleamine-2,3-dioxygenase expression in these cells. Furthermore, GL inhibited IL-4 production in T-bet-deficient CD4(+) T cells and down-regulated the suppressor of cytokine signaling-3 (SOCS-3), also in the absence of STAT-3 in T cells, in the lung in a murine model of asthma. In addition, we found reduced amounts of pSTAT-5 in the lung of GL-treated mice that correlated with decreased release of IL-2 by lung OVA-specific CD4(+) T cells after treatment with GL in vitro also in the absence of T-bet. Thus, GL treatment in vivo and in vitro emerges as a novel therapeutic approach for allergic asthma by modulating lung DC phenotype and function resulting in a protective response via CD4(+)FoxP3(+) regulatory T cells locally.

Vitamin E content in fish oil emulsion does not prevent lipoperoxidative effects on human colorectal tumors.

OBJECTIVE: The anticancer action exerted by polyunsaturated fatty acid peroxidation may not be reproduced by commercially available lipid emulsions rich in vitamin E. Therefore, we evaluated the effects of fish oil (FO) emulsion containing α-tocopherol 0.19 g/L on human colorectal adenocarcinoma cells and tumors. METHODS: HT-29 cell growth, survival, apoptosis, and lipid peroxidation were analyzed after a 24-h incubation with FO 18 to 80 mg/L. Soybean oil (SO) emulsion was used as an isocaloric and isolipidic control. In vivo, nude mice bearing HT-29 tumors were sacrificed 7 d after an 11-d treatment with intravenous injections of FO or SO 0.2 g ∙ kg(-1) ∙ d(-1) FO or SO to evaluate tumor growth, necrosis, and lipid peroxidation. RESULTS: The FO inhibited cell viability and clonogenicity in a dose-dependent manner, whereas SO showed no significant effect compared with untreated controls. Lipid peroxidation and cell apoptosis after treatment with FO 45 mg/L were increased 2.0-fold (P < 0.01) and 1.6-fold (P = 0.04), respectively. In vivo, FO treatment did not significantly affect tumor growth. However, immunohistochemical analyses of tumor tissue sections showed a decrease of 0.6-fold (P < 0.01) in the cell proliferation marker Ki-67 and an increase of 2.3-fold (P = 0.03) in the necrotic area, whereas malondialdehyde and total peroxides were increased by 1.9-fold (P = 0.09) and 7.0-fold (P < 0.01), respectively, in tumors of FO-treated compared with untreated mice. CONCLUSION: These results suggest that FO but not SO has an antitumor effect that can be correlated with lipid peroxidation, despite its vitamin E content.

Human epidermal stem cell function is regulated by circadian oscillations.

Human skin copes with harmful environmental factors that are circadian in nature, yet how circadian rhythms modulate the function of human epidermal stem cells is mostly unknown. Here we show that in human epidermal stem cells and their differentiated counterparts, core clock genes peak in a successive and phased manner, establishing distinct temporal intervals during the 24 hr day period. Each of these successive clock waves is associated with a peak in the expression of subsets of transcripts that temporally segregate the predisposition of epidermal stem cells to respond to cues that regulate their proliferation or differentiation, such as TGFβ and calcium. Accordingly, circadian arrhythmia profoundly affects stem cell function in culture and in vivo. We hypothesize that this intricate mechanism ensures homeostasis by providing epidermal stem cells with environmentally relevant temporal functional cues during the course of the day and that its perturbation may contribute to aging and carcinogenesis.

CCL17 Promotes Intestinal Inflammation in Mice and Counteracts Regulatory T Cell-Mediated Protection From Colitis.

BACKGROUND & AIMS: Priming of T cells by dendritic cells (DCs) in the intestinal mucosa and associated lymphoid tissues helps maintain mucosal tolerance but also contributes to the development of chronic intestinal inflammation. Chemokines regulate the intestinal immune response and can contribute to pathogenesis of inflammatory bowel diseases. We investigated the role of the chemokine CCL17, which is expressed by conventional DCs in the intestine and is up-regulated during colitis. METHODS: Colitis was induced by administration of dextran sodium sulfate (DSS) to mice or transfer of T cells to lymphopenic mice. Colitis activity was monitored by body weight assessment, histologic scoring, and cytokine profile analysis. The direct effects of CCL17 on DCs and the indirect effects on differentiation of T helper (Th) cells were determined in vitro and ex vivo. RESULTS: Mice that lacked CCL17 (Ccl17(E/E) mice) were protected from induction of severe colitis by DSS or T-cell transfer. Colonic mucosa and mesenteric lymph nodes from Ccl17-deficient mice produced lower levels of proinflammatory cytokines. The population of Foxp3(+) regulatory T cells (Tregs) was expanded in Ccl17(E/E) mice and required for long-term protection from colitis. CCR4 expression by transferred T cells was not required for induction of colitis, but CCR4 expression by the recipients was required. CCL17 promoted Toll-like receptor-induced secretion of interleukin-12 and interleukin-23 by DCs in an autocrine manner, promoted differentiation of Th1 and Th17 cells, and reduced induction of Foxp3(+) Treg cells. CONCLUSIONS: The chemokine CCL17 is required for induction of intestinal inflammation in mice. CCL17 has an autocrine effect on DCs that promotes production of inflammatory cytokines and activation of Th1 and Th17 cells and reduces expansion of Treg cells.

