Relationship between postpartum uterine involution and biomarkers of inflammation and oxidative stress in clinically healthy mares (Equus caballus).

To test the hypothesis that delayed/impaired uterine involution could be associated with oxinflammation, we studied the progression of the uterine involution in association with some biomarkers of inflammation and oxidative stress in clinically healthy mares (N = 26) during early postpartum. The examination of the reproductive tract was performed on Days 7 and 21 after foaling. Uterine involution was assessed considering: a) the increase of the gravid uterine horn diameter (GUHD) compared with diameter recorded before pregnancy during the previous breeding season; b) the level of endometrial edema (EE); c) the degree of accumulation of intrauterine fluid (IUFA); d) the status of the cervix (CS). Inflammation and oxidative stress were studied by measuring serum amyloid A (SAA), cortisol, DHEA, AOPP, protein carbonyl groups, malondialdheyde (MDA) and thiols in plasma on Days 7 and 21. By Day 21 after parturition, a significant improvement (P < 0.01) was observed for GUHD and EE; while IUFA increased in six animals. Plasma SAA and DHEA concentrations were higher when the clinical parameters indicated a lower degree of uterine involution. On Day 7, the cortisol/DHEA ratio was lower in animals with higher degree of EE. Plasma AOPP and MDA concentrations were significantly lower (P < 0.05) in animals with the lower GUHD. On Day 21, plasma MDA concentrations were significantly lower (P < 0.05) in animals with the lower IUFA. Our data suggest that a mild condition of inflammation and oxidative stress occur in mares with delayed/impaired uterine involution.

Myogenic differentiation of human rhabdomyosarcoma cells induced in vitro by antineoplastic drugs.

The effect of various antineoplastic drugs (1-beta-D-arabinofuranosylcytosine, 5-azacytidine, cisplatin, dactinomycin, epirubicin, vincristine, and the activated metabolite of cyclophosphamide, mafosfamide) on cell differentiation in vitro was investigated using a human alveolar rhabdomyosarcoma cell clone, RMZ-RC2. These cells are able to differentiate spontaneously from small mononuclear proliferating elements to terminal, extremely elongated multinuclear structures resembling myotubes; morphological differentiation is accompanied by the expression of myosins, in particular the embryonic isoform, which was used in this study as a specific marker of myogenic differentiation. The proportion of differentiated myosin-positive cells, which was around 10-15% in control cultures 10-15 days after seeding, was increased by some drug treatments up to 30-40%; the proportion of multinuclear elements was also increased. 1-beta-D-arabinofuranosylcytosine and 5-azacytidine were the most effective drugs, while dactinomycin had no effect; other molecules ranked in between. Since significant increments were usually observed after treatment with drug doses inhibiting cell growth, the kinetic behavior of the absolute number of myosin-positive cells or nuclei was analyzed to assess whether some effects could be due to a negative selection of proliferating, undifferentiated cells. This appeared to be the case for vincristine and epirubicin, while 1-beta-D-arabinofuranosylcytosine, 5-azacytidine, and, to a lesser degree, mafosfamide and cisplatin actually seemed to increase differentiation ability.

Interferon-mediated enhancement of metastasis. Are MHC antigens involved?

The relationship between major histocompatibility complex (MHC) antigens and metastasis was investigated on B16 melanoma variants. B16 cell lines express low amounts of murine MHC (H-2) antigens. A high expression can be induced in line B16-A by in vitro treatment with immune interferon (IFN-gamma) or by in vivo transplant in allogeneic mice. The increase of H-2 antigens correlated with an enhancement of lung colonization in young syngeneic mice. The higher metastatic capacity of B16-A cells with induced high levels of H-2 antigens was observed also in adult mice and in young mice pretreated with cyclophosphamide. These results were confirmed investigating the behaviour of a mutant B16 clone (B78H1) which was selectively resistant to the H-2-inducing action of IFN-gamma: lung colonization ability was not increased by IFN pretreatment. The study of variants derived from individual B16-A lung colonies revealed a wide range of H-2 levels. Variants with a low expression had a low colonization ability; one out of two variants with a high H-2 expression also was poorly colonizing. IFN-gamma-mediated H-2 expression appeared to act as an enhancer, rather than a determinant of B16 metastatic capacity.

Are colony-stimulating factor-producing cells facilitated in the metastatic process?

The relationship between production of Colony-Stimulating Factor (CSF) and metastasis has been investigated in the TS/A murine model. CSF production was determined in TS/A cell variants isolated through serial in vivo selection of lung metastatic nodules induced by intravenous or subcutaneous injection of tumor cells (artificial and spontaneous metastases, respectively). All the cell variants selected for high artificial metastatic ability produced higher amounts of GM-CSF in vitro and stronger haematological alterations in vivo than cells obtained by serial selection of spontaneous metastases. Our data suggest that the late, rather than the early, steps of the metastatic process could be enhanced by GM-CSF production.

Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum.

