Indoleamine 2,3 dioxygenase as a prognostic and follow-up marker in melanoma. A comparative study with LDH and S100B.

BACKGROUND: To date, lactate dehydrogenase (LDH) and S100B remain the most useful biomarkers for follow-up of melanoma patients. In recent years, indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme, has been proposed as a new potential tumour biomarker for melanoma. However, further studies are needed to confirm the usefulness of IDO expression as an independent prognostic factor. OBJECTIVE: To explore the potential association between serum IDO levels and melanoma stage at diagnosis and recurrence, and to compare the results to those obtained with LDH and S100B. In addition, we also investigated a possible cut off for IDO level as a prognostic factor for overall survival. METHODS: IDO, LDH and S100B levels were measured in serum samples of 186 patients in all melanoma stages at diagnosis and twice a year thereafter. A cut-off point for IDO levels was calculated using receiver operating characteristic curves to explore the association between these levels and the likelihood of lymphatic spread. Survival curves were estimated for patient groups stratified by IDO level (higher or lower than the cut off), using the Kaplan-Meier method. RESULTS: At diagnosis, serum IDO levels were significantly higher in stages IB, II, III and IV, whereas S100B levels were significantly higher in stages III and IV, and LDH levels were only higher in stage IV. In relapsed patients, significant increases were found in levels of all three markers. Finally, overall survival was significantly longer in patients with IDO levels below a cut off of 1.65 µM at diagnosis than in those with higher levels (91.3 vs. 71.0% at 36 months). CONCLUSION: In melanoma patients, serum IDO levels are significantly associated with disease stage, relapses and overall survival. These results indicate IDO could be a useful serum prognostic marker for melanoma.

Crystallinity assessment and in vitro cytotoxicity of polylactide scaffolds for biomedical applications.

Bioresorbable polylactides are one of the most important materials for tissue engineering applications. In this work we have prepared scaffolds based on the two optically pure stereoisomers: poly(L: -lactide) (PLLA) and poly(D: -lactide) (PDLA). The crystalline structure and morphology were evaluated by DSC, AFM and X-ray diffraction. PLLA and PDLA crystallized in the α form and the equimolar PLLA/PDLA blend, crystallized in the stereocomplex form, were analyzed by a proliferation assay in contact with mouse L-929 and human fibroblasts and neonatal keratinocytes for in vitro cytotoxicity evaluation. SEM analysis was conducted to determine the cell morphology, spreading and adhesion when in contact with the different polymer surfaces. The preserved proliferation rate showed in MTT tests and the high colonization on the surface of polylactides observed by SEM denote that PLLA, PDLA and the equimolar PLLA/PDLA are useful biodegradable materials in which the crystalline characteristics can be tuned for specific biomedical applications.

HR007: a family of biomaterials based on glycosaminoglycans for tissue repair.

Most new advances in tissue engineering (TE) focus on the creation of adequate microenvironments that may accelerate the repair processes of damaged tissues. Extracellular matrix (ECM) of Wharton's jelly (WJ) from umbilical cords is very rich in sulphated GAGs (sGAGs) and hyaluronic acid (HA), components which have special properties that could positively influence the regeneration of several types of tissue. Previously, we described the methodology for the extraction and purification of GAGs from WJ and, importantly, the separation of sGAGs and HA to develop various scaffolds for regenerative medicine. In this new study we hypothesized that the biomaterials obtained, called HR007s, would be excellent candidates for two different applications, chondral and dermal repair. First, we have confirmed that the GAGs obtained are biocompatible, as they do not cause cytotoxicity, haemolysis or an inflammatory response. Second, we have developed three-dimensional (3D) structures through the combination of different ratios of GAGs and their subsequent stabilization, which can be properly adapted to target tissues, cartilage or skin. Finally, we have combined these scaffolds with adipose mesenchymal stem cells (ASCs) or fibroblasts for application to chondral or dermal defects, respectively, with the goal of promoting fast reparative processes. The results show that HR007 scaffolds induce cell proliferation, enhance the expression of specific gene markers, increase the production of tissue ECM proteins and have chemotactic effects over the studied cells. In summary, the bioactive properties of HR007 scaffolds make them promising candidates for use in regenerative medicine, at least for chondral and dermal repair. Copyright © 2015 John Wiley & Sons, Ltd.

