Hepatic damage and oxidative stress induced by Griseofulvin in mice.

Erythropoietic Protoporphyria (EPP) is a disease associated with ferrochelatase deficiency, which produces accumulation of protoporphyrin IX (PROTO IX) in erythrocytes, liver and skin. In some cases, a severe hepatic failure and cholestasis was observed. Griseofulvin (Gris) develops an experimental EPP with hepatic manifestations in animals. The aim of this work was to further characterize this model studying its effect on different metabolisms in mice Gris feeding (0-2.5%, 7 and 14 days). PROTO IX accumulation in liver, blood and feces, induction of ALA-S activity, and a low rate of Holo/Apo tryptophan pyrrolase activity was produced, indicating a reduction of free heme pool. The progressive liver injury was reflected by the aspect and the enlargement of liver and the induction of hepatic damage. Liver redox balance was altered due to porphyrin high concentrations; as a consequence, the antioxidant defense system was disrupted. Heme oxygenase was also induced, however, at higher concentrations of antifungal, the free heme pool would be so depleted that this enzyme would not be necessary. In conclusion, our model of Protoporphyria produced liver alterations similar to those found in EPP patients.

Histologic aspects of concomitant resistance induced by nonimmunogenic murine tumors.

Concomitant resistance to a second tumor implant was induced in both conventional and nude BALB/c mice by two nonimmunogenic syngeneic tumors of spontaneous origin, an epidermoid carcinoma and a lymphoid leukemia. In the secondary tumor, which was significantly inhibited by concomitant resistance, histologic examination revealed the presence of well-preserved tumor cells without any sign of necrosis and without any host cell infiltration, contrasting with classical immunologic rejection. Tumor cell proliferation as evaluated by the number of mitoses per high-power field was significantly inhibited in the secondary tumor as compared with the corresponding controls. No effect of concomitant resistance could be detected on primary tumor growth.

Tumor necrosis can facilitate the appearance of metastases.

The non-metastatic murine mammary adenocarcinoma M3 and its metastatic variant MM3 were used to evaluate the role of intratumoral necrosis in cell detachment and metastasis. Accelular extracts from necrotic areas of both tumors increased in vitro cellular detachment from M3 but not from MM3 fragments. Furthermore, the in vivo inoculation of the necrotic extracts within non-metastatic M3 tumors gave rise to pulmonary metastases. Histological studies revealed in M3 a central necrosis limited by an uninterrupted peripheral ring of well preserved cells, while in MM3 necrotic and non-necrotic areas alternated. It is concluded that the distribution of necrosis within the primary tumor by facilitating cell detachment is, at least in part, responsible for the development of metastases.

Expression of gelatinase/type IV collagenase in tumor necrosis correlates with cell detachment and tumor invasion.

We have previously observed that acellular extracts from necrotic areas (NE) of the non-metastatic murine mammary adenocarcinoma M3, enhance in vitro cell detachment and spontaneous lung metastases. In the present study, using different proteinase inhibitors along with NE, only the calcium chelator EDTA could significantly abrogate the enhanced cell detachment from M3 produced by NE. The typical cleavage products of type IV collagenase were detected inside the tumor necrotic area, mainly in association with necrobiotic cells, as evaluated by Western blot analysis and immunohistochemical assays. Zymography revealed the presence of 72- and 92-kDa gelatinase/type IV collagenase in NE. Moreover, NE increased the in vitro invasive ability of cultured M3 cells. The use of specific antibodies against both 72- and 92-kDa type IV collagenases in the invasion assay showed that only the latter was able to revert the enhanced invasiveness to the baseline. It can be concluded that tumor necrosis is an important source of gelatinase/type IV collagenase, mainly in its 92 kDa form, and plays a major role in tumor invasion.

The time of tumor cell division and death depends on the site of growth.

We show here, for the first time, in two very different murine tumors, a mammary one (ectoderm) and a lung one (endoderm), that: tumors have day/night differences of spontaneous apoptosis additional to the well-known circadian rhythm of mitosis. The times of maximal and minimal mitosis and apoptosis changed for a tumor cell line when growing in different organs (as metastasis) or anatomical sites. Both tumor lines, have identical circadian curves when growing in a specific organ or anatomical site. The peaks of apoptosis match with the valleys of mitosis and vice versa.

Two different types of concomitant resistance induced by murine tumors: morphological aspects and intrinsic mechanisms.