To identify bacterial traits related to adhesion ability in human bifidobacteria, 13 strains of Bifidobacterium longum isolated from human gastric juice and intestine were studied. Strains were tested for their capability to adhere to Caco-2 cells and classified as adhesive (Adh+) or non-adhesive (Adh-). Adh+ and Adh- strains were then investigated for their autoaggregation ability and surface hydrophobicity. Comparing the properties of Adh+ and Adh-, we observed that strains were able to adhere to cell monolayers if they autoaggregate and manifest a good degree of hydrophobicity as determined by microbial adhesion to hydrocarbons. These two traits could be used for preliminary screening to identify potentially adherent isolates.

Various effects on transposition activity and survival of Escherichia coli cells due to different ELF-MF signals.

Previous assays with weak sinusoidal magnetic fields (SMF) have shown that bacteria that had been exposed to a 50 Hz magnetic field (0.1-1 mT) gave colonies with significantly lower transposition activity as compared to sham-exposed bacteria. These experiments have now been extended by using a pulsed-square wave magnetic field (PMF) and, unexpectedly, it was found that bacteria exposed to PMF showed a higher transposition activity compared to the controls. The increase of the transposition activity was positively correlated with the intensity of the magnetic fields (linear dose-effect relation). This phenomenon was not affected by any bacterial cell proliferation, since no significant difference was observed in number and size of PMF-exposed and sham-exposed colonies. In addition, the cell viability of E. coli was significantly higher than that of the controls when exposed to SMF, and lower than that of the controls when exposed to PMF. Under our experimental conditions it was shown that exposure to PMF stimulates the transposition activity and reduces cell viability of bacteria, whereas exposure to SMF reduces the transposition mobility and enhances cell viability. These results suggest that the biological effects of magnetic fields may critically depend on the physical characteristics of the magnetic signal, in particular the wave shape.

Extremely low frequency magnetic fields affect transposition activity in Escherichia coli.

The aim of this study was to verify whether extremely low frequency (ELF) magnetic fields (MF) could affect transposition activity like some environmental stress factors such as heat shock or UV irradiation. Using an Escherichia coli Lac Z(-) strain transformed with a plasmid containing a Tn 10 derivative element expressing beta-galactosidase only after transposition, it was possible to determine the events of transposition evaluating the rate at which the colonies developed dark coloured papillae (Lac Z(+)). We found that those bacteria that had been exposed for a long time (58 h) to a 50 Hz low intensity MF (0.1-1 mT) gave colonies with significantly lower transposition activity compared to sham-exposed bacteria. Such reduction in transposition activity was positively correlated to the intensity of the MF, in a dose-effect manner. This phenomenon was not affected by bacterial cell proliferation, since no significant differences were observed in number, diameter and perimeter between sham-exposed and MF-exposed colonies.

Autoaggregation and adhesion ability in a Bifidobacterium suis strain.

On the basis of autoaggregation ability, two different phenotypes (Agg+ and Agg-) were selected from a strain (BSu895) of Bifidobacterium suis. The relationship between autoaggregation and adhesion of bacteria to intestinal tissue was investigated by observing the adhesivity of the two phenotypic variants to ileum and colon tissue pieces collected from six new-born piglets. The results suggest that there is a good relationship between autoaggregation and adhesion as variant Agg+ (autoaggregating) has a stronger adhesion ability than Agg- (non-autoaggregating).

Human rhabdomyosarcoma cells in nude mice as a model for metastasis and differentiation.

We investigated the ability of alveolar (RMZ-RC2) and of embryonal (RD) human rhabdomyosarcoma cell lines to grow and metastasize in nude mice. Both cell lines produced local tumors, but failed to give rise to spontaneous metastases. When RD cells were injected intravenously into nude mice pretreated with cyclophosphamide (in order to depress primarily natural killer activity), several large lung colonies were obtained. Renal localization was also observed. In vitro cultures obtained from tumors or secondaries retained the ability to differentiate in the myogenic pathway shown by the parental cell lines. Intravenous injection into nude mice could therefore constitute an interesting experimental system to study metastatic ability and differentiation properties of human rhabdomyosarcoma cells.

Growth and metastasis in allogeneic hosts. Lack of H-2-negative or somatic hybrid variant selection.

In vitro cultured B16 melanoma cells, which were previously found to have an impaired expression of H-2Kb and Db (as evaluated by antisera absorption assay), were used to study growth and metastasis in allogeneic mice in relation to H-2 expression. The possible emergence of somatic hybrids with host cells was also examined. B16-A cells grown subcutaneously in allogeneic BALB/c mice (H-2d) did not show a lack of H-2b expression, nor the acquirement of H-2d antigens was found. Spontaneous lung metastases were found in about 30% of BALB/c mice with progressing B16 tumor. Single lung metastases showed higher H-2b levels than cells from the respective tumors, and did not express host H-2 antigens. Tumor take was concomitant to the rise of an anti-H-2b humoral response: disease outcome was independent from the serum titer. B16-A cells injected intravenously in BALB/c mice gave rise to lung colonies; different colonies showed a wide range of H-2b antigens levels and no H-2d expression. Lung colonization capacity in normal allogeneic BALB/c mice was significantly higher than in syngeneic C57BL/6 mice. Both syngeneic and allogeneic mice, when pretreated with cyclophosphamide (CY) to reduce natural killer cell activity, showed a significant increase in lung colonization by B16-A cells. CY-treated C57BL/6 mice showed significantly higher numbers of lung colonies than CY-treated BALB/c mice. In conclusion, B16 growth and metastasis in the allogeneic environment do not seem to be determined by a selection of H-2-negative variants or by the emergence of somatic hybrids with host cells.

Tumorigenic and metastatic ability of SV40-transformed BALB/c cell lines and MHC antigen expression.

Tumorigenic and metastatic potential were studied in relation to class I MHC expression in four different SV40-transformed BALB/c cell lines. All the lines studied, tumorigenic or not, expressed both H-2Kd and Dd, so MHC antigens did not seem to be involved in the control of SV40-transformed cells' growth in vivo. Lung metastases were observed in all tumour-bearing mice. Cells cultured after in vivo passage, obtained either from tumour tissue or from individual lung metastases, still expressed similar levels of H-2d antigens, thus suggesting that tumour growth and metastasis do not occur through the selection of variants with altered MHC expression.

Colony-stimulating activity from the new metastatic TS/A cell line and its high- and low-metastatic clonal derivatives.

We investigated the presence of colony-stimulating factor (CSF) in supernatants obtained from TS/A, a new metastatic murine cell line, and from its high-and low-metastatic clonal derivatives (E and F clones, respectively). TS/A cells produced a CSF in vitro that induced proliferation and differentiation of murine monocytic and granulocytic progenitors in agar cultures. In TS/A-bearing mice remarkable splenomegaly, blood granulocytosis and thymus depletion were observed along with a stimulatory activity in serum and a strong proliferative activity both in spleen and in bone marrow populations. Conditioned media from E clones showed an in vitro colony-stimulating activity greater than those of F clones. Mice injected subcutaneously with cells of all clones studied showed granulocytosis, splenomegaly and thymus depletion, although to varying degrees. However, no direct correlation between granulocytosis-splenomegaly and the number of spontaneous lung metastases was observed.

RMZ: a new cell line from a human alveolar rhabdomyosarcoma. In vitro expression of embryonic myosin.

The RMZ cell line was established from a bone marrow metastasis of a human alveolar rhabdomyosarcoma. Since the beginning of the in vitro culture, RMZ cells showed differentiation-related morphological heterogeneity: actively proliferating polygonal or spindle-shaped cells were observed along with a few multinucleated myotube-like structures and giant cells, frequently multinucleated. All these cell types were still present after over 40 passages. A set of clonal derivatives has been obtained from the second in vitro subculture. All the clones showed the same morphological heterogeneity of the parental cells, but differed from one another in the degree of differentiation. Multinucleated myotube-like structures were strongly stained by anti-desmin antibody; most mononuclear cells were weakly stained. About 80% of RMZ and cloned cells were scored as desmin-positive in cytocentrifuged preparations. The expression of embryonic myosin heavy chain, specifically recognized by the monoclonal antibody BF-G6, was found in RMZ cell line and was localised in the myotube-like structures. Only a few giant cells and rare mononucleated polygonal cells were stained. The average proportion of BF-G6 positive cells in cytocentrifuged preparations was of about 6% of the total RMZ cells. In the two RMZ clones studied, the expression of embryonic myosin was correlated to the proportion of myotube-like structures: a BF-G6 positivity of 35% was found in the most differentiated one.

In vivo and in vitro production of haemopoietic colony-stimulating activity by murine cell lines of different origin: a frequent finding.

The production of colony-stimulating factor (CSF) by murine transformed cells was investigated in 10 cell lines derived from spontaneous or chemically induced tumours and from cells transformed by SV40 or Moloney-MSV; histologic types included carcinomas, sarcomas and melanoma. Nine of 10 supernatants contained CSF activity as judged by in vitro proliferation and differentiation of normal murine monocytic and granulocytic progenitors in agar cultures. Tumours induced with CSF-producing cells caused alterations of haemopoiesis which can include leukocytosis, granulocytosis and splenomegaly. Haemopoietic alterations were also evident in the absence of a local tumour in mice bearing large experimental lung metastases. Production of CSF seems to be a frequent finding among murine cell lines, and its biological and immunological consequences on host-tumour relationships should be taken into account.

Different metastatic aggressiveness by murine TS/A clones: ultrastructure, extracellular glycoproteins and type IV collagenolytic activity.

Cell ultrastructure, extracellular matrix glycoproteins, and type IV collagenolytic activity have been examined in four murine TS/A clones characterized by different metastatic aggressiveness. In vitro, highly metastatic clones (E) exhibited slightly less differentiated ultrastructure, and high type IV collagenolytic activity, while low metastatic clones (F) showed a more differentiated cytotype, with either high and low collagenolytic activity. Type IV collagen, laminin, and fibronectin were expressed without correlation with the metastatic efficiency; keratin was slightly more evident in E than in F cells. The morphological differences between E and F clones were less evident in the tumors produced by subcutaneous injection, and markedly reduced in the relative metastases composed only of the less differentiated cytotype; differences were also reduced in cells cultured on extracellular-matrix-coated flasks. The present study shows that no general correlation is observed between the studied parameters and metastatic aggressiveness.