EWS-FLI-1 modulates miRNA145 and SOX2 expression to initiate mesenchymal stem cell reprogramming toward Ewing sarcoma cancer stem cells

Introduction: Cancer stem cells (CSC) display plasticity and self renewal properties reminiscent of normal tissue stem cells but the events responsible for their emergence remain obscure. We have recently identified CSC in Ewing sarcoma family tumors (ESFT) and shown that they arise from mesenchymal stem cells from the bone marrow. Objective of the study: To analyze the mechanisms underlying cancer stem cell development in ESFT. Methods: Primary human mesenchymal stem cells (MSC) isolation from adult and pediatric bone marrow. Retroviral delivery of fusion protein (EWS-FLI1) to primary MSC, and transcriptional and phenotypical analysis. Results: We show that the EWS-FLI-1 fusion gene, associated wit 85-90% of ESFT and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2 and NANOG in human pediatric MSC (hpMSC) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSC expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWSFLI- 1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Conclusion: Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a cancer stem cell phenotype.

Regenerative Medicine at the Single Cell Level

Adult stem cells are instrumental for renewal, regeneration, and repair. Self-renewal and the capacity to generate a tissue for an extended period of time (theoretically a life time) are fundamental properties of adult stem cells that allow longterm tissue reconstruction from a single stem cell as experimentally demonstrated with the bone marrow and the skin. Moreover, human epidermal stem cells (holoclones) can be extensively expanded and manipulated in culture before they are transplanted. We have taken advantage of these unique capacities to demonstrate the feasibility of a single epidermal stem cell approach for ex vivo gene therapy using recessive dystrophic epidermolysis bullosa (RDEB) as a model system. We have demonstrated that is possible to reconstruct a functional epidermis and anchoring fibers from the progeny of a single RDEB epidermal stem cell transduced with a Col7a1 cDNA by means of a SIN retrovirus. Demonstrations of safe proviral insertion, absence of tumorogenicity and of dissemination of the transduced engrafted cells meet regulatory affairs safety requirements.

VP22 light controlled delivery of oligonucleotides to ocular cells in vitro and in vivo.

PURPOSE: To study VP22 light controlled delivery of antisense oligonucleotide (ODN) to ocular cells in vitro and in vivo. METHODS: The C-terminal half of VP22 was expressed in Escherichia coli, purified and mixed with 20 mer phosphorothioate oligonucleotides (ODNs) to form light sensitive complex particles (vectosomes). Uptake of vectosomes and light induced redistribution of ODNs in human choroid melanoma cells (OCM-1) and in human retinal pigment epithelial cells (ARPE-19) were studied by confocal and electron microscopy. The effect of vectosomes formed with an antisense ODN corresponding to the 3'-untranslated region of the human c-raf kinase gene on the viability and the proliferation of OCM-1 cells was assessed before and after illumination. Cells incubated with vectosomes formed with a mismatched ODN, a free antisense ODN or a free mismatched ODN served as controls. White light transscleral illumination was carried out 24 h after the intravitreal injection of vectosomes in rat eyes. The distribution of fluorescent vectosomes and free fluorescent ODN was evaluated on cryosections by fluorescence microscopy before, and 1 h after illumination. RESULTS: Overnight incubation of human OCM-1 and ARPE-19 cells with vectosomes lead to intracellular internalization of the vectosomes. When not illuminated, internalized vectosomes remained stable within the cell cytoplasm. Disruption of vectosomes and release of the complexed ODN was induced by illumination of the cultures with a cold white light or a laser beam. In vitro, up to 60% inhibition of OCM-1 cell proliferation was observed in illuminated cultures incubated with vectosomes formed with antisense c-raf ODN. No inhibitory effect on the OCM-1 cell proliferation was observed in the absence of illumination or when the cells are incubated with a free antisense c-raf ODN and illuminated. In vivo, 24 h after intravitreal injection, vectosomes were observed within the various retinal layers accumulating in the cytoplasm of RPE cells. Transscleral illumination of the injected eyes with a cold white light induced disruption of the vectosomes and a preferential localization of the "released" ODNs within the cell nuclei of the ganglion cell layer, the inner nuclear layer and the RPE cells. CONCLUSIONS: In vitro, VP22 light controlled delivery of ODNs to ocular cells nuclei was feasible using white light or laser illumination. In vivo, a single intravitreal injection of vectosomes, followed by transscleral illumination allowed for the delivery of free ODNs to retinal and RPE cells.

