Itshak Zusman

Melatonin and colon carcinogenesis: III. Effect of melatonin on proliferative activity and apoptosis in colon mucosa and colon tumors induced by 1,2-dimethylhydrazine in rats

Forty-eight two-month-old outbred female LIO rats were injected weekly with a single dose of 1,2-dimethylhydrazine (DMH; 21 mg/kg of body weight) administered s.c. for 15 consecutive weeks. From the day of the 1st injection of the carcinogen the part of rats were given five days a week during the night time (from 18.00 h to 08.00 h) melatonin dissolved in tap water, 20 mg/l. 10 rats were treated similarly with solvents and served as control. The experiment was terminated 6 months after the first injection of the carcinogen. Colon tumors (mainly adenocarcinomas) developed in a hundred percent of rats exposed both to DMH or to DMH plus melatonin. However, descending colon carcinomas were observed in 65 % of rats exposed to DMH plus melatonin against 100% in those exposed to DMH alone (p < 0.01). The multiplicity of colon tumors was also reduced in rats under the influence of melatonin. This effect is correlated with the significant inhibitory effect of the pineal hormone on mitotic index and with stimulating effect of melatonin on the relative number of apoptotic cells (TUNEL-method) in colon tumors. Long-term treatment with melatonin was followed also by the decrease in the area of lymphoid infiltrates in the colon mucosa of tumor-bearing rats.

Olive oil consumption during pregnancy and lactation in rats influences mammary cancer development in female offspring.

This study examined the effects of variety and quantity of dietary fat consumed by rats during pregnancy and lactation on female offsprings response to chemically induced mammary cancer. Groups of six female rats were fed diets containing 7% corn oil (7-CO), 15% CO (15-CO), 7% olive oil (7-OO), or 15% OO (15-OO) for 5 wk prior to, and during, pregnancy and lactation. Female offspring (n = 15 per group) were fed a 7-CO diet, and mammary cancer was induced with 7,12-dimethylbenz[a]anthracene (DMBA). Three months following cancer induction tumor incidence and size were recorded, and markers of apoptosis, serum estrogen concentrations, and hepatic phase II enzymes were measured. Tumor incidence was 47% in offspring born to mothers fed the 7-OO diet, rose to 67% in 7-CO and 15-OO offspring, and reached 86% in 15-CO. A trend toward smaller tumors was observed in the 7-OO group, and offspring of mothers fed high-fat diets had significantly more tumors. Estradiol levels at the end of lactation were significantly lower in mothers fed 7-OO but were similar in all groups of offspring. In tumor tissue, Bcl-2 expression was highest in the 15-CO offspring, and Bak expression was significantly higher in rats exposed to OO. A distinct trend toward increased caspase-3 expression (20 kDa) was observed in the 7-OO offspring, and both low-fat diets significantly elevated caspase activity. In healthy mammary tissue, rats exposed to low-fat diets had significantly higher caspase-3 (32-kDa) levels, and caspase-3 activity was significantly higher in the healthy tissue from both OO groups. Hepatic quinone reductase activity was significantly lower in offspring of mothers fed the low-fat diets. These results indicate that perinatal exposure to OO may have a protective effect against future development of mammary cancer in female offspring, whereas high-fat diets fed to pregnant and lactating rats, in particular CO, may be deleterious.

An immunohistochemical study of the secretory immune system in human fetal membranes and decidua of the first trimester of pregnancy.

Problem: We analyzed the presence and distribution of components of the secretory immune system (SIS) in human fetal membranes (amnion, yolk sac, chorion) and decidua from the first trimester of pregnancy.

Pathology of Lymphoid Organs in Low-Birth-Weight Human Fetuses Subjected to Antigen-Induced Influences: A Morphological and Morphometric Study

To evaluate the pathology of lymphoid organs in low-birth-weight (LBW) human fetuses obtained after premature birth, morphological and morphometric features were studied. The ages of fetuses ranged from 22 to 32 weeks and their weights from 400 to 1180 g. In fetuses at 22-23 weeks without antigenic effects, the lymphoid organs were seen to be well developed and their differentiation was similar to that of full-term fetuses. In older unaffected fetuses (up to 32 weeks), a significant increase in size of the lymphoid organs and a rise in the rate of the differentiation of lymphoid cells were observed. Fetuses exposed to antigen-related diseases underwent morphological changes in lymphoid organs presumably as a consequence of the primary fetal immune reaction. These changes were characterized by a high increase in the number of lymphoblasts and partly of macrophages in the spleen and lymph nodes. Reactive centers in spleen follicles and in lymph nodes, and plasmocytes in all the lymphoid organs, were absent. The main reaction to severe antigenic influences was decompensation of the lymphoid organs manifested morphologically by their devastation as a result of a decrease in the number of small lymphocytes and, in severe cases, of lymphoblasts and macrophages. Exposure of fetuses to antigen-related diseases thus appears to cause marked changes in the normal ontogenesis of lymphoid organs.

Secretory Component, J Chain, and Immunoglobulins in Human Embryos and Fetuses of the First Trimester of Pregnancy: Immunohistochemical Study

In our previous studies, we described the development of the secretory (mucosal) immune system (SIS) in human fetuses in the second trimester of pregnancy. In the present study, we examined the presence and distribution of components of this system in human embryos and early fetuses in the first trimester. An immunohistochemical study was performed on 17 embryos and 9 fetuses (4 to 12 wk of development) using antibodies against secretory component (SC), joining (J) chain, immunoglobulins (IgA, IgM, IgG), subsets of T and B lymphocytes, and macrophages. Cells positive for SC, J chain, and IgG were found in epithelial tissues from wk 4 of pregnancy. In the internal organs, such as the myocardium and endocardium, capillary endothelium, epithelium of the kidney tubules and some others, only J chain and immunoglobulins were seen. IgA was weakly reactive in tissues where SC and/or J chain were presented. IgM was very weak or absent. Among the cellular components of the SIS, only macrophages were seen in 4-wk-old embryos. CD3+ and CD20+ lymphocytes were found at wk 7 to 8. IgA- and IgM-positive lymphocytes appeared at the end of wk 9. The SIS is widespread in embryonic and early fetal periods and begins to function before the appearance of the common immune system in the developing organism. The first functional components of the SIS, such as IgG and IgA observed in this study, are most probably of maternal origin.

Lymphoid-Epithelial Secretory Immune System in Human Fetuses in the Second Trimester of Gestation

The development of the secretory immune system (SIS) in the respiratory, digestive, and urogenital tracts and other organs of fetuses in the second trimester of gestation is described. Tissues of all internal organs of human fetuses (n = 36) that had died between 13 and 25 weeks of gestation were studied immunohistochemically for the presence of secretory component (SC), J chain, IgA, IgM, IgG, macrophages, and different subsets of lymphocytes. We found protein elements of the SIS in fetuses during the entire second trimester in the epithelium of the digestive, respiratory, and urinary tracts; in hepatocytes; in the epithelium of the bile duct, renal tubules, and all the urinary tract; in the salivary glands, pancreas, and thyroid; in the epithelium of the Fallopian tubes and uterus; in the epididymis and the rete testes; in the skin; and in other organs. Immunocompetent cells, including IgA- and IgM-secreting cells, were located in these organs under the epithelium and sometimes between epithelial cells. In fetuses with acute infection, the number of immunocompetent cells was higher, reflecting a whole–immune system reaction, including the SIS. We conclude that the fetal SIS is a ramified, defensive immune system that is distributed throughout most organs of epithelial origin in second-trimester fetuses, and that it reacts against intrafetal infiltration by foreign antigens.

Assessment of the nucleolar organizer regions by automated image analysis in benign and malignant colonic tumours and adjacent tissues in rats

An automated computer image analysis technique was used to study the morphological parameters of argyrophilic nucleolar organizer regions (AgNORs) in chemically induced rat colonic tumours of different grades. Different nuclear parameters were of different diagnostic value. For distinguishing tumorous tissue from normal tissue all the parameters studied were capable of serving as diagnostic markers. Malignant processes could, however, be more reliably detected by means of the area of the nucleus, nuclear shape factor, area of AgNOR and ratio of AgNOR area to nuclear area. In macroscopically normal tissue adjacent to a tumour, the values of all the AgNOR parameters studied were similar to those in tumorous tissues. It can be concluded that the initial stages in tumorigenesis are accompanied by changes in all of the nucleus and AgNOR parameters, but as malignancy develops, only some of these parameters continue to change. Close correlations between nuclear parameters in malignant tissue suggest that for diagnostic purposes only nuclear and AgNOR areas should be used.

Immunoprotection of gonads and genital tracts in human embryos and fetuses: immunohistochemical study.

PROBLEM: The immune protection of genital organs in embryogenesis has not been sufficiently studied. The purpose of this study was to investigate the development of the secretory immune system (SIS) in the gonads and genital tracts of human embryos and fetuses.
 MATERIALS AND METHODS: Developing gonads at different stages and genital tracts from 18 embryos and 39 fetuses in the first to third trimester of gestation were analyzed for presence of different component of SIS: secretory component (SC), joining (J) chain, IgA, IgM, IgG, macrophages, and subsets of lymphocytes. The material was divided into two groups: cases not subjected to foreign antigenic effects (group I, n=31) and those under antigenic attack (chorioamnionitis, group II, n=26).
 RESULTS: In embryos and fetuses of group I, SC, J chain, and IgG were seen in the epithelium of mesonephric and paramesonephric ducts, proliferating coelomic epithelium, epithelium of the uterine tubes and uterus, epithelium of the vas deferens, epididymis, and rete testis. IgA and IgM appeared in 6‐week‐old embryos. J chain, IgA, IgM, and IgG, but not SC, were found in the primary oocytes and oogonia, spermatogonia, and interstitial cells. An abundance of macrophages was seen in 4‐week‐old embryos. T and B lymphocytes first appeared in 6–7‐week‐old embryos. In embryos and fetuses of group II, reactivity of immunoglobulins (Igs) decreased until they disappeared altogether.
 CONCLUSIONS: Components of SIS were seen in genital organs in 4–5‐week‐old embryos and were present during the whole intrauterine period. We suggest the presence of two forms of immune protection of fetal genital organs. One form contains SC, J chain, and Igs and is present in the genital tract epithelium. The second form contains only J chain and Igs and is present in germ cells of gonads. The loss of Igs in cases with chorioamnionitis reflects the functional participation of the SIS of genital organs in response to antigen attack.

Response of T and B lymphocytes in the spleen, lymph nodes and mammary tumors in rats treated with human soluble tumor-associated antigens and commercial human albumin

We showed previously that soluble tumor-associated antigens (sTAA) isolated from breast cancer patients could suppress chemically-induced tumorigenesis in rats in comparison to the effect of commercial human albumin (CHA). Herein we analyze the possible mechanism of those findings. The following groups of mammary tumor-bearing rats were used in the studies: i) control rats treated with saline; ii) rats treated with CHA; and iii) rats treated with human sTAA. Different zones of the spleen, regional lymph nodes and tumors and their cellular content (B and T cells) were analyzed using the methods of morphometry and immunohistochemistry. Treatment of tumor-bearing rats with CHA resulted in a significant decrease in the size of the germinal center of the follicles. The number of B lymphocytes in the mantle layer of the follicles, the marginal zone and red pulp decreased significantly. The number of CD8+ T cells also decreased in the marginal zone and red pulp, whereas the number of CD4+ T cells increased in the periarterial lymph sheath (PALS) and the red pulp. Reaction of the spleen to vaccination with sTAA manifested in a significant increase in the size of most areas of the white pulp and in the number of B lymphocytes. In lymph nodes from control rats or those treated with CHA, CD8+ lymphocytes mainly accumulated in the paracortical zone. In rats treated with sTAA, CD8+ lymphocytes accumulated also in the medulla. The number of CD4+ T cells in these rats sharply increased and accumulated mainly in the medulla around the vessels. The total number of lymphocytes was changed differently in different areas of tumors (peripheral vs. at depth). The number of CD8+ cells significantly increased at depth of tumors, and also the ratio in the number of these cells at depth of tumors compared to a periphery increased. No difference was found in response of lymph cells to different types of treatment. All findings indicated a strict antitumor effect of vaccination with the sTAA, which prevents the development of insufficiency of the immune system when an intensive immune reaction takes place.