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In-vivo single cell clonal analysis in the adult mouse kidney has previously shown lineage-restricted clonal proliferation within varying nephron segments as a mechanism responsible for cell replacement and local regeneration. To analyze ex-vivo clonal growth, we now preformed limiting dilution to generate genuine clonal cultures from one single human renal epithelial cell, which can give rise to up to 3.4 * 10 6 cells, and analyzed their characteristics using transcriptomics. A comparison between clonal cultures revealed restriction to either proximal or distal kidney sub-lineages with distinct cellular and molecular characteristics; rapidly amplifying de-differentiated clones and a stably proliferating cuboidal epithelial-appearing clones, respectively. Furthermore, each showed distinct molecular features including cell-cycle, epithelial-mesenchymal transition, oxidative phosphorylation, BMP signaling pathway and cell surface markers. In addition, analysis of clonal versus bulk cultures show early clones to be more quiescent, with elevated expression of renal developmental genes and overall reduction in renal identity markers, but with an overlapping expression of nephron segment identifiers and multiple identity. Thus, ex-vivo clonal growth mimics the in-vivo situation displaying lineage-restricted precursor characteristics of mature renal cells. These data suggest that for reconstruction of varying renal lineages with human adult kidney based organoid technology and kidney regeneration ex-vivo, use of multiple heterogeneous precursors is warranted.

Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

Identification of tissue-specific renal stem/progenitor cells with nephrogenic potential is a critical step in developing cell-based therapies for renal disease. In the human kidney, stem/progenitor cells are induced into the nephrogenic pathway to form nephrons until the 34 week of gestation, and no equivalent cell types can be traced in the adult kidney. Human nephron progenitor cells (hNPCs) have yet to be isolated. Here we show that growth of human foetal kidneys in serum-free defined conditions and prospective isolation of NCAM1(+) cells selects for nephron lineage that includes the SIX2-positive cap mesenchyme cells identifying a mitotically active population with in vitro clonogenic and stem/progenitor properties. After transplantation in the chick embryo, these cells-but not differentiated counterparts-efficiently formed various nephron tubule types. hNPCs engrafted and integrated in diseased murine kidneys and treatment of renal failure in the 5/6 nephrectomy kidney injury model had beneficial effects on renal function halting disease progression. These findings constitute the first definition of an intrinsic nephron precursor population, with major potential for cell-based therapeutic strategies and modelling of kidney disease., (© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.)

In the human fetal kidney (HFK) self-renewing stem cells residing in the metanephric mesenchyme (MM)/blastema are induced to form all cell types of the nephron till 34(th) week of gestation. Definition of useful markers is crucial for the identification of HFK stem cells. Because wilms' tumor, a pediatric renal cancer, initiates from retention of renal stem cells, we hypothesized that surface antigens previously up-regulated in microarrays of both HFK and blastema-enriched stem-like wilms' tumor xenografts (NCAM, ACVRIIB, DLK1/PREF, GPR39, FZD7, FZD2, NTRK2) are likely to be relevant markers. Comprehensive profiling of these putative and of additional stem cell markers (CD34, CD133, c-Kit, CD90, CD105, CD24) in mid-gestation HFK was performed using immunostaining and FACS in conjunction with EpCAM, an epithelial surface marker that is absent from the MM and increases along nephron differentiation and hence can be separated into negative, dim or bright fractions. No marker was specifically localized to the MM. Nevertheless, FZD7 and NTRK2 were preferentially localized to the MM and emerging tubules (<10% of HFK cells) and were mostly present within the EpCAM(neg) and EpCAM(dim) fractions, indicating putative stem/progenitor markers. In contrast, single markers such as CD24 and CD133 as well as double-positive CD24(+)CD133(+) cells comprise >50% of HFK cells and predominantly co-express EpCAM(bright), indicating they are mostly markers of differentiation. Furthermore, localization of NCAM exclusively in the MM and in its nephron progenitor derivatives but also in stroma and the expression pattern of significantly elevated renal stem/progenitor genes Six2, Wt1, Cited1, and Sall1 in NCAM(+)EpCAM(-) and to a lesser extent in NCAM(+)EpCAM(+) fractions confirmed regional identity of cells and assisted us in pinpointing the presence of subpopulations that are putative MM-derived progenitor cells (NCAM(+)EpCAM(+)FZD7(+)), MM stem cells (NCAM(+)EpCAM(-)FZD7(+)) or both (NCAM(+)FZD7(+)). These results and concepts provide a framework for developing cell selection strategies for human renal cell-based therapies.

Problem: While relying on previous publications, our aim was to examine the morphologic changes, induced in early rat embryos by intra-uterine exposure to the low-molecular weight fraction of boiled human serum containing antiphospholipid antibodies (APLA) that had been obtained from women with antiphospholipid syndrome (APS)., Method of Study: Human APLA-positive sera were pooled, boiled, centrifuged and separated by ultrafiltration. The molecular weight fraction lower than 30 kDa was used for the experiments. One hundred and fifty microlitres was injected into one uterine horn of 12 pregnant rats, 5 or 6 days after fertilization, while similarly prepared normal human serum or saline were injected into the contralateral horn. The rats were subsequently sacrificed. Serial sections, obtained from all uterine horns, were stained histologically and immunohistochemically. Normal embryos developed in the control uterine horns, while embryos in the experimental horns were destroyed rapidly., Results: Signs of apoptosis appeared 2 hr following the injection, and 4 hr later all the embryonic cells were apoptotically destroyed. There was only partial damage to cytotrophoblasts and intermediate trophoblasts., Conclusion: These findings support the existence of a novel factor in the APLA-positive serum, causing a detrimental effect to the conceptus, without any relation to the antiphospholipid antibodies.

Problem: Morphologic changes in the placental barrier in spontaneous early abortions under the maternal-embryonic immune conflict, and the role of maternal immunoglobulins (Igs) in these changes., Materials and Methods: We examined chorionic villi and other tissues obtained from 54 aborts between weeks 3.5 and 8 of pregnancy. Material was divided into two groups. Group 1 (control) contained 15 medically recommended and spontaneous early aborts with no signs of inflammations or pathologic immune processes. Group 2 contained 39 spontaneous early aborts with acute chorionic villitis. Immunohistochemical and morphometric methods were used to study the Igs, different types of immunocompetent cells, and apoptosis-related components of the placental barrier., Results: Acute villitis was found to be characterized by the destruction of all components of the chorionic villi, thrombovasculitis with apoptosis of the endothelium of capillaries and erythroblasts, mucous swelling of the basal membrane, and coagulation of the blood proteins. Due to destruction of the capillaries, the number of avasculate villi increased, and the average number of capillaries per villus decreased. The extremely high number of phagolysosomes with IgG and IgA in the villous monocytes in the group 2 indicates an increase in the phagocytic activity of monocytes against maternal Igs and may reflect the presence of mother-embryo immune conflict. Apoptosis of monocytes and a high number of promonocytes were seen accompanied by a high concentration of p53 protein. A large disturbance in the trophoblast occurred with disappearance of bcl-2 and the appearance of Fas ligand., Conclusion: Massive destruction of maternal Igs in embryonic monocytes and acute villitis in the placental barrier are manifested during the mother-embryo immune conflict, and this may be one of the reasons of spontaneous early abortions.

Catecholamines are important regulators of homeostasis, yet their functions in hematopoiesis are poorly understood. Here we report that immature human CD34+ cells dynamically expressed dopamine and beta2-adrenergic receptors, with higher expression in the primitive CD34+CD38(lo) population. The myeloid cytokines G-CSF and GM-CSF upregulated neuronal receptor expression on immature CD34+ cells. Treatment with neurotransmitters increased the motility, proliferation and colony formation of human progenitor cells, correlating with increased polarity, expression of the metalloproteinase MT1-MMP and activity of the metalloproteinase MMP-2. Treatment with catecholamines enhanced human CD34+ cell engraftment of NOD-SCID mice through Wnt signaling activation and increased cell mobilization and bone marrow Sca-1+c-Kit+Lin- cell numbers. Our results identify new functions for neurotransmitters and myeloid cytokines in the direct regulation of human and mouse progenitor cell migration and development.

Two-year-old mice of the long-living transgenic mice of the alphaMUPA strain were previously found to show higher tumor resistance than the their initial wild-type (WT) strain (Tirosh, 2003). To better understand the mechanism underlying the differences in tumorigenesis rates between the two mouse lines, the rate of tumorigenesis and survival effects were studied in alphaMUPA mice and parental WT mice exposed to dimethylbenz(a)anthracene (DMBA). Each animal received three intragastric feedings of DMBA, each one week apart, at doses of 2, 1, and 1 mg dissolved in 0.2 ml corn oil; thus, the total amount of the carcinogen was 4 mg/mouse. Control mice received corn oil. The alphaMUPA mice exhibited distinctly higher survival rates in experimental chemically-induced tumorigenesis compared to their WT counterparts: 93% vs. 67%, p =2.7. The rate of tumorigenesis differed between the mouse lines (yield was 1.5 and 2.1), owing to a distinct tendency toward decreased tumor frequency in the skin and forestomach in the alphaMUPA mice. The experimental duration was also significantly higher for transgenic mice: 35.9 +/- 1.2 weeks compared to 30.5 +/- 1.3 weeks in WT mice, p <0.01. The lungs, forestomach and skin were target organs for the carcinogenic effect of DMBA. Our observations suggest that aging promotes the rate of spontaneous and induced tumorigenesis.

The potential preventive effect of the synthetic pineal peptide Epitalon (Ala-Glu-Asp-Gly) on spontaneous tumorigenesis in mice was studied. One-year-old female C3H/He mice were kept for 6.5 months under standard conditions. Epitalon was injected at a dose of 0.1 microg, 5 times a week. Long-term exposure to Epitalon in small doses did not show any toxic effect. Treatment with Epitalon decreased the number of tumor-bearing mice with malignant tumors and prevented the development of metastases. Spontaneous tumors of the reproductive organs (mammary glands and ovaries) were predominant in both groups of mice (control and experimental). The mammary gland tumors were different variants of invasive ductal carcinomas. In the ovaries, granulosa-cell tumors were found. Tumors were in the minority in other organs and had benign characteristics. In control mice, metastases were found in 3 out of 9 tumor-bearing mice, all of them being from tumors of the reproductive organs. Treatment with Epitalon slowed down the development of metastases from spontaneous tumors, and no metastases were found in the experimental mice. These data highlight the antimetastatic effect of Epitalon as part of its oncostatic properties.

We analyzed the morphological changes accompanying the development of cancer in the mouse forestomach. The main aim of the study was to evaluate whether cancer in this area of the stomach arises de novo or undergoes a series of precancerous changes. Tumors were induced by the 1,2-dimethylbenz(a)antracene (DMBA) at a total dose of 4 mg/mouse. The suspected areas of the stomach were studied morphologically in 79 mice. Benign tumors (squamous-cell papillomas) and malignant tumors (squamous-cell carcinomas) were found in 40 mice. Tumors arose in all cases together with differential changes in the forestomach epithelium. These changes were seen as irregular diffuse hyperplasia or focal proliferation, with or without differential signs of dysplasia. Destruction of the basal epithelial membrane indicated transformation of the process into malignant invasive carcinoma. Thus, in chemically induced cancer of the forestomach, squamous-cell carcinoma develops as the final stage of morphologically recognizable precancerous changes in the epithelial layer. De novo formation of such tumors in the forestomach was not observed.

In our previous studies, we showed that soluble tumor-associated antigens (sTAA) of 66 kDa and 51 kDa have distinct tumor-preventive effects on chemically induced mammary cancer in rats and are able to repair the damage caused by tumorigenesis in its early stages. In the present study, we investigated whether these proteins can prevent the development of chemically induced tumors in mice. The study was performed on C3H/He mice which have the ability to develop many spontaneous tumors with age. Forty-four, 6-week-old mice were exposed twice at a 2-week interval to the carcinogen 9,10-dimethyl-1,2-benz(alpha)anthracene (DMBA), at a dose of 2 mg/mouse administered intragastrically. Two months later, the mice were divided into two groups. One group received sterile saline twice a week at a dose of 0.2 ml/mouse, intraperitoneally (i.p.). The other group received sTAA twice a week at a dose of about 10 microl in 0.2 ml of sterile saline/mouse, i.p. Periodically, all mice were checked for the presence of tumors. The experiment was terminated at week 35. Vaccination with sTAA increased the time of involvement of mice in the experiment, prevented the tumorigenic effect of DMBA, and inhibited further development of existing tumors.

Previously, we showed that the 66 and 51 kDa soluble tumor-associated antigens (sTAAs) have distinct suppressive effects on chemically induced mammary cancer in rats, both alone and in combination with the hormone-related anticancer drug tamoxifen. Here, we describe the effects of both sTAA and tamoxifen on the histological structure of ovaries in mammary tumor-bearing 30- to 34-week-old rats. Central ovary sections were pooled, the number of the healthy and degenerated follicles were counted, and the size of the corpora lutea was estimated. In follicular development primordial, primary, preantral and antral stages were recognized. Only healthy follicles with visible nuclei were counted. Follicular degeneration was estimated as the number of atretic follicles with follicular remnants. Treatment with tamoxifen alone or in combination with sTAA significantly increased the number of primordial follicles and atretic follicles in the ovaries, and promoted the formation of small follicular cysts. Total area of the corpora lutea decreased. sTAA participated in this process by increasing apoptosis in degenerated follicles.

We studied the effect of ascending infections of the birth canal on the transport of maternal immunoglobulins (Igs) through the placental barrier in humans. The study was performed on 41 human placentas obtained from embryos (n=21) and fetuses (n=20) who had died from different causes, including those connected with ascending infections of the birth canal, and seven placentas obtained after normal delivery at term. Different morphological and immunohistochemical methods were used. The transfer of Igs through the placental barrier is a complex process that involves tissues (trophoblast, stroma of the trophoblastic villi, and capillaries), cells (monocytes and erythroblasts) and molecular components (at least six types of transfer receptors and biologically active components). We found that the intensification of transfer of different types of maternal Igs (IgG, IgA, IgM) is accompanied by certain morphological and functional changes in the placental barrier. In normal development without infection, the transfer of IgG is steady and the process most intensive, while the transfer of IgA was evaluated in 75% of the cases, and of IgM in only 10%. Inflammation of the birth canal was accompanied by an increase in the transport of IgG in early embryogenesis, which was maintained throughout intrauterine development. In cases with moderate infection, transfer of IgG and IgA was found in all cases studied, while transfer of IgM was seen in 45% of the cases. In cases with massive infection, transfer of all three types of Igs was seen, the most intensive being of IgG and the least of IgM. Ascending infection of the birth canal changes dramatically the transport of Igs through the placenta and can be dangerous and even fatal for the embryo or fetus.

The role of protein components of the secretory immune system (SIS), such as the polymeric immunoglobulin receptor/secretory component (pIgR/SC), immunoglobulins (Igs) and joining (J) chain, in human intrauterine development was reviewed. These components are already present in 3.5- to 4-week-old embryos, and found in all tissues and organs of epithelial origin. The SIS is made up of two parts: the SIS of mucous membranes and their derivatives (mucosal or secretory immune system), and the SIS of barrier structures (barrier immune system). During organogenesis, SC disappears from the cells of organs that lose their exocrine Ig-secretion function, such as the hypophysis, pancreatic islands and adrenal glands. In cells and tissues of mesenchymal origin, SC is absent from the start, i.e. during their initial development. As examples of the barrier immune system, blood-tissue and tissue-tissue barriers, such as the chorion of the placenta, the epithelium of the choroid plexuses in the brain, as well as other barrier structures to Ig transfer were considered. Besides the SC and J chain, Fc receptors, cellular and tissue structures participate in this process. Three stages were described in Ig transfer: i) passing from the maternal blood into intervillous spaces and the trophoblast, ii) shifting in the intravillous stroma and its cells, and iii) excretion into embryonic (fetal) blood through the endothelium of the trophoblastic villous capillaries. Igs of maternal origin, mainly IgG and least abundant IgA, pass through the placental barrier in healthy embryos. Following a massive antigenic attack, the increased exocrine secretion of IgG, IgA, and IgM to a lesser extent, are already seen in embryos, reflecting increased functional activity of the SIS. Thus, in human intrauterine development, the SIS is a very early immune defensive system, which presents and acts before the appearance of the common lymphoid system.

This study examined whether soluble 66 and 51 kDa tumor-associated antigens (sTAA), isolated from the serum of rats with mammary cancer, possess specific suppressive effects on chemically-induced mammary tumorigenesis in syngeneic counterparts. Dimethylbenzanthracene (DMBA, 10 mg/rat, two administrations) was used to induce mammary tumors in 8-week-old Sprague Dawley rats. After the appearance of numerous tumors, preparations of sTAA (50 to 60 microg/rat in 0.5 ml sterile PBS) obtained from breast cancer patients (heterologous sTAA) or from syngeneic mammary tumor-bearing rats (syngeneic sTAA) were administered weekly for 12 weeks. The following groups of mammary tumor-bearing rats were studied: groups 1 and 3, control rats treated with saline; group 2, rats treated with heterologous sTAA; and group 4, rats treated with syngeneic sTAA. The experiment was terminated when tumors in 50% of the rats became ulcerous. The treatment with both types of sTAA significantly decreased, compared to controls and initial values, the yield and total area of the tumors. We conclude that syngeneic sTAA have tumor-suppressive properties, which are very similar to those in heterologous sTAA.

The mechanism of human stem cell expansion ex vivo is not fully understood. Furthermore, little is known about the mechanisms of human stem cell homing/repopulation and the role that differentiating progenitor cells may play in these processes. We report that 2- to 3-day in vitro cytokine stimulation of human cord blood CD34(+)-enriched cells induces the production of short-term repopulating, cycling G1 CD34(+)/CD38(+) cells with increased matrix metalloproteinase (MMP)-9 secretion as well as increased migration capacity to the chemokine stromal cell-derived factor-1 (SDF-1) and homing to the bone marrow of irradiated nonobese diabetic severe/combined immunodeficiency (NOD/SCID) mice. These cycling G1 cells enhance SDF-1-mediated in vitro migration and in vivo homing of quiescent G0 CD34(+) cells, which is partially abrogated after inhibition of MMP-2/-9 activity. Moreover, the engraftment potential of quiescent G0 SCID repopulating cells (SRCs) is also increased by the cycling G1 CD34(+)/CD38(+) cells. This effect is significantly abrogated after incubation of cycling G1 cells with a neutralizing anti-CXCR4 antibody. Our data suggest synergistic interactions between accessory cycling G1 CD34(+)/CD38(+) committed progenitor cells and quiescent, primitive G0 CD34(+)/CD38(-/low) SRC/stem cells, the former increasing the motility and engraftment potential of the latter, partly via secretion of MMP-9.

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.

We analyzed the role of T- and B-lymphocytes in the antitumor effects of the anticancer drug tamoxifen and soluble tumor-associated antigens (sTAA) on rat mammary carcinogenesis. Studies were performed on the spleen from the following groups of mammary tumor-bearing rats. i) Rats in group 1 were not exposed to DMBA and served as age-related controls. Rats in other groups were exposed to DMBA and received different types of treatment; ii) rats in group 2, received no additional treatment, and served as carcinogen-related controls; iii) rats in group 3 were treated with the commercial hormone-dependent anticancer drug tamoxifen by weekly subcutaneous (s.c.) injections of 10 mg dissolved in 0.5 ml distilled water per rat; iv) rats in group 4 were vaccinated s.c. weekly with a preparation of sTAA (50 micro l/rat) dissolved in 0.5 ml of phosphate-buffered saline; v) rats in group 5 were treated with tamoxifen and were also vaccinated with a preparation of sTAA. Different zones of the spleen were measured and their T- and B-cell contents were analyzed immunohistochemically. The treatment with tamoxifen significantly increased the total number of lymphocytes in the follicles, PALS (periarterial lymph sheath) and red pulp relative to all other groups. The combined treatment with tamoxifen and sTAA increased the areas of white pulp, the PALS, and marginal zone. The number of B-cells was higher in the marginal zone of spleens from age-related controls, as well as from rats treated with sTAA and those treated with tamoxifen and sTAA. The number of CD4+ lymphocytes in the PALS was higher in rats treated with sTAA and tamoxifen, and notably so in those treated with sTAA alone. The number of CD8+ lymphocytes was significantly lower in the PALS of spleens from all tumor-bearing rat groups compared to the unexposed age-related control rats. We suggest that the tumor-suppressive effect of sTAA and tamoxifen is accompanied by the activation of B- and T-lymphocyte production.

The role of joining (J) chain, one of the protein components of the secretory immune system (SIS), in the immune reactions of the human embryo and fetus was analyzed on the basis of data from the literature and our previous studies. All organs and structures, including extra-corporeal ones, of 18 embryos (4-8 weeks of development) and 45 fetuses (9-38 weeks) were studied using methods of pathomorphology, immunohistochemistry and morphometry. This approach enabled us to analyze the problem in the whole organism throughout its embryonic and fetal development. J chain, as well as polymeric immunoglobulin (Ig) receptor-secretory component (pIgR/SC) and Igs, are already widely distributed in 4-week-old embryos before the appearance of the common immune system. The whole complex of protein components of the SIS was seen in mucous layers, and in blood-tissue and tissue-tissue barrier structures. Therefore, we can consider two parts of the SIS: mucosal and barrier. Already in embryos, an increase in the functional activity of the SIS following massive antigenic attack in cases of acute chorioamnionitis reflects the increased exocrine secretion of Igs. The J chain appears to participate in the endocytosis but not exocytosis of Igs. J chain and Igs, but not pIgR/SC, were present in cells of the heart, endocrine glands, gonads and some other organs. The exocrine secretion of Igs, the main function of the SIS, is absent in these organs, and, they are therefore, not considered part of the SIS.

Trafficking of human CD34+ stem/progenitor cells (HSCs/HPCs) is regulated by chemokines, cytokines, proteolytic enzymes, and adhesion molecules. We report that the adhesion receptor CD44 and its major ligand, hyaluronic acid (HA), are essential for homing into the bone marrow (BM) and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice and engraftment by human HSCs. Homing was blocked by anti-CD44 monoclonal antibodies (mAbs) or by soluble HA, and it was significantly impaired after intravenous injection of hyaluronidase. Furthermore, stromal cell-derived factor-1 (SDF-1) was found to be a rapid and potent stimulator of progenitor adhesion to immobilized HA, leading to formation of actin-containing protrusions with CD44 located at their tips. HPCs migrating on HA toward a gradient of SDF-1 acquired spread and polarized morphology with CD44 concentrating at the pseudopodia at the leading edge. These morphologic alterations were not observed when the progenitors were first exposed to anti-CD44 mAbs, demonstrating a crosstalk between CD44 and CXCR4 signaling. Unexpectedly, we found that HA is expressed on human BM sinusoidal endothelium and endosteum, the regions where SDF-1 is also abundant. Taken together, our data suggest a key role for CD44 and HA in SDF-1-dependent transendothelial migration of HSCs/HPCs and their final anchorage within specific niches of the BM.

This study examined whether the soluble 66- and 51 kDa tumor-associated antigens (sTAA), isolated from the serum of breast cancer patients, possess specific suppressive effects on chemically-induced rat mammary tumorigenesis in comparison to commercial human albumin. Dimethylbenzanthracene (DMBA, 10 mg/rat, 2 administrations) was used to induce mammary tumors in 8-week-old Sprague Dawley rats. After the appearance of many large tumors, preparations of sTAA (50-60 micro g/rat in 0.5 ml sterile PBS) or commercial human albumin (HA, in the same doses as sTAA) were administered weekly, for 10-14 more weeks. The following groups of mammary tumor-bearing rats were studied: i) control non-treated rats, ii) rats treated with HA, iii) rats treated with sTAA. The experiment was terminated when tumors in 70% of the rats became ulcerous. The treatment with sTAA significantly decreased, compared to controls, the yield and total area of the tumors. In rats treated with sTAA, the appearance of new tumors stopped at week 5 as compared to week 7 in rats treated with HA and week 10 in control rats. In rats treated with sTAA, the time of appearance of ulcerous tumors increased to 8 weeks, as compared to 6 weeks in controls and in rats treated with HA. Duration of the experiment increased from 11 weeks in controls to 12 weeks in rats treated with HA and to 14 weeks in rats treated with sTAA. We conclude that sTAA have tumor-suppressive properties, which are well-defined if the treatment is begun on small tumors.

A major problem after clinical hematopoietic stem cell transplantations is poor T-cell reconstitution. Studying the mechanisms underlying this concern is hampered, because experimental transplantation of human stem and progenitor cells into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice usually results in low T-lymphocyte reconstitution. Because tumor necrosis factor alpha (TNFalpha) has been proposed to play a role in T-lineage commitment and differentiation in vitro, we investigated its potential to augment human T-cell development in vivo. Administration of TNF to irradiated NOD/SCID mice before transplantation of human mononuclear cells from either cord blood or adult G-CSF-mobilized peripheral blood (MPBL) led 2-3 weeks after transplantation to the emergence of human immature CD4(+)CD8(+) double-positive T-cells in the bone marrow (BM), spleen, and thymus, and in this organ, the human cells also express CD1a marker. One to 2 weeks later, single-positive CD4(+) and CD8(+) cells expressing heterogenous T-cell receptor alpha beta were detected in all three organs. These cells were also capable of migrating through the blood circulation. Interestingly, human T-cell development in these mice was associated with a significant reduction in immature lymphoid human CD19(+) B cells and natural killer progenitors in the murine BM. The human T cells were mostly derived from the transplanted immature CD34(+) cells. This study demonstrates the potential of TNF to rapidly augment human T lymphopoiesis in vivo and also provides clinically relevant evidence for this process with adult MPBL progenitors.

In this communication, we report for the first time, that immunization of cancer patients with autologous soluble tumor-associated antigens (sTAA) isolated from their own serum prevents the toxic side effects of chemotherapy, improves the patients' clinical status, and has therapeutic effects without chemotherapy. In 2001 and 2002, two cancer patients were treated, during chemotherapy, with autologous sTAA. Another benign tumor-bearing patient was treated with a medicinal herb and autologous sTAA. Doses for subcutaneous injections varied between 2.5 and 3 mg of sTAA in 0.5 ml of sterile distilled water. Injections were performed twice a week or at weekly intervals. In each case, the clinical status of the patient became more stable and healthier. Toxic side effects caused by chemotherapy decreased or even disappeared. No additional toxic side effects were observed after vaccination with sTAA. In the studied cases, a polyp disappeared and a metastatic brain tumor began to encapsulate. No metastases were seen in the case with colon adenocarcinoma. We concluded that vaccination of patients with autologous sTAA prevents the toxic side effects of chemotherapy in cancer patients and improves their clinical status. In the case with the benign tumor, this vaccination activated the host's immune system, prevented progress of the disease and even promoted tumor disappearance. We suggest that immunotherapy with autologous sTAA provides significant clinical benefits in cancer patients and appears to be an important new adjuvant treatment of cancer.

This study examined whether the soluble 66 and 51 kDa tumor-associated antigens (sTAA) could promote suppression by the anticancer drug 5-fluorouracil (5-Fu) of chemically induced mammary tumorigenesis, and which, if any, morphological changes in the immune organs accompany this treatment. Dimethylbenzanthracene (DMBA, 8 mg/rat, twice) was used to induce mammary tumors. After the appearance of many large tumors, the preparations of sTAA and 5-Fu, alone or in combination, were administered in weekly doses, for 4 weeks. The following groups of mammary tumor-bearing rats were studied: 1) control non treated rats, 2) rats treated with sTAA, 3) rats treated with 5-Fu, 4) rats treated with 5-Fu and sTAA. The experiment was terminated when tumors in 70% of control rats became ulcerous. Treatment with sTAA alone significantly decreased tumor yield and their total area relative to controls. Both of these parameters showed an even larger significant decrease after treatment with 5-Fu, and the most marked decrease was obtained after the combined treatment with 5-Fu and sTAA. Results demonstrated that not only do sTAA have tumor-suppressive properties, they also enhance the anticancer effects of 5-Fu and prevent its toxic side effects. Morphologically, the treatment with sTAA was manifested in a significant increase in the size of the spleen follicles and mantle layer compared to control rats with large tumors. The treatment with 5-Fu decreased the sizes of almost all areas of the spleen compared to control rats, whereas the combined treatment with 5-Fu and sTAA increased all these parameters to the levels found in rats treated with sTAA alone. The total areas of the cortex and paracortex in the lymph nodes increased after treatment with sTAA. Treatment with 5-Fu alone resulted in a significant decrease of these areas which, as seen in the spleen, increased after combined treatment with 5-Fu and sTAA. Similar changes were seen in the areas of the separate lymph node zones. We concluded that the addition of sTAA to conventional tumor chemotherapy regimens has a remarkable synergistic effect on mammary tumors leading to curative antitumor responses of the host's immune organs.

The effect of the synthetic pineal peptide Epitalon (Ala-Glu-Asp-Gly) on proliferative activity in colon tumors, and in mucosal epithelial cells adjacent to and located far from tumors was studied in rats. To evaluate the effect of Epitalon on different stages of carcinogenesis, different treatment regimens were used: during the tumor initiation stage, during the tumor-promotion stage, or during the entire process of tumor development. Eighty 2-month-old male LIO rats were exposed weekly to five subcutaneous injections of 1,2-dimethylhydrazine (DMH) at a single dose of 21 mg/kg body weight. Rats were divided into four groups. Control rats (group 1) received saline at a dose of 0.1 ml during the entire experiment. Rats in group 2 were treated with Epitalon at a dose of 1 micro g, five times a week, for 6 months, from the first injection of DMH till the end of the experiment. Rats in group 3 were treated with Epitalon after termination of the carcinogen injections. Rats in group 4 were treated with Epitalon only during the period of DMH exposure (for the first 5 weeks of the experiment). DMH induced proliferation of the secretory epithelium, and this phenomenon was accompanied by a decrease in the size of the stromal area and the area of lymph infiltration in colon tumors and in the colon mucosa adjacent to the tumors (group 1). Epitalon attenuated this effect, especially when the treatment was continued throughout the experiment (group 2). It increased the stromal areas, as well as that of lymphoid infiltration in the colon mucosa adjacent to the tumors. The intensity of lymphoid infiltration was activated in both the colon mucosa adjacent to a tumor and in the tumor. Mitotic activity of tumor cells was significantly inhibited by Epitalon when the treatment was given throughout the experiment (group 2). In parallel, a high level of apoptosis was seen in the same group. Thus, the strongest inhibitory effect of Epitalon on carcinogenesis in the colon mucosa was manifested when the treatment was continued throughout the experiment.

Several proteins, such as polymeric immunoglobulin (Ig) receptor/secretory component (pIgR/SC), immunoglobulins (Igs) and joining (J) chain, and cellular components of the secretory immune system (SIS) of the human embryo and fetus were analyzed and compared with reviewed information concerning SIS organization and function. All organs and structures, including extracorporeal ones, of 18 embryos (4-8 weeks of development) and 45 fetuses (9-38 weeks) were studied using methods of pathomorphology, immunohistochemistry and morphometry. This approach enabled us to analyze the problem in the whole organism during its entire development. SC, Igs and J chain were already widely distributed in 4-week-old embryos and were later seen in the mucosa and glands of the digestive, respiratory and urogenital tracts, ovaries, testis, endocrine glands, extracorporeal tissues, blood-brain and other blood-organ barrier structures, as well as in serous membranes. The presence of protein transport and later of cellular components suggests an active role for SIS not only in mucous membranes, but also in blood-tissue barriers. Loss of morphological contact between epithelial structures and mucous membranes during organogenesis of some endocrine organs (hypophysis, pancreatic islets, etc.) is followed by the disappearance of SC as a result of cessation of Ig exocytosis. Secretion of Igs increased in the epithelium and glands of the digestive and respiratory tracts following massive antigenic attack in cases of acute chorioamnionitis. All of this demonstrates that SIS is a widely branching immune system which appears and acts early in the human embryo, before the lymphoid system is formed, using IgG and IgA of maternal origin. IgA and IgM-synthesized lymphocytes appear in 9-week-old fetuses.

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., Materials and Methods: Specimens from 17 embryos (4-8 weeks of pregnancy) and nine fetuses (9-12 weeks) were divided into those that had not been exposed to massive foreign antigenic effects (Group I, n = 18) and those that had suffered acute chorioamnionitis (Group II, n = 8)., Results: Positive immunostaining for secretory component (SC), joining (J) chain, IgG, IgA, and macrophages was seen from 4 to 5 weeks of development and then during the whole first trimester of pregnancy in the syncytio- and cytotrophoblasts, the amniotic epithelium, the yolk sac endoderm and decidual cells. Macrophages with J chain, IgG and IgA were found in embryonic tissues on week 4, whereas lymphocytes, including those synthesizing IgA and IgM, appeared only at the end of the first trimester of pregnancy. In the decidua, lymphocytes and macrophages were recognized during the whole period of study. In cases with chorioamnionitis (Group II), reactivity of IgG and IgA in the mentioned cells of fetuses decreased sharply while the rate of immunoreactivity of SC and J chain as well as the number of T and B lymphocytes did not change. In the decidua, the number of immune reactive cells sharply increased with the appearance of plasma cells., Conclusions: In the fetal membranes and decidua all the SIS components are present. We suggest that two SIS, maternal and fetal, participate in the formation of the barrier between a mother and the fetus. Both these systems have different origin and cellular content as well as different immune reactions.

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.

We studied whether feeding pregnant female rats different high fat diets affects structural zones in the spleen and lymph nodes, involved in production of T and B cells, as well as cell kinetics and apoptosis in some offspring with mammary glands tumors. Rat mothers were fed either a 7% or 15% corn-oil or a 7% or 15% olive-oil diet. At four weeks of age, female offspring (n=10-15 per group) were transferred to 7% corn oil diet. Five-week old offspring were exposed twice to the carcinogen, dimethylbenz(a)antracene (10 mg/rat/week). Three months later, tumors were counted and sized, and samples from the spleen, axillary lymph nodes and tumors collected for immunohistochemical analyses. Feeding the mothers with both the 7% and 15% olive-oil diets significantly increased the number of tumor-free rats in offspring. Tumors were characterized with active mitosis, intensive lymphoid infiltration inside a knot and high rates of apoptosis, particularly in tumors obtained from rats whose mothers were fed the 15% olive-oil diet. In the spleen, the 15% olive-oil diet significantly increased the areas of the follicles and germinal centers but only in tumor-free rats. In tumor-bearing rats, areas of germinal centers increased compared to the 7% olive-oil diet. The 15% olive-oil diet increased all areas of the lymph nodes in tumor-free rats, while in tumor-bearing rats, this diet increased the areas of the cortex and mantle layer. We conclude that exposure to various diets in utero and during lactation affects the immune system. In addition, the promotion of apoptosis may play a key role in the mechanisms involved in the transplacental effects on mammary tumor development as seen using a 15% olive-oil diet, similar to the high fat diets of Mediterranean countries.

The aim of this review was to analyze the role of cell kinetics and apoptosis in the cellular mechanism underlying the effects of soluble tumor-associated antigens (sTAA) on chemically-induced mammary cancer in rats treated with the anticancer drug cyclophosphamide (CPA). Mammary gland cancer was induced with dimethylbenz(a)anthracene (DMBA), and tumors, the spleen, thymus, bone marrow and lymph nodes were studied by morphometric and immunohistochemical methods. We suggest that inhibition of the functional activity of the immune system is one of the main reasons for the toxic effects of CPA, and that the tumor-suppressive antitoxic effects of sTAA result from their activation of T-lymphocyte production in this system, particularly in the spleen and lymph nodes. The results of our experiments have shown that vaccination with sTAA actively promotes generation of the host's antitumor immune response. This is manifested in inhibited proliferative activity of the tumor cells, stimulated production of T lymphocytes and an increased rate of apoptosis among tumor cells.

We showed in a previous study that soluble low-molecular-mass tumor-associated antigens (sTAA) promote the anti-tumor effect of the anticancer drug cyclophosphamide (CPA) on rat mammary carcinogenesis. In this report, we analyzed the possible mechanism underlying this phenomenon. Studies were performed on the bone marrow and thymus from the following groups of rats: i) control rats, ii) rats treated with sTAA, iii) rats treated with CPA, iv) rats treated with CPA and sTAA. The cellular content of the bone marrow and thymus (CD4+ and CD8+ lymphocytes) was analyzed morphometrically and immunohistochemically. In the bone marrow, CPA caused significant substitution of cellular components with fatty tissue whereas sTAA repaired this process. We found that CPA affects mainly the process of myelogenesis whereas sTAA protect the production of lymphocytes. In the thymus, CPA alone or in combination with sTAA repaired the inhibition effect of DMBA on synthesis of CD4+ and CD8+ thymocytes. sTAA further increased the amount of CD8+ T lymphocytes in the medulla of the thymus. Data in the literature as well as the findings presented here demonstrate that the tested treatment, including vaccination with sTAA, actively promotes the generation of the host's antitumor immune response.

The aim of this study was to evaluate the possible role of apoptosis-related proteins (ARP: Fas and Fas ligand, bcl-2, p53) in the progress of tumorigenesis in breast cancer. Epithelial tumor cells and lymphocytes were analyzed immunohistochemically for the rate of lymphoid infiltration and presence of ARP in 50 human breast tumors of different types. The tumors included: i) fibrocystic disease (n=12); ii) benign fibroadenoma (n=11); iii) carcinoma in situ (n=8); iv) invasive ductal carcinoma with high lymphoid infiltration (n=12); and v) invasive ductal carcinoma with lymphoid depletion (n=7). Both fibrocystic disease and fibroadenomas had low amounts of lymphocytes and very little lymphoid infiltration. In cancer in situ, expansion of lymphoid infiltrates and increased density of lymphocytes resulted in a rise in the total number of lymphocytes, reflecting intensification of the immune response. In carcinomas with high lymphoid infiltration, a significant increase in the number of Fas and FasL and p53-positive cells was found. The number of bcl-2-positive tumor cells in these tumors was not changed, whereas the number of bcl-2-positive lymphocytes decreased. In carcinomas with lymphoid depletion, the opposite picture was found as a result of deep subcompensation of the lymph system. ARP are present in a significantly higher number of lymphocytes than of the epithelial tumor cells. This indicates that the cells initiating apoptosis in tumors are themselves damaged by the process. The intense apoptosis in lymphocytes in malignant tumors may be one of the reasons for the progress of breast tumors.

The aim of this study was to analyze the roles of proliferative activity, lymphoid infiltration and apoptotic rate in the cellular mechanism underlying the promotion effects of soluble tumor-associated antigens (sTAA) in mammary cancer in rats treated with the anticancer drug cyclophosphamide (CPA). Studies were performed on the following groups of rats: i) control tumor-bearing rats, ii) tumor-bearing rats treated with sTAA, iii) tumor-bearing rats treated with CPA, iv) tumor-bearing rats treated with CPA and sTAA. Mammary gland cancer was induced with dimethylbenz(a)anthracene (DMBA), and the rate of lymphoid infiltration, T cell content (CD4+ and CD8+ cells), and mitotic and apoptotic indices in tumors were evaluated. In control tumor-bearing rats, high lymphoid proliferation, as well as a high number of CD8+ cells, was found in tumors. Treatment with sTAA further significantly increased the total number of lymphoid cells and the number of CD8+ lymphocytes. CPA sharply decreased the production of all lymphoid cells studied, especially of CD4+ lymphocytes. The combined treatment of CPA and sTAA increased the number of lymphoid cells, although they did not reach control levels. The mitotic index significantly decreased as a result of the treatment with CPA alone and especially after the combined treatment with CPA and sTAA. The results of our experiments demonstrate that vaccination with sTAA actively promotes the generation of the host's antitumor immune response. This is manifested in inhibited proliferative activity of tumor cells, stimulated production of T lymphocytes and increased rate of apoptosis among tumor cells.

The objective of this study was to examine whether splenectomy alters the tumorigenic response of rats to chemically-induced mammary tumors and to determine the role played by the parathymic lymph nodes (PTLN) in the antitumor immune response. Female rats were splenectomized and then exposed to 9,10-dimethyl-1,2-benz(a)anthracene (DMBA) to induce mammary tumors. Splenectomy decreased the rate of tumor appearance. The latent period for appearance of the first tumors in splenectomized rats was 12.0 +/- 0.9 week compared to 9.7 +/- 0.5 week in intact controls. The latent periods for appearance of all tumors including those with malignancy were similar for both rat groups. Splenectomy caused a significant increase in the size of the PTLN as well as changes in their structure. The cortex became thick, better formed and enriched with lymph elements of follicles and a wide venous net. CD8+ cells infiltrated the paracortex whereas CD4+ cells were located around the blood vessels. In conclusion, splenectomy inhibits the early stages of tumorigenesis, but does not prevent the progress of carcinogenesis. Different responses of splenectomized (operated) and intact rats to the effect of DMBA can be explained by an increase in the nonspecific resistance of splenectomized rats as a result of the operation. PTLN actively participate in this compensatory reaction of the immune system of animals.

We have shown previously that soluble low-molecular-mass tumor-associated antigens (sTAA) promote the anti-tumor effect of the anticancer drug cyclophosphamide (CPA) on rat mammary carcinogenesis. In this study, we analyzed the possible mechanism underlying this phenomenon. Studies were performed on tumors obtained from the following groups of mammary tumor-bearing rats: i) control rats, ii) rats treated with sTAA, iii) rats treated with CPA, iv) rats treated with CPA and sTAA. All analyzed tumors represented different types of invasive duct carcinomas. The rate of lymphoid infiltration and T cell content (CD4+ and CD8+ cells) of tumors were analyzed immunohistochemically. In parallel, mitotic index was evaluated in tumor cells. In tumor-bearing rats, high lymphoid proliferation was found at the periphery of tumors, and to a lesser extent deep inside the tumors. In control tumors, CD4+ T cell content was very low whereas CD8+ cells were highly abundant, especially at the tumor periphery. Treatment with sTAA significantly increased the total number of lymph cells and the number of CD8+ lymphocytes inside the tumors. Cytoplasmic vacuolization, decreased mitotic index and various degrees of fibrosis were the most distinct changes in tumors treated with CPA alone. CPA also sharply decreased the activity of all lymph cells studied, especially of CD4+ lymphocytes which could no longer be observed following this treatment. The combined treatment of CPA and sTAA increased the number of lymph cells, although they did not reach control levels. Inhibition of mainly CD4+ lymphocyte synthesis of CPA was confirmed by the low CD4/CD8 ratio, which increased slightly after the combined treatment with CPA and sTAA. Findings in the present study demonstrate that vaccination with sTAA actively promotes the generation of the host's antitumor immune response.

The objective of the present study was to analyze the function of lymphoid elements in the tumorigenesis of human breast cancer, similar to their elucidation in human ovarian cancer in our previous work. The lymphocytic and macrophageal content of lymphocytes and macrophages was analyzed immunohistochemically and morphometrically in 49 human breast tumors of different types. The following types of tumors were studied: 1) fibrocystic disease, 2) fibroadenoma, 3) carcinoma in situ, 4) infiltrating ductal and lobular carcinoma with high lymphoid infiltration, and 5) infiltrating ductal and lobular carcinoma with lymphoid depletion. The first two had little lymphoid infiltration and few lymphocytes (mainly T cells), while carcinoma in situ had extensive lymphoid infiltration and increased lymphocytic density, the consequence of a sharp rise in total lymphocytes reflecting the intensified immune response. In ductal and lobular infiltrating carcinoma with high infiltration, T cells were in large excess of B cells (81% and 87% vs. 11%) and CD8+ lymphocytes were the predominant type of T cells (up to 90%), in both tumoral parenchyma and stroma. In infiltrating carcinoma with lymphoid depletion, the total lymphocyte and macrophage count and areas of lymphoid infiltrates decreased, relative to highly infiltrated carcinomas, as signs of deep subcompensation of the lymphoid system. The host's reaction to disease was reflected in high correlations between the densities of the lymphoid cellular elements as tumorigenesis evolved. We suggest that the stromal immunocompetent cells are a reservoir of T killers that eventually cross into the parenchyma and join T helpers and B lymphocytes in the immune antitumor response. In later stages of cancer the response is exhausted, as manifested in lymphoid subcompensation.

In a previous study, we showed that soluble low-molecular-mass tumor-associated antigens (sTAA) promote the anti-tumor effect of the anticancer drug cyclophosphamide (CPA) on rat mammary carcinogenesis. In this report, we analyzed the underlaying mechanisms. Studies were performed on the spleen and lymph nodes from the following groups of mammary tumor-bearing rats: i) control rats, ii) rats treated with sTAA, iii) rats treated with CPA, i.v.) rats treated with CPA and sTAA. Different zones of the spleen and lymph nodes were measured and their T cell content (CD4(+) and CD8(+) cells) was analyzed immunohistochemically. CPA decreased the size and cell content of follicles, splenic areas related to the production of B cells, of the marginal zone and to a lesser extent of the periarterial lymph sheath, and decreased the number of CD4(+) and, at a lower rate, of CD8(+ )T cells in the spleen. Addition of sTAA restored activity in the splenic zones producing these cells. Similar effects of CPA and sTAA were found in lymph nodes with accumulation of B lymphocytes in the primary and secondary follicles and of T lymphocytes, including both CD4(+) and CD8(+) cells, in the paracortical zone. We suggest that inhibition of the functional activity of the immune system is one of the main reasons for the toxic effects of chemotherapeutic drugs such as CPA and that the tumor-suppressive antitoxic effects of sTAA result from their activation of B- and T-lymphocyte production in this system, particularly in the spleen and lymph nodes.

This study examined whether the soluble tumor-associated antigens (sTAA) of 66 kDa and 51 kDa could promote suppression of chemically-induced rat mammary tumorigenesis by the hormone-related anticancer drug tamoxifen and prevent the drug's toxic side-effects. Dimethylbenzanthracene (DMBA, 10 mg/rat, 3 administrations) was used to induce mammary tumors in 8-week-old Wistar rats. Then, for 13-17 more weeks, preparations of sTAA (50 microg/rat) and tamoxifen (10 mg/rat) were administered, separately or in combination, on a weekly basis. The experiment was continued for 18 weeks and was terminated when the number of dead rats reached 50% in each group. Treatment with tamoxifen inhibited tumor growth and their malignance: the number of rats without malignant tumors significantly increased compared to controls, 27.3% and 5.6%, respectively. Treatment with sTAA resulted in a significant increase in the number of regressed tumors to 10.1% compared to 0% and 1.4% in control and tamoxifen-treated rats, respectively. Moreover, the period of 50% survival increased from 13 weeks in tamoxifen-treated rats to 17 weeks, and as a result, rats treated with sTAA were involved in the experiment for an average 14.3 weeks compared to 10 and 10.4 weeks in control and tamoxifen-treated groups. In rats treated simultaneously with tamoxifen and sTAA, the time of appearance of each new tumor increased from 4.5 weeks to 6.6 weeks with a significant increase to 14.3% in the number of regressed tumors. The period to 50% survival increased to 18 weeks, and these rats were involved in the experiment for up to 16.4 weeks. The number of rats without malignant tumors increased to 22.2% and the time of appearance of malignancy increased to 9.6 weeks, as compared to 7.3 weeks in controls. The results demonstrated that sTAA have tumor-suppressive properties, and also enhance the anticancer effects of tamoxifen and prevent its toxic side-effects.

The objective of this study was to examine how splenectomy affects the immune response, particularly T cells, in chemically-induced mammary tumors. Female rats were splenectomized and then exposed to 9,10-dimethyl-1,2-benz(a)anthracene (DMBA) to induce mammary tumors. Splenectomy significantly decreased the rate of tumor appearance and their malignant transformation. The tumor latency period in splenectomized rats was 12.0+/-0.9 weeks compared to 9.7+/-0.5 wk in intact controls, and malignancy appeared in 45% of splenectomized rats, compared to 70% in controls. By the end of the experiment, the total number of tumors and their size were similar in both groups. Blood CD4+ and CD8+ T cell concentrations were similar in tumor-bearing and tumor-free splenectomized animals, but in both groups CD4- and CD8- lymphocytes decreased sharply compared to control animals. In tumor-bearing rats, splenectomy also resulted in significantly more circulating natural killer cells. The spleens of tumor-bearing control rats had significantly fewer CD4+ and CD8+ lymphocytes and more CD4- and CD8- lymphocytes and natural killer cells than did their blood. In conclusion, splenectomy inhibits the early stages of tumorigenesis and reduces the rate of malignant transformation of benign tumors, but does not prevent the progress of carcinogenesis. Differences between splenectomized (operated) and intact rats to the effect of DMBA can be explained by an increase in non-specific resistance of splenectomized rats as a result of operation.

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.

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.

This study evaluated whether the feeding of rats with a 15% orange-pulp diet affects the lymphatic system and the tumorigenic response in rats exposed to a high dose of carcinogen. Five-week-old Sprague Dawley rats were divided into 2 groups fed a control chow diet or the same diet with 15% orange pulp. All rats were injected with 1,2-dimethylhydrazine (DMH) (20 mg/kg) weekly for 6 weeks. At 8 months, tumors, spleens and descending colon were taken from each group for analyses. Feeding rats the 15% orange-pulp diet did not reduce the tumor number but modified the number of adenocarcinomas found in the orange-pulp group compared to controls: 66.7% vs. 93.7%. The number of endophytic tumors was also significantly lower in the experimental group: 6.3% vs. 32.3% in controls. DMH affected the size of the splenic structures. The size of follicles and germinal centers decreased significantly in tumor-bearing rats compared to tumor-free rats. This effect was changed in rats fed the orange-pulp diet. In tumor-bearing rats from this group, only the area of the marginal zone decreased and the red pulp increased compared to tumor-free rats. The size of germinal centers significantly increased compared to tumor-bearing rats in controls. The total number of lymphoid cells decreased in germinal centers of spleens obtained from control tumor-bearing rats compared to tumor-free rats. DMH alone significantly increased the total number of cells in the colon mucosa of the rats fed the control diet. In tumor-bearing rats exposed to the carcinogen and fed the 15% orange-pulp diet, the total number of cells and the number of Ki-67+ cells increased in the depth of tumors whereas the number of CD8+ T cells increased in the colon mucosa, at the border of tumors and its depth. The caspase-3 protein a cysteine protease was elevated in tumors from rats fed the orange-pulp diet. Although the 15% orange-pulp diet did not change the number of tumors in the tumor-bearing rats, feeding rats orange pulp significantly decreased the number of endophytic tumors and increased the number of exophytic tumors. Increased activity of T cell killers in tumors and higher level of proteins involved with apoptosis following consumption of the orange pulp indicate a clear tumor suppressor effect of these dietary fibers.

There is increasing evidence that tumors express putative target molecules in a therapeutic immune reaction. Identification of immunogenic tumor-associated antigens (TAA) may enable the development of new modes of vaccination with the addition of immunotherapy as a potentially powerful weapon in the fight against cancer. In the present review, the authors' observations on the role of the soluble low-molecular-mass heat shock proteins and tumor-associated antigens, named as a complex of STAA, in the prevention and therapy of chemically-induced tumorigenesis are analyzed and compared with data from the literature. It has been shown that STAA have both tumor-preventive and tumor-suppressive effects on chemically-induced cancers of the colon, skin and mammary glands in rats and mice. These effects were shown to be connected with activation of the host's immune system, especially that which is responsible for the activity of T and B lymphocytes. These findings have led to a wave of new trials involving cancer immunotherapy. Understanding the mechanisms of antitumor immunity and identifying relevant tumor-specific antigens is expected to improve vaccine strategies and provide for a successful cancer therapy in the future.

We studied whether feeding pregnant female rats a 15% olive-oil diet affects the activity of lymph cells in the spleen and tumors in offspring with chemically-induced colon tumors. Rat mothers were fed either a 7% corn-oil or a 15% olive-oil diet. Five-week-old male offspring were divided into 3 groups. A control group was fed the 7% corn-oil diet similar to their mothers. The experimental group I was fed the 7% corn-oil diet whereas their mothers were fed the 15% olive-oil diet. The experimental group II was fed the same 15% olive-oil diet as their mothers. Experimental rats were injected weekly for 8 weeks with the carcinogen, 1,2-dimethylhydrazine (DMH), 20 mg/kg b.w. Results of experiments were studied 6 months later. The area of zones in the spleen responsible for producing B and T lymphocytes were measured and the number of cells counted. The activity of lymphoid elements of the spleen and of tumors were studied using immunohistochemical methods for evaluating the synthesis of CD8(+) lymphocytes and proliferative activity of lymphocytes in spleens and tumors. Feeding pregnant or lactating mothers with the 15% olive-oil diet had no marked tumor-protective effect on chemically-induced colon cancer in offspring. Diet-dependent changes were found at the cellular level. In the spleen of control offspring, the presence of a tumor was accompanied by an increase in the number of Ki-67(+) cells and CD8(+) lymphocytes in the red pulp. In experimental group I, DMH significantly increased the total cell number and the number of CD8(+) lymphocytes in the red pulp of the spleen in both tumor-bearing and tumor-free rats. In experimental group II, the total number of lymph cells and the number of CD8(+) lymphocytes increased compared to offspring fed a control diet. Tumor formation activated the proliferative activity of lymph elements. The total number of cells in infiltrates of the colon mucosa decreased in tumor-bearing rats compared to tumor-free counterparts, and this was seen in all three dietary groups of rats. In tumors from offspring of experimental group II, only the number of CD8(+) lymphocytes increased compared to those in offspring of experimental group I. The findings indicate that feeding mothers the 15% olive-oil diet had a cancer-inhibiting role in offspring, predominantly changes at the cellular level.

The objective of this study was to describe the development of the secretory immune system (SIS) in the placenta of 32 human fetuses who had died from different causes during the second trimester of pregnancy. The distribution of SIS protein elements, including the secretory component (SC), J-chain, IgA, IgM, IgG, as well subsets of lymphocytes and macrophages, were studied by immunohistochemical methods. Both the fetal and maternal parts of the placenta were found to contain these elements. In the fetal part, the immunoglobulins, SC and J chain were located in the syncytio- and cytotrophoblast of the chorion and in the epithelium of the amnion. The villous stroma contained a small amount of different subsets of lymphocytes. Macrophages accounted for up to 45% of the stromal cells of the villi and contained IgG and J-chain. In the maternal part of the placenta, the SIS proteins were in the decidual cells. Relatively few lymphocytes and macrophages were observed in the decidual stroma. Our data suggest that the placenta has two different SIS, one in the fetal part and the other in the maternal part. They differ in their structure and orientation: the maternal SIS protects the mother against paternal antigens from the fetus, while the fetal SIS protects the fetus against macromolecules and infectious agents penetrating from the mother. The placenta represents the largest extracorporal immune system that is functionally active during the whole second trimester of gestation. We suggest that the concept of placental barrier should be expanded to include both fetal and maternal parts of the SIS, fetal membranes and the decidual tissue.

This study examined whether the soluble tumor-associated-antigens (TAA), of 66 kDa and 51 kDa, could promote suppression by anticancer drugs of chemically-induced mammary tumorigenesis. Dimethylbenzanthracene (DMBA, 10 mg/rat, twice) was used to induce mammary tumors. Then, for nine more weeks, the preparation of TAA and cyclophosphamide (CPA), alone or in combination with TAA, were administered in weekly doses. Twenty weeks after DMBA exposure, the mammary tumor yield was 2.4, 2.8 and 2.9 in the experimental groups compared to 3.5 in the controls. Seventy-five percent of the rats in the control group, but only 37% of TAA, 50% of the CPA, and 30% of the CPA and TAA treated animals had malignant tumors. In the experimental groups, 6.5%, 25% and 38%, respectively, of the tumors regressed, compared to 3% in controls. In the groups receiving CPA or TAA, regression was observed in the fifth week of treatment, and in the group receiving combined treatment, already in the first week. The size of the tumors in control rats increased during the last 10 weeks 3.6 times, in the CPA treated rats 1.15 times, but in those receiving CPA plus TAA it decreased by 0.7 times. The results of our experiment demonstrated that TAA have distinct tumor-suppressive properties, and can enhance the anticancer effects of CPA.

The effect of extremely large hepatomas on splenic lymphoid elements and apoptosis-related proteins in rats were studied. Hepatoma cells were inoculated subcutaneously into 6-week-old rats, and 4 months later the quantities of T and B cells, macrophages, and cells positive for Fas, Fas ligand (FasL) and interleukin-2 (IL-2) were immunohistochemically evaluated in spleens. Grafting of hepatoma cells caused hyperplasia of the spleen and development of giant tumors that could reach one-third of the rat's body weight. A 7-fold increase in the weight of the spleen was mainly due to proliferation of B lymphocytes and macrophages in the red pulp, while the relative quantity of CD4+ and CD8+ T cells decreased. Extremely small amount of Fas+ and FasL+ lymphocytes were present in the marginal zone, the follicles, red pulp, and occasionally in the PALS. All the splenic zones were abundant with IL-2+ cells, while macrophages and siderophages were present mainly in the red pulp and in the marginal zone of the white pulp. We suggest that all these changes are compensatory processes of the host's lymphatic system.

We examined the presence and distribution of components of the secretory immune system (SIS) in fetal endocrine organs and their embryonic precursors. Specimens from 16 embryos (4 to 8 weeks of development) and 32 fetuses (9 to 38 weeks) were divided into those that had not been exposed to massive foreign antigenic effects (Group I, n=28) and those that had suffered from chorioamnionitis (Group II, n=20). An immunohistochemical study was performed using antibodies against the secretory component (SC), joining (J) chain, IgA, IgM, IgG, subsets of T and B lymphocytes, