Evaluation of serum levels of cytokines and intercellular adhesion molecule-1 (ICAM-1) in astrocytic tumours.

Soluble form of intercellular adhesion molecules (sICAM) are increased in serum of many inflammatory diseases and tumours: the expression of such molecules is regulated by cytokines. In the present paper serum levels of interleukin-2 (IL-2), soluble interleukin-2 receptor (sIL-2R) and sICAM-1 were evaluated in patients with glioma compared with different tumours (lung and kidney carcinoma) in order to investigate the compromise of the immune system in these malignancies and to understand the host defence mechanisms. 14 cases of astrocytomas (WHO grade II, III), 20 cases of glioblastomas (GBL, WHO grade IV), 5 cases of lung carcinoma and 6 cases of kidney carcinoma were studied; the results were compared with 15 healthy controls. IL-2, sIL-2R, sICAM-1 concentrations were assessed by an enzyme-linked immunosorbent assay (ELISA) technique. The results were analyzed by Student's t test. Our findings showed that serum levels of IL-2 and sIL-2R were increased in all cancer patients; on the contrary, sICAM-1 serum levels were not significantly increased in GBL and astrocytoma patients. The increased values of IL-2 and sIL-2R are in agreement with a depression of the immune reactivity in patients with glioblastoma and astrocytoma, as reported in literature. On the contrary the levels of sICAM-1 are unchanged in astrocytic tumours while patients with kidney carcinoma presented the higher levels and an unfavourable prognosis.

Hemopoiesis in the liver of adult tumor-bearing mice.

In the liver of adult mice bearing an Ehrlich carcinoma on the leg, progressively hypoxic and displaying reactive hepatitis but not metastatic dissemination, extramedullary hemopoiesis was detected. Electron microscopy revealed mainly erythropoietic islands and scattered megakaryocytes in maturation stages up to the platelet-releasing phase. Erythropoietic cells expressed an embryonic-type of hemoglobin, which is more adequate to oxygenate hypoxic environments than the adult type. They were positive for the peroxidase reaction due to the presence of hemoglobin and could furthermore be visualized by the blue-excited red autofluorescence of protoporphyrin IX. Extramedullary hemopoiesis, one of the various examples of reactivation of fetal features in the liver associated with carcinogenesis, is supposed to be compensatory for the loss of blood cells induced by the tumor. Reviewing this process has the purpose of raising the question whether the fetal features are better adapted than adult ones to the metabolic and physiological characteristics of a tumor-influenced organism.

Increase in liver pigmentation during natural hibernation in some amphibians.

The amount/distribution of liver melanin in 3 amphibian species (Rana esculenta, Triturus a. apuanus, Triturus carnifex) was studied during 2 periods of the annual cycle (summer activity-winter hibernation) by light and electron microscopy, image analysis and microspectrofluorometry. The increase in liver pigmentation (melanin content) during winter appeared to be correlated with morphological and functional modifications in the hepatocytes, which at this period were characterised by a decrease in metabolic activity. These findings were interpreted according to the functional role (e.g. phagocytosis, cytotoxic substance inactivation) played by the pigment cell component in the general physiology of the heterothermic vertebrate liver and, in particular, in relation to a compensatory engagement of these cells against hepatocellular hypoactivity during the winter period.

Comparison of K(+)-p-nitrophenyl phosphatase activity in the urinary bladder of the frog Rana esculenta during hibernation and active life.

We have studied effects of hibernation on the frog urinary bladder, an organ involved in water and ion transepithelial transport and taking part in osmoregulation. We have demonstrated K(+)-p-nitrophenyl phosphatase activity (an enzyme involved in ion and water transport) both in active and hibernating frogs. Most of the reaction product deposition was found on basolateral membranes of granular cells of the urinary bladder epithelium during all seasons. Therefore, it seems likely that this organ, unlike organs studied previously (skin, kidney and lung), maintains its function in the osmoregulatory process during hibernation.

Tumor interstitial fluid: misconsidered component of the internal milieu of a solid tumor.

The tumor interstitial fluid (TIF) is a fluid phase present in the extracellular space of all tumors whose importance in oncology is seldom recognized. In order to stimulate other researchers to give it the due importance, a review of the available data (including our own) is provided. An hypothesis is presented for the genesis, fate and role of the TIF in the processes of invasion, growth and metastatization. Open questions regarding the TIF's role in tumor response to therapy are raised.