Nanoparticles for gene delivery to retinal pigment epithelial cells.

PURPOSE: To evaluate the safety and potential use of poly(lactic) acid (PLA) and poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) as vectors for gene transfer to RPE cells. METHODS: Experiments were conducted with primary bovine RPE cells and with the ARPE-19 human RPE cell line. Rhodamine loaded NPs were used to study factors influencing the internalization process by the various RPE cells: concentrations of NPs, duration of contact time, stage of cell culture and ambient temperature. The extent of NPs internalization was evaluated by fluorescence and phase microscopy. Potential NP toxicity was measured by the trypan blue exclusion dye test and the MTT method. Green fluorescent protein (GFP) plasmid or red nuclear fluorescent protein (RNFP) plasmid were sequestered in NPs. The ability ot these "loaded" NPs to generate gene transfection and protein expression in RPE cells was assessed both in vivo and in vitro by fluorescence and confocal microscopy. RESULTS: The extent of NP internalization in cultured cells increases with their concentration reaching a plateau at 1 mg/ml and a contact time of up to 6 h. Temperature and culture stage did not influence the in vitro internalization process. No toxic effects on RPE cells could be detected when these were incubated with up to 4 mg/ml of NPs. In human and bovine RPE cells incubated with GFP loaded NPs, cytoplasmic green fluorescence was observed in 14+/-1.65% of the cultured cells. Incubation with RNFP loaded NPs yielded a nuclear red fluorescence in 18.9+/-1.6% of the cells. These percentage levels of expression initially detected after 48 h of incubation remained unchanged during the following 8 additional days in culture. No significant differences in the extent of cytoplasm or nuclear fluorescence expression were observed between bovine or human RPE cultured cells. In vivo, a preferential RNFP expression within the RPE cell layer was detected after intra vitreous injection of RNFP plasmid loaded NPs. CONCLUSIONS: The ability of PLGA NPs to sequester plasmids, their nontoxic characteristics, and rapid internalization enables gene transfer and expression in RPE cells. These findings may be of potential use when designing future gene therapy strategies for ocular diseases of the posterior segment.

Carcinoembryonic antigen expression, antibody localisation and immunophotodetection of human colon cancer liver metastases in nude mice: a model for radioimmunotherapy.

Colorectal cancer frequently disseminates through the portal vein into the liver. In this study, outbred Swiss nude mice were adapted to facilitate the induction of liver metastases by a pre-grafting treatment with 6 Gy total body irradiation and i.v. injection of anti-asialo GM1 antibody. One day later, cultured LS 174T human colon cancer cells were injected into the surgically exposed spleen, which was resected 3 min later. In 48 of 65 mice, a few to several hundred liver metastases were macroscopically observed at dissection 3 to 4 weeks after transplantation. Ten of 10 mice, followed-up for survival, died with multiple large confluent liver metastases. By reducing the radiation dose to 4 or 0 Gy, or omitting the anti-asialo GM1 antibody injection, only 60%, 37% or 50% of mice, respectively, had visible metastases 3 weeks after transplantation. Carcinoembryonic antigen (CEA) measured in tumour extracts was in the mean 25.6 micrograms/g in liver metastases compared with 9.2 micrograms/g in s.c. tumours. Uptake of radiolabelled anti-CEA monoclonal antibody (MAb) in the metastases 12, 24 and 48 hr after injection gave a mean value of 39% of the injected dose per gram of tissue (ID/g). In comparison, MAb uptake in s.c. and intrasplenic tumours or lung metastases gave a mean percentage ID/g of 20, 18 and 15, respectively. Laser-induced fluorescence after injection of indocyanin-MAb conjugate allowed direct visual detection of small liver metastases, including some that were not visible under normal light. Preliminary results showed that mice, pre-treated with 4 Gy irradiation and the anti-asialo GM1 injection, were tolerant to radioimmunotherapy with a total dose of 500 muCi 131I labeled anti-CEA intact MAbs given in 3 injections.