Synthesis and characterization of a biocompatible chitosan-based hydrogel cross-linked via 'click' chemistry for controlled drug release.

A chemically cross-linked chitosan-based hydrogel was successfully synthesized through Diels-Alder (DA) reaction and characterized. The final product was obtained after different steps; on the one hand, furan-modified chitosan (Cs-Fu) was synthesized by the reaction of furfural with the free amino groups of chitosan. On the other hand, highlighting the novelty of the present research, maleimide-functionalized chitosan (Cs-AMI) was prepared by the reaction of a maleimide-modified aminoacid with the amino groups of chitosan through amide coupling. The two complementary chitosan derivatives were cross-linked to the final hydrogel network. Both modification reactions were confirmed by FTIR and 1H NMR, obtaining a degree of substitution (DS) of 31% and 26% for Cs-Fu and Cs-AMI, respectively. The as-designed hydrogel was analyzed in terms of microstructure, swelling capacity and rheological behaviour. The hydrogel showed pH-sensitivity, biocompatibility and inhibitory bacterial activity, promising features for biomedical applications, particularly for targeted-drug delivery.

Selective implantation and growth in rats and mice of experimental liver metastasis in acinar zone one.

The functional zonation of liver tissue provides a framework for studying the implantation and growth of metastatic colonies within given zones of the hepatic acini. A very exact method for calibrating the position of metastatic foci in the hepatic acini was made possible by using the succinate-dehydrogenase reaction, which reveals the functional differences of the acinar zones. In mouse livers, metastasis was induced by intrasplenic injection of both high and low metastatic-capacity B16 melanoma and Lewis lung carcinoma cells. In rats, liver metastasis resulting from rhabdomyosarcoma was studied by injecting these cells into the s.c. tissue. All cases of metastasis occurred in hepatic acinar zone 1, and no significant differences were detected resulting from the type of tumor, its metastatic potential, or the procedure used for obtaining the metastasis. In subsequent experiments, metastasis was induced after first altering the zonal distribution of the hepatic extracellular matrix; distribution of the sinusoidal macrophages; and the sinusoidal diameter. However, even under these conditions, metastasis continues to occur exclusively in hepatic acinar zone 1. Thus, metastatic predilection for hepatic acinar zone 1 cannot be explained solely in terms of hemodynamic causes or the influence of extracellular matrix. Although it may still turn out that these elements play some secondary role in the phenomenon, our research points to the sinusoidal endothelial cells as a factor directly responsible for metastatic predilection for zone 1.

Generation of tunable glycosaminoglycan hydrogels to mimic extracellular matrices.

Biomaterials and, especially, scaffolds may function as temporary extracellular matrix (ECM), mimicking in vivo environmental structures and facilitating cell growth and tissue regeneration. ECM is composed mostly of glycosaminoglycans (GAGs) and proteins, the ratio of GAGs, hyaluronic acid (HA):sulphated GAGs (sGAGs) being characteristic of each type of tissue. Umbilical cord (UC) and particularly Wharton's jelly (WJ) have been proposed as good sources for obtaining GAGs. In this work, we present a novel methodology for the extraction, purification and separation of GAGs from UC obtained from two different species, human and pig. The new methodology is based on enzymatic digestion of WJ, precipitation of GAGs with organic solvents, purification steps and chromatographic separation of GAGs using ion exchange columns. This novel process allows highly purified HA and sGAGs to be obtained from human and pig WJ. The composition of sGAGs and molecular weight of HA were very similar in the two species and GAGs are haemocompatible and non-cytotoxic. Finally, these new biomaterials have significant bioactive properties, increasing proliferation rates of two cell lines, human adipose mesenchymal stem cells (ASCs) and fibroblasts. In summary, the separation of HA and sGAGs, linked to the improvement in the GAG quantification method described in this paper, opens new avenues for the formulation of natural biomaterials with various ratios of GAGs, mimicking tissue matrix for different tissue-engineering applications. Copyright © 2014 John Wiley & Sons, Ltd.