Concomitant resistance (CR) is the phenomenon according to which a tumor-bearing host inhibits the growth of a secondary implant of the same tumor at a distant site. Confirming and extending previous results of our laboratory, histological studies have revealed that two temporally separate peaks of CR can be detected throughout tumor evolution. The first peak induced by immunogenic small tumors, in euthymic but not in nude mice, is associated with extensive necrosis of the secondary tumor implant and a profuse infiltration of polymorphonuclear granulocytes and mononuclear cells resulting in its final destruction; these features correspond to a typical immunological rejection. The second peak of CR induced by both immunogenic and non-immunogenic large tumors, in euthymic as well as in nude mice, is characterized by a dormant tumor stage with scarce or null mononuclear infiltration, associated with a significant reduction of tumor mitotic index and of the number of PCNA+ cells along with an increase in apoptosis and an arrest in S phase. In previous reports we suggested that a 1000 D serum fraction from mice bearing large tumors could be responsible for the induction of this dormant tumor stage. In this study tumor cells incubated in vitro with that serum factor mimicked the inhibition and cellular alterations observed in vivo in the secondary tumor inhibited by the second peak of CR. Moreover, the passive transfer of this factor by the intra-peritoneal (i.p.) route induced an in vivo inhibition of an i.p. tumor reproducing the image characteristic of the second peak of CR. This represents a direct proof that this serum factor can restrain tumor growth in vivo and that it is, most probably, the effector of the second peak of CR.

Inhibition of metastases by a serum factor associated to concomitant resistance induced by unrelated murine tumors.

Murine lung metatases growing undisturbed by the primary tumor were significantly inhibited by the concomitant resistance induced by a secondary subcutaneous implant of two unrelated tumors. Such inhibition was T-independent since it was also observed in nude mice; its full expression was dependent on the presence of the secondary tumor implant and it was exerted on both macroscopic and microscopic established metastases and not on the process of tumor cell dissemination from the primary tumor. Direct and indirect mechanisms seemed to be involved, the former affecting the metastatic cells per se by causing a decrease in proliferation and an increase in apoptosis while the latter affected neo-vascularization. These antitumor and antiangiogenic effects could be attributed to a serum factor induced by the unrelated tumors generating concomitant resistance. This factor proved to be heat, acid and alkaline resistant and dialysable; it was recovered in an HPLC column with maximum absorption at 215 and 266 nm; it was anionic at neutral pH, exhibiting free carboxil groups and one or more molecules of tyrosine, with a molecular weight between 870 and 1300 Dalton. Intravenous administration of this factor significantly inhibited lung metastases, decreasing mitosis and increasing apoptosis similar to that observed in the presence of the unrelated tumors.

[Antitumor concomitant immunity: a possible metastasis control mechanism]. / "Inmunidad" concomitante antitumoral: un posible mecanismo de control de las metástasis.

Concomitant immunity (CI) is defined as the lack or retardation or proliferation of a secondary tumor implant at a distant site; it has been given an immunological interpretation. Our experiments were designed to investigate CI in association with spontaneous tumors with or without metastases. In BALB/c mice, various syngeneic tumors, of undetectable immunogenicity, induced CI, preventing the development of a secondary implant of the same and occasionally of another tumor, indicating some degree of nonspecificity. This CI could also be observed in young and adult nude mice with high and low NK level, and in those treated with silica, discarding and participation of T lymphocytes, NK cells and macrophages, respectively. The lack of development of the secondary implant was not due to tumor rejection--contrarily to observations in allogeneic systems with immunogenic tumor--but to cytostasis. This "dormant tumor" state is observed together with the absence of host cell infiltration. The creation of a local inflammatory reaction abrogated CI, resulting in rapid tumor growth. On the other hand, an inflammatory reaction created by a foreign body inhibited the development of a tumor implant in the contralateral flank and tumor growth could be inhibited by piroxicam; furthermore, the tumor itself diminished the inflammatory reaction created by a foreign body at a distant site. Considering metastases as natural secondary implants, two mammary adenocarcinomas with a common origin were compared, one with 0 and the other with 100% metastatic incidence. The non-metastatic tumor induced stronger and earlier CI against both tumors and prevented the development of experimental and spontaneous metastases; moreover; its surgical extirpation led to the appearance of lung metastases.(ABSTRACT TRUNCATED AT 250 WORDS)

[Fine-needle aspiration puncture of abdominal masses using echographic monitoring. Cytological diagnosis and experience with biological markers]. / Puncion aspiración con aguja fina de masas abdominales bajo control ecográfico. Diagnóstico citológico y experiencia con marcadores biológicos.