Ultrastructural changes of neutrophils following IL-2 treatment in vivo.

An ultrastructural analysis of peripheral blood cells of cancer patients following recombinant interleukin-2 (rIL-2) treatment at high doses (rIL-2, 18 x 10(6) I.U./m2/day) has been performed. Characteristic patterns of intense phagocytosis (neutrophils with large vaculoes containing electron-dense bodies) were observed in granulocytes of the treated patients with respect to the basal situation. These characteristics were particularly evident in the advanced stages of the therapy (24 and 48 h). The state of neutrophil activation was sometimes accompanied by the appearance of a close net of cell membranes, probably derived from lamellopodia.

Characterization of the metabolism of perinecrotic cells in solid tumors by enzyme histochemistry.

Hypoxic tumor cells resist most therapies and cause tumor regrowth when their environment improves. Identifying the adaptation strategies to hypoxia would help develop better tailored cancer therapies. Ehrlich carcinomas implanted on mice were analyzed histochemically for the following enzyme activities: lactate, succinate and glucose-6-phosphate dehydrogenases, dihydrofolate reductase, purine nucleoside phosphorylase, xanthine oxidoreductase, and acid phosphatase. With the exception of xanthine oxidoreductase, which was not active in tumor cells, and of succinate dehydrogenase the activity of which was not significatively altered, all other activities were much higher in perinecrotic cells with respect to cells close to blood vessels. These data suggest the integration of metabolic paths allowing purine and lipid biosyntheses. Degradation products from the necrosis are presumed to be employed as surrogates of blood-borne nutritive substances by cells distant from the vascularization.

Morphofunctional characterization of lymphokine activated killer (LAK) cells in vitro.

In order to characterize the cellular subsets of peripheral blood lymphocytes cultured with interleukin-2 (IL-2), a study in vitro was developed. Both conditioned medium from PHA-stimulated lymphocytes and recombinant IL-2 (rIL-2) were employed. The following aspects were examined: the morphological pattern of IL-2 stimulated lymphocytes; the ability of these cells to recognize, bind, attack and destroy tumoral lines; their cytochemical and immunological (CD) feature. The results suggest that, in our experimental conditions, these cells showed a characteristic "hand mirror shape", cytotoxic antitumor activity, lack of hydrolytic enzymes and CD3+, CD4+ phenotype.

Enzyme histochemical studies on tumor blood vessels.

The oxygenation, the growth rate and the metastatic potential of a solid tumor depend on its vascularization and, in particular, on angiogenesis; a therapeutic approach affecting angiogenesis has been suggested as an alternative to conventional ones. Especially the study of the metabolism in the cells of the vessel wall should be a useful prerequisite for this approach. In this connection, an enzyme histochemical study was performed to characterize the blood vessels in a solid tumor (Ehrlich carcinoma). The following enzymes were considered: (a) alkaline phosphatase, involved in the transcellular phosphate transport and in the response to inflammatory and growth promoting factors; (b) dihydrofolate reductase, involved in the metabolism of tetrahydrofolate (for the synthesis of nucleic acids and the metabolism of serine and glycine); (c) purine nucleoside phosphorylase, involved in the degradation of purines and, in particular, of extracellular ATP and ADP; (d) xanthine oxidoreductase, engaged in the same degradation path and leading to the formation of urate, a strong antioxidant. Various patterns of enzyme activities were observed in the vessel wall. In particular, thin linear capillaries (presumed to be host capillaries penetrating the tumor) were identified for the intense positivity of alkaline phosphatase, dihydrofolate reductase and purine nucleoside phosphorilase; tortuous capillaries with variable diameters (presumed to be induced by angiogenesis from the host vessels) were negative for the alkaline phosphatase and expressed an heterogeneous pattern for the dihydrofolate reductase. All the data suggest a different vessel behaviour concerning the response to cytokines and to inflammatory stimuli.

Immunohistochemistry of dihydrofolate reductase in methotrexate-sensitive and -resistant human cell lines by flow cytometry: a comparison with the cytochemical tetrazolium salt method.

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an enzyme involved in the metabolism of nucleic acids; it is also an important target for folate antagonists such as methotrexate (MTX). The distribution and expression of DHFR both in human HeLa BU-25 cell line and in methotrexate-resistant (MTX-R) variant, deriving from the human VA2-B cell line (having the DHFR gene amplified) was studied by tetrazolium salt method and by flow cytometric analysis. The immunohistochemical labelling of DHFR was achieved by using the streptavidinbiotinilated complex technique. DHFR activity was low in the human HeLa BU-25 cell line, while it was very high in the MTX-R cell line; the activity level increased with the increasing concentration of the MTX. The results obtained with cytochemical and immunohistochemical technique were compared. These findings showed that the hyperproduction of DHFR is strictly related with the cells having the DHFR gene amplified. Since MTX resistance is a common finding in the cells of patients with acute leukaemia, these studies may be extended to tumour-bearing patients at onset and following chemotherapy with methotrexate.

Stroma formation in Ehrlich carcinoma. I. Oedema phase. A mitosis burst as an index of physiological reoxygenation?

Tumor stroma induction has been shown closely to resemble wound repair process, both involving the replacement of a fibrin gel by vascularized connective tissue. In such a process the initial phase of hyperpermeability of blood vessels leads to diffuse oedema. It is here reported that cell loosening and a remarkably high mitotic burst were observed in Ehrlich carcinoma in regions in contact with the exudate, particularly at the perinecrotic (hypoxic) region. This suggests both an enhanced cell detachment from the tumour parenchyma and an improvement of the microenvironment, the exudate thus appearing as beneficial to the malignant cells contributing to the reoxygenation of formerly hypoxic regions. The temporary and well-localized concentration of mitoses in inner tumour areas has perhaps been disregarded by the pathologists engaged in mitosis counting for tumour grading. Peripheric and intraparenchymal concentrations of mast cells, lipid pools and platelets were seen in apparently key geometric disposition for controlling fibrin deposition and angiogenesis. Hypoxia is known to cause resistance to oxygen-dependent treatments and to facilitate cell detachment; normal fibroblasts respond and survive under hypoxic conditions by exhibiting features of the malignant phenotype. During reoxygenation, gene instability, cellular heterogeneity and increased drug resistance and metastatic spread have been reported. A reoxygenation process can also be deduced from several other histochemical and morphological patterns observed in this study. The findings here reported thus suggest that the oedema phase is a crucial phase regulating growth, invasion and dissemination of tumour cell populations, that should be specifically addressed therapeutically.

Histochemical probes for the detection of hypoxic tumour cells.

Hypoxia is thought to be a major cause of failure in cancer treatment. In this paper, we report methods transposable to clinical practice, for identifying hypoxic tumour cells. They consist of histochemical tests for revealing lactate dehydrogenase activity, endogenous lactate and accumulation of neutral fat. An ascites tumour (Yoshida hepatoma) and a solid tumour (Ehrlich carcinoma) were used as the experimental models. A gel film technique was used for visualizing lactate dehydrogenase and "nothing dehydrogenase" (or endogenous lactate). The fluorescent dyes Nile Red and Acridine Orange were used to demonstrate lipid accumulation and to visualize the tumour morphology, respectively. Tumour cells at the edge of areas of necrosis and at a distance of about 130 microns from a blood vessel were presumed to be hypoxic and showed the following features: 1) a dark blue granular pattern of lactate dehydrogenase (LDH) activity, ascribed to intense activity of the LDH5 and/or LDHk isoenzymes bound to membranous structures; 2) an intense granular positivity of "Nothing Dehydrogenase" due to high concentrations of endogenous lactate; 3) neutral lipid droplets emitting an intense yellow fluorescence in Nile Red-stained preparations; 4) a yellow cytoplasmic fluorescence in Acridine Orange-stained sections, attributable to a low cellular RNA content. Electron microscopy revealed moderately osmiophilic lipid globules in close association with damaged mitochondria. Better oxygenated cells showed: (a) a reddish-blue diffuse pattern of LDH, ascribed to moderately active soluble LDH isoenzymes containing H subunits; (b) almost no "Nothing Dehydrogenase" positivity; (c) no cytoplasmic lipid droplets; and (d) an intense orange-red fluorescence in the cytoplasm of Acridine Orange-stained specimens, due to high concentrations of cellular RNA. Nile Red fluorescence showed that the lipids of the solid tumour membranes were more hydrophobic than in the normal surrounding tissue. This suggests that there are abnormal domains of neutral lipids in the tumour cell membranes. In solid tumours, cells with the characteristics attributable to hypoxia were usually observed on the edge of necrosis of cuff-like formations. In very advanced growth stages, however, they were also seen surrounding (and occasionally clogging) blood vessels, or in tentacular formations coming from a necrosis border and polarized towards the vessels. Lipid-loaded cells were also seen in blood vessels distant from the tumour. These observations point towards a chemotactic process of hypoxic cells towards better environments.

A qualitative and quantitative cytochemical assay of dihydrofolate reductase in erythroid cells.

The distribution and intensity of dihydrofolate reductase (DHFR) cytochemically demonstrable was studied in erythroid cells. Cells of normal human bone marrow, of human erythroleukaemia (M6), and cells of the Friend (MEL) clone 745A murine erythroleukaemia (also after differentiation with dimethylsulphoxide, DMSO) were stained according to Gerzeli and de Piceis Polver (1969) technique; quantification of the reaction product was made using a Vickers M86 microdensitometer. The enzyme activity progressively decreased during the normal differentiation of the erythropoietic series while persisted at high levels in erythroleukaemia cells. It can be suggested that in the 1st case, the cytochemical pattern of dihydrofolate reductase may be a useful added tool for studying the erythroid differentiation. In the 2nd case, the increased level of this enzyme may be related to an amplification of the gene of DHFR in the malignant transformation.

Tetrahydrofolate dehydrogenase cytochemistry in acute lymphoblastic leukemia.

We studied the cytochemical distribution of tetrahydrofolate dehydrogenase (FH4D), an enzyme involved in nucleic acid metabolism and thus in cell proliferation and differentiation processes, in bone marrow blasts from 37 cases of acute lymphoblastic leukemia (ALL), of whom 23 were pediatric patients. 26 cases were analyzed at onset, 11 in relapse. The ALL cases were immunologically classified as T (10), common (20), B (3) and null (4). In each subgroup the majority of lymphoblasts were positive, with heterogeneous positivity patterns and variable degrees of enzyme activity. Most T lymphoblasts were characterized by focal localization of FH4D, whereas in common blasts reactivity - usually less strong - was either focally localized or scattered with several fine granules. Finally, many B and null blasts showed diffuse positivity. A quantitative evaluation of FH4D activity using cytophotometric technique (Vickers M86) demonstrated higher degrees of reactivity in leukemic blasts than in normal lymphocytes. Moreover, slightly different levels of reactivity were observed in relation to immunological phenotype, age and stage of the disease. Therefore we think that FH4D is a useful additional marker for ALL characterization.

Qualitative and quantitative study of dihydrofolate reductase in myelodysplastic syndromes.

Dihydrofolate reductase (FH2-R) was studied cytochemically in the bone marrow erythroblasts of 20 normal controls and 46 patients with myelodysplastic syndromes (MDSs) classified according to FAB, prior to therapy. The reaction product was quantified for the same samples with a Vickers M86 microdensitometer. The enzyme activity progressively decreased during the normal differentiation of the erythroid cells and persisted at high levels in MDS cells. The high level of FH2-R may be related to the malignant transformation of the cells, or to increased compensatory erythropoietic activity of ineffective erythropoiesis, or to both.

Leucine aminopeptidase activity in blood and hemopoietic organs of different vertebrates. A histochemical comparative study.

Leucine aminopeptidase distribution was examined in the cells of peripheral blood and hemopioetic organs of different Vertebrate classes using histoenzymatic methods. Various degrees of staining intensity were observed in leukocytes of the distinct Vertebrates: in particular, leukocytes of fresh water Osteichthyes and Mammals looked strongly positive, whereas leukocytes of Amphibians, Reptiles, and Birds were barely reactive without great differences among species. The observations should be related to enzyme molecular structure and to kinetics and substrate specificity.

Ultrastructural patterns of the alpha-naphthyl butyrate esterase activity in normal and leukaemic peripheral blood leucocytes.

The activity of alpha-naphthyl butyrate esterase was investigated at the ultrastructural level in normal human peripheral blood and in a few cases of hairy cell leukaemia, B-chronic lymphocytic leukaemia and acute monocytic leukaemia. A membrane reactivity was detected in most normal monocytes and lymphocytes. The activity in monocytes was very strong and was inhibited by NaF. It was NaF-resistant and less intense in lymphocytes. The reaction product was localized in the cytoplasm only in a small percentage of lymphocytes. In lymphocytes and monoblasts from pathological samples the pattern of reactivity was similar to that found in their normal counterparts, except for a lower intensity. The hairy cells showed a discrete distribution of the NaF-resistant reaction product on their cell surface. The different patterns of enzyme distribution are discussed critically.