Effects of all-trans retinoic acid on tumor recurrence and metastasis.

OBJECTIVE: All-trans-retinoic acid (ATRA) promotes cell differentiation. We have studied its effect on the local recurrence and metastatic spreading of an experimental rhabdomyosarcoma in rats. DESIGN: syngenic rhabdomyosarcoma cells (S4MH) were inoculated s.c. in male WAG/RijCrl rats. After 25 days tumors were excised and a 40% hepatectomy was performed for all animals. Ten days later the rats were sacrificed and a thorough necropsy was performed. The animals were randomly allocated to receive daily doses of ATRA (5 mg/kg, i.p.) or its solvent (Clinoleic/ethanol 90/10), starting three days before surgery until the end of the experiment. RESULTS: ATRA reduced the incidence of local recurrence from 70 to 33% (p < 0.05), but the tumor size was not altered (1.8 vs. 2.0 cc). Regarding inguinal metastasis, there was a six-fold decrease (0.2 vs. 1.2 cc; p < 0.05) in mean tumor volume, although the rate of this proliferation increased sharply (86 vs. 29%; p < 0.05) for treated animals. The volume of the retroperitoneal tumor masses also decreased with ATRA (0.7 vs. 5.1 cc; p < 0.05), but the difference in rate was not significant (71 vs. 67%). Lung metastases, which were present in 100% of control animals, were found in only 33% of treated rats, while the mean number of metastatic foci dropped from 26.7 to 5.7 (p < 0.05). CONCLUSION: Protocols including retinoid administration prior to and following primary tumor excision could help in controlling both recurrence and metastatic progression in surgically treated rhabdomyosarcoma.

Interleukin-2 increases intracellular glutathione levels and reverses the growth inhibiting effects of cyclophosphamide on B16 melanoma cells.

Glutathione (GSH) plays an essential role in the metabolism of melanoma. As changes in intracellular GSH content can modify the processes of cell proliferation and detoxification, this could determine the therapeutic response to some cancer treatment strategies. The purpose of this study was to test the effects of treatment with interleukin-2 (IL-2), alone and in combination with cyclophosphamide (CY), on survival of mice bearing B16 melanoma liver metastases, and to determine the influence of these therapeutic agents on the GSH metabolism of B16 cells. In the in vivo test system, B16 melanoma liver metastases were induced in C57BL/6 mice which were subsequently treated with IL-2, CY and CY plus IL-2. Survival time was used to determine the response to treatment. In the in vitro system, we evaluated the effects of IL-2, acrolein (an active metabolite of CY responsible for GSH depletion) and acrolein plus IL-2 on GSH levels and proliferation of B16 melanoma cells. Results indicated that, in vivo, all treatments increased mouse survival times with respect to control mice. However, the addition of IL-2 to CY therapy decreased survival time compared with treatment with CY alone. In vitro, whereas acrolein produced a GSH depletion and inhibited B16 cell proliferation, IL-2 increased GSH content and cell proliferation rate compared with untreated cells. Moreover, addition of IL-2 to cells preincubated with acrolein increased GSH levels and proliferation with respect to acrolein alone. In summary, the data suggest that GSH plays a critical role in the growth-promoting effects of IL-2 on B16F10 melanoma cells and in the antagonistic effect of IL-2 on CY inhibitory activity on these tumor cells.

Effects of L-2-oxothiazolidine-4-carboxylate on the cytotoxic activity and toxicity of cyclophosphamide in mice bearing B16F10 melanoma liver metastases.

Glutathione (GSH) is the major non-protein thiol in cells that plays a critical role against damage from electrophilic agents such as alkylating drugs. Selective therapeutic GSH elevation in normal but not in tumour cells has been suggested as a means of protecting host tissues against more intense doses of chemotherapy. The present study investigated the response of B16 melanoma to treatment with the cysteine pro-drug L-2-oxothiazolidine-4-carboxylate (OTZ), alone and in combination with cyclophosphamide (CY). We found that OTZ decreased the GSH levels and proliferation rate of B16 melanoma cells in vitro, sensitizing them to the cytotoxic action of the activated metabolite of CY, acrolein (AC). In contrast to OTZ, the cysteine deliverer N-acetylcysteine (NAC) enhanced B16 melanoma cell proliferation by increasing GSH levels, and markedly decreased the sensitivity of these tumour cells to AC. In vivo studies showed the antitumoral activity of OTZ in B16 melanoma liver metastasis-induced mice, increasing their life span. We also observed that, whereas with CY treatment the GSH levels in peripheral blood mononuclear cells (PBMCs) were reduced and a dose-dependent leukopenia was produced, OTZ significantly increased PBMC GSH content, reducing toxicity and enhancing the survival of mice bearing established melanoma liver metastases treated with lethal dose CY. These results suggest a critical role for OTZ in protecting against alkylator agent-induced immunosuppression, which may allow the dose escalation of these cytostatic drugs to improve their therapeutic benefit in the treatment of malignant melanoma.

In vitro and in vivo comparison between the effects of treatment with adenosine triphosphate and treatment with buthionine sulfoximine on chemosensitization and tumour growth of B16 melanoma.

In this study we compare the effects of treatment with external sodium adenosine 5'-triphosphate (ATP) with the effects of L-buthionine-SR-sulfoximine (BSO) on B16 melanoma growth and on the modulation of the cytotoxic antimelanoma activity of cyclophosphamide (CY). We evaluated the in vitro effects of treatment with ATP or BSO on intracellular glutathione (GSH) levels, mitochondrial membrane potential (delta psi(m)) and the proliferation rate of the B16F10 melanoma cell line. Compared with untreated cells, delta psi(m) and GSH levels were already significantly decreased (25% and 57% reduction, respectively) after the first hour of incubation in culture cells exposed to 3 mM ATP. After 24 and 48 h a major reduction was observed in delta psi(m) (nearly 30%). GSH levels were also maximally depleted at 24 h (approximately 75%) and partially recovered (up to 37% of levels of control) after ATP was removed from the medium. At 24 and 48 h, the proliferation rate was decreased 1.4- and 1.7-fold, respectively, compared with control cells. Treatment with 50 microM BSO produced a time-dependent decrease in GSH levels (0.5, 21, 48 and 97.3% reduction at 1, 4, 8 and 24 h, respectively), but up to 54% of the levels of control cells was recovered after BSO was removed from the medium. In contrast to ATP, neither delta psi(m) nor proliferation rate was significantly modified in the first 24 h with BSO treatment. At 48 h, delta psi(m) was reduced by nearly 27%, and cell proliferation decreased 1.2-fold compared with controls. When the in vitro cytotoxic effect of low dose acrolein (an active metabolite of CY) in combination with BSO or ATP was analysed, a synergistic effect was found between BSO and acrolein, with a dose modification factor (DMF) of 1.98, but the antiproliferative effects of acrolein plus ATP were only approximately additive (DMF = 1.05). In addition, in in vivo studies differential effects were found between ATP and BSO. Specifically, whereas BSO alone significantly increased the survival time of mice bearing B16 melanoma liver metastases, and enhanced the cytotoxic effect of CY on this tumour model, no therapeutic benefits could be observed with ATP treatment, either alone or in combination with diethyl maleate (a GSH-depleting agent) and CY. In conclusion, our findings show that in our experimental system, both extracellular ATP and BSO have growth-inhibitory properties against B16 melanoma in vitro. In vivo, however, only BSO produces a chemosensitizing effect, whereas ATP has not proved useful as a biological modifier of chemotherapy.

L-2-Oxothiazolidine-4-carboxylate reverses the tumour growth-promoting effect of interleukin-2 and improves the anti-tumour efficacy of biochemotherapy in mice bearing B16 melanoma liver metastases.

The efficacy of sequential chemoimmunotherapy involving interleukin-2 (IL2) in metastatic melanoma is limited, in part, by the severe toxicity associated with most therapeutic regimens. Glutathione (GSH), the most prevalent intracellular non-protein thiol, plays an important role in protecting against cellular injury caused by various anticancer agents. GSH is also involved in the IL2-induced proliferative activity of immune system cells and some melanoma cells expressing IL2 receptors, such as B16 melanoma cells. The present study investigated the effect of selective manipulation of GSH using the cysteine prodrug l-2-oxothiazolidine-4-carboxylate (OTZ) on the response of B16 melanoma to sequential biochemotherapy with cyclophosphamide (CY) and IL2. We found that OTZ, by depressing GSH levels, abrogates the in vitro growth-promoting effects of IL2 on B16 melanoma cells. The combination of OTZ plus IL2 in vivo also showed antitumour activity in mice bearing B16 melanoma liver metastases, significantly increasing their life span. Schedule dependency between both compounds was found; OTZ given intermittently in combination with daily IL2 administration was found to be the best therapeutic schedule. We also observed that whereas IL2 or OTZ alone added to CY resulted in a lower or non-significant improvement in the life span of the mice compared with tolerated doses of CY alone, the addition of both OTZ and IL2 to CY produced a significantly greater increase in survival than CY alone, and markedly protected mice against CY-induced toxicity, which allowed the administration of otherwise lethal doses of CY, with the CY activity/toxicity ratio being increased by four-fold.

Tumor-host interaction in non-random metastatic pattern distribution.

In the clinical evolution of malign tumors, prognosis depends on whether metastasis develops or not. Biologically speaking, the formation of metastasis implies the existence of tumor cells capable of successfully performing all the steps in the metastatic process: local invasion, lymphatic or hematogenous dissemination, arrest in the microvascular bed of an organ, extravasation and growth of a secondary colony. Clinical observations have demonstrated that for each primary tumor there is a colonization pattern determined by the characteristics of the microvascular endothelium and the functional environment of the target organ. Moreover, the formation of metastasis depends on at least two additional factors: a) tumor cell-tumor cell and tumor cell-host cell relations modulated by intercellular contact and/or soluble paracrine or autocrine growth factors; b) the antitumor efficiency of the immune system, mediated primarily by the action of NK/LAK cells, macrophages and cytolytic T-lymphocytes, whose activity is in turn regulated by a complex of cytokines, including interferons, tumor necrosis factors and interleukins. In this work, we first review certain aspects of tumor biology that are specifically involved in tumor cell-host cell interactions determining non-random metastatic pattern distribution, and then review the implication of certain cytokines in the regulation of tumor proliferation.

[Prognostic value of nuclear DNA of 106 colorectal tumors, determined with microspectrocytophotometry]. / Valor pronóstico del ADN nuclear de 106 tumores colorrectales, medido por microespectrocitofotometría.

OBJECTIVE: The aim of this study was to look for correlations between nuclear DNA content of colo-rectal tumors, and such clinical parameters as age, sex, location, CEA, histological grade and Duke's stages. EXPERIMENTAL DESIGN: A prospective study is carried out on surgical patients, subjected to standard criteria of radicality. Nuclear DNA content was quantified in tumoral cells by microcytophotometric techniques. PATIENTS: 106 patients with colo-rectal cancer. Patients with colonic perforation, other concomitant neoplasia, non-curative surgery or receiving adjuvant therapies were excluded from the study. Five patients died during the postoperative period and one was lost. RESULTS: Histological grade: 28% G1, 35% G2 and 37% G3. Dukes': 8% A, 40% B, 32% C and 20% D. DNA quantification has rendered 45% as euploid and 55% as aneuploid. There was no statistical correlation between ploidy and location, age, sex or CEA. However, there is a clear preponderance of euploid tumors in G1 (23 vs. 5), while the aneuploid tumors double the euploid ones (24 and 25 vs. 12 and 12) in G2 and G3. A similar result was found comparing ploidy and Dukes: euploid tumors reach 77% both in stages A and B, while they drop to 24% and 14% in stages C and D. It has also been found that euploid tumors show a longer period of survival free of recurrence. CONCLUSIONS: Evidence has been found supporting a prognostic value for tumoral DNA quantification in colo-rectal cancer. A longer follow-up is required to study absolute survival of the patients.

[Development of an experimental model for the study of hepatic metastasis]. / Desarrollo de un modelo experimental para el estudio de metástasis hepáticas.

An experimental model for the induction of hepatic metastasis by means of the subcutaneous injection of rhabdomyosarcoma cultured cells (S4MH) is described. The growth of the primary tumor and the dissemination process (local, ganglionar and hematogenous) are studied. The particular ability of the tumor to produce liver metastasis is assessed. The microscopic foci may be found in all of the specimens on day 25th post-injection. The maximum metastatic load is achieved by day 35 th. In contrast, the ganglionar and pulmonary metastasis were only found on the last step of the process.

A study of the mechanical properties and cytocompatibility of lactide and caprolactone based scaffolds filled with inorganic bioactive particles.

The mechanical properties of highly porous (90% porosity) poly(l-lactide) (PLLA), poly(ε-caprolactone) (PCL) and poly(l-lactide/ε-caprolactone) (PLCL) were investigated. Young's modulus of non-porous PLLA, PCL and PLCL dropped from 2263.4, 183.7 and 5.7 MPa to 16.8, 1.0 and 1.0 MPa, respectively, for their ~90% porous counterparts. Elongation at break of PCL decreased noticeably with porosity fraction while PLCL maintained a highly elastomeric character and strain recovery capacity even in the presence of pores. Inorganic bioactive particles (hydroxyapatite or bioglass) were added to confer bioactivity to the aforementioned synthetic bioresorbable polymers, and their effect on the mechanical properties was also investigated. Addition of 15 vol.% of inorganic bioactive particles increased the Young's modulus of highly porous PLLA from 16.2 to ~30 MPa. On the contrary, the difference between Young's modulus of filled and unfilled PCL and PLCL scaffolds was not statistically significant. Finally, an in vitro study of the cytocompatibility and adhesion of adipose derived stem cells (ADSCs) was conducted. The observed viability and excellent adhesion of these cells to both porous and non-porous templates indicate that the employed materials can be good candidates for application in tissue engineering.

Removal of N-glycans from cell surface proteins induces apoptosis by reducing intracellular glutathione levels in the rhabdomyosarcoma cell line S4MH.

Expression of determined Asn-bound glycans (N-glycans) in cell surface glycoproteins regulates different processes in tumour cell biology. Specific patterns of N-glycosylation are displayed by highly metastatic cells and it has been shown that inhibition of N-glycan processing restrains cell proliferation and induces cell death via apoptosis. However, the mechanisms by which different N-glycosylation states may regulate cell viability and growth are not understood. Since malignant cells express high levels of intracellular glutathione (GSH) and a reduction of intracellular GSH induces cell death via apoptosis, we investigated whether GSH was involved in the induction of apoptosis by removal of cell surface N-glycans. We found that removal of N-glycans from cell surface proteins by treating the rhabdomyosarcoma cell line S4MH with tunicamycin or N-glycosidase resulted in a reduction in intracellular GSH content and cell death via apoptosis. Moreover, GSH depletion caused by the specific inhibitor of GSH synthesis BSO induced apoptosis in S4MH cells. This data indicates that adequate N-glycosylation of cell surface glycoproteins is required for maintenance of intracellular GSH levels that are necessary for cell survival and proliferation.

Coincident implantation, growth and interaction sites within the liver of cancer and reactive hematopoietic cells.

We have examined the anatomical-functional sites within mouse liver where phenylhydrazine (PHZ)-induced hematopoietic foci, and M5076 reticulum cell sarcoma, B16F10 melanoma and Lewis lung-carcinoma cells specifically develop as colonies after intrasplenic injection. Cancer foci occurred predominantly in the 2.4 to 4.0 segment of the sinusoidal pathway, corresponding to hepatic acinar zone I. No significant differences were detected between different types of tumor, including their different tendencies to spontaneously metastasize liver, or as a result of the different procedures used for obtaining foci or metastases. In addition, PHZ-treatment of mice previously injected with tumor cells, resulted in double colonization of the liver tissue by both hematopoietic and cancer cells, predominantly in zone I. This spatial coincidence indicates that non-cancer-specific mechanisms operate in zone I, either promoting implantation and/or growth of cell colonies or, alternatively, inhibiting these processes in the region surrounding the central vein (Rappaport zone 3). Our observations failed to reveal mutual displacement of cancer or hematopoietic foci by potential competition for development sites in zone I. Enumeration and diameter measurements of cancer foci in PHZ-treated animals showed that the presence of hepatic hematopoietic foci coincided with a significant increase in the hepatic metastasis volume. However, the fact that no significant differences in pulmonary metastases occurred in both the PHZ-treated and control mice given tail-vein injection of cancer cells, and that PHZ reduces cancer cell proliferation in vitro, reveal evidence of local interactions with hematopoietic foci which promote growth of cancer foci in liver.

Estimating anatomical-functional position coordinates in liver tissue.

Hepatocyte enzyme activity was demonstrated by examining adult C57BL/6 mouse liver cryostat sections under a succinate dehydrogenase (SDH) histochemical reaction, and quantified by microspectrophotometry and microdensitometry. The hepatocyte SDH activity gradient along the path between the portal veins (PV) and efferent terminal hepatic venules (THV) was analyzed by measuring the concentration of the chromophore precipitated in 10 consecutive hepatic parenchymal domains located along imaginary lines drawn across the entire PV-to-THV distance. The profiles of intensity or of normalized relative optical density obtained on a high number of lines were correlated with distance values along the PV-to-THV pathway, enabling us to establish a general mathematical function relating SDH activity (chromophore concentration) to position values on a scale of 0 to 10 corresponding to the theoretical PV-to-THV distance. The equation can be used to interpolate the SDH activity surrounding any intrahepatic object located between the PV and the THV, thus making it possible to calculate the object's anatomical-functional position coordinates in the liver acinus. To demonstrate how this method is used, we have calibrated the intrahepatic position of hemopoietic foci induced in the liver tissue of adult mice treated with phenylhydrazine (PHZ), and show that these foci are located on coordinate 3.31 (maximum range 1.25-4.86) of the sinusoidal domain-that is, on the borderline between Rappaport's acinar zones 1 and 2.

Functional variations in liver tissue during the implantation process of metastatic tumour cells.

We have examined several properties of sinusoidal cells in the unaffected tissue of micrometastasis-containing livers. Tumour cells from either B16 melanoma (B16F10) or Lewis lung carcinoma (LLC) were injected intrasplenically in syngeneic mice and sacrificed on the 7th day. Light and scanning electron microscopy (SEM) showed tumour cells in hepatic veins and sinusoids in close contact with endothelial walls and macrophages. Following quantitative analysis of SEM images from sinusoidal walls it was found that endothelial fenestrae from B16F10 or LLC-colonized livers were diffusely reduced both in size and density/microns 2 throughout the sinusoid wall, although especially affected zone 3 segments. Following the intrasplenic injection of 1 microns fluorescent latex particles 1 h prior to sacrifice of the mice a significant reduction of the latex particle uptake by sinusoidal cells was detected in B16F10-colonized livers (27% of controls) which was in contrast to the significant increase in LLC-colonized mice (180% of controls). Despite the focal character of the tumour cell implantation process, hepatic sinusoidal cells reacted diffusely to metastatic cells. However, over liver acini, endothelial cell changes were mainly expressed in zone 3 while phagocytic properties mainly varied in zone 1 and depending on the tumour type. Although the significance of these sinusoidal changes on metastatic development is unclear, data suggests that "soil" conditions in the liver are different before and after being metastasized by tumour cells.