43 fine needle aspiration biopsies of abdominal masses were performed under echographic guide. Cytologic diagnosis was done with routine techniques. Tumor, lineage and origin biologic markers were detected by immunocytochemical methods. Sensibility, specificity and accuracy of this procedure were evaluated. Advantages of the method are discussed.

Primary synchronous bilateral breast cancer: epidemiological approach.

Twenty-eight (1.69%) cases of primary synchronous bilateral breast cancer (PSBC) out of 1,654 new cases were studied. PSBC compared with unilateral cases had a significantly higher (p less than 0.001) first degree family history of breast cancer; high frequency of subareolar location; no predominance of lobular and non-invasive types; no significantly different percentage of pathological stage I presentation. As there is no complete agreement on what constitutes a PSBC, studies should be carried out to formulate a more precise definition of this entity.

Deferoxamine reduces tissue injury and lethality in LPS-treated mice.

We studied the effect of deferoxamine (DFX), an iron chelator, which can also act as a free radical scavenger, in an experimental murine model of sepsis. In vivo studies demonstrated that pretreatment of mice with DFX reduces tumor necrosis factor alpha (TNF-alpha) serum levels and increases the rate of survival of mice inoculated with lethal doses of lipopolysaccharide (LPS) or Escherichia coli O111:B4. By using the iron chelated form of DFX (ferrioxamine) the same results were obtained, suggesting that in this model, DFX could act as a free radical scavenger. On the other hand, DFX prevents mortality induced either by LPS or murine recombinant TNF-alpha in D(+)-galactosamine (GalN)-sensitized mice. These protective actions of DFX correlate with an attenuated tissue damage observed in lungs, livers and kidneys of LPS-treated animals and GalN-sensitized mice inoculated with TNF-alpha.

Adenomas of the colon: overexpression of p53 protein and risk factors.

BACKGROUND AND STUDY AIMS: p53 gene mutation and the ensuing overexpression of its protein is one of the steps related to colorectal carcinogenesis. This study analyzed the relationships between immunohistochemically detected p53 protein accumulation in colonic adenomas and morphological and clinical indicators of risk of malignant transformation and relapse. PATIENTS AND METHODS: A total of 100 endoscopically resected sporadic colonic adenomas (nonpolyposis) from 79 patients were retrospectively studied by using archival paraffin tissue blocks. p53 protein immunohistochemically detected was related to morphological adenoma risk factors (size, histological type and dysplasia) and in patients to neoplastic colonic pathology (NCP), such as previous adenomas/carcinomas or coexistent adenomas. RESULTS: There was a correlation between p53 expression and the grade of dysplasia but not with size or histological type in adenomas. NCP data was present in 52.5% of patients with p53 positive adenomas, and only in 25.6% of patients with p53 negative adenomas. This difference remains even in those with low-grade dysplastic lesions. When coexistent adenoma and previous adenoma/carcinoma data were analyzed separately, similar results were obtained. CONCLUSIONS: The presence of previous and/or coexistent NCP was more frequent in patients with p53 expressing adenomas, even in low-grade dysplastic lesions, which probably could be a high risk subpopulation. Its follow-up may be eventually reviewed.

Piroxicam treatment protects mice from lethal pulmonary challenge with Pseudomonas aeruginosa.

The effect of treatment with the nonsteroidal anti-inflammatory agent piroxicam on leukocyte migration to the lungs was investigated after aerosol administration of sublethal doses of Pseudomonas aeruginosa to mice. Piroxicam decreased, in a dose-related fashion, the polymorphonuclear leukocyte recruitment to, and the degree of perivascular and peribronchial infiltration in, the lungs. Piroxicam treatment also protected the animals in a dose-dependent manner from challenge with lethal doses of P. aeruginosa. The effect of piroxicam was not related to direct action of the drug on the microorganisms. Piroxicam treatment maintained the animal's pulmonary defenses against infection while diminishing inflammatory responses against P. aeruginosa, an occurrence decreasing the potential for tissue damage due to phagocytes migrating from circulation.

Demonstration of cytokeratins by immunoperoxidase staining in prostatic tissue.

The presence and distribution of cytokeratins (CK) have been investigated using an epidermal keratin antiserum in various dilutions and the PaP (peroxidase-antiperoxidase) and avidin-biotin-peroxidase (ABC) immunohistochemical methods. A total of 44 samples of prostatic tissue were divided into alcohol-fixed (22 cases) and formaldehyde-fixed (22 cases). Each group included 12 non-malignant lesions (hyperplasias and prostatitis) and 10 adenocarcinomas. The best results were achieved with the ABC method in alcohol-fixed tissues, while formaldehyde-fixed tissues gave poor staining despite the use of different enzymes to unmask antigenic determinants. With similar dilutions of the specific antiserum the PaP method gave less intense staining. Cytokeratins were detected in basal and columnar cells, in areas of transitional and squamous metaplasia and in normal transitional epithelium. Columnar cells showed strong staining in the supranuclear portion. Adenocarcinomas gave positive staining for cytokeratins varying from weak to strong. The intensity of staining showed no correlation with the degree of differentiation of the tumor. Different degrees of intensity were frequently observed within the same tumor. High dilutions of the specific antiserum (greater than 1/400) failed to stain carcinomas or stained them poorly, whereas they still stained normal or hyperplastic tissues. Gland-forming tumors showed a highly polarized labelling with the strongest staining in the luminal portion of the cell. The conclusion is that all epithelial prostatic tissues, benign and malignant, express cytokeratins.

Short communication. Role of agar beads in the pathogenicity of Pseudomonas aeruginosa in the rat respiratory tract.

Sterile agar beads plus Pseudomonas aeruginosa injected intratracheally produced local infection in rats, similar to that described for the injection of agar beads containing the same pathogen. It is suggested that it is not necessary for P. aeruginosa to be inside the beads to induce lung infection.

Plasma cell granuloma of the bladder: a case report.

We report a case of plasma cell granuloma of the bladder. Few cases of extrapulmonary localization have been described and none has been reported in the bladder. Immunocytochemical techniques identifying kappa and lambda immunoglobulin light chains confirmed the polyclonal nonneoplastic nature of the lesion.

Role of concomitant resistance in the development of murine lung metastases.

An attempt was made to explain the distinct lung metastatic patterns of 2 mammary adenocarcinomas with a common BALB/c origin: M3, which does not induce spontaneous metastases, and MM3 with an almost 100% incidence. No difference between the 2 tumors was detected with respect to host mononuclear cell content, degree of immunogenicity or lung-colony-forming ability. Conversely, there was a marked difference in the capacity to induce concomitant resistance: M3-bearing mice induced stronger and earlier resistance against i.v. challenge of both M3 and MM3 tumor cells than MM3-bearing mice; this resistance was expressed as lower number of lung metastases and lower tumor-cell proliferation in metastatic nodules. M3 was also able to control the development of spontaneous metastases: metastases developed in all M3-excised mice, compared with none in M3-bearing mice, while MM3-bearing mice also bearing a secondary M3 tumor developed fewer metastases than mice bearing MM3 only. This anti-metastatic effect does not appear to depend on classical immunological mechanisms since no difference could be detected between the 2 tumors in response to T cells, NK, macrophages or antibodies.

Association of human papillomavirus infection and vulvar intraepithelial neoplasia: a morphological and immunohistochemical study of 30 cases.

Thirty cases of vulvar intraepithelial neoplasia (VIN) were analyzed in order to determine the frequency of association with human papillomavirus (HPV) infection, and the relationship between this association and patient's age, extent of vulvar lesions, and coexistence with cervicovaginal neoplasia. The presence of condyloma or moderate to marked koilocytosis, now considered as morphological evidence of HPV infection, was observed in 66.6% of our cases. A search for HPV antigens, using the peroxidase-antiperoxidase (PAP) method, was performed in 13 selected cases, and positive staining was detected in 3 of them. The presence of HPV infection correlates with a mean age of 48.8 years, 50% of multicentricity of VIN and coexistence with cervical neoplasia in 30% of the cases, as opposed to a mean age of 55.5 years, 10% of multicentricity of VIN and absence of cervical neoplasia in patients without HPV infection. The demonstration of multiple foci of early stromal invasion in a 43-year-old woman, with multicentric VIN lesions associated with HPV infection, indicates that, even in the presence of such clinicopathological features, the risk of developing stromal invasion should be considered. Considerations are made in relation with the presence of HPV antigen in morphological normal epithelium adjacent to the lesion. Therapeutic implications were also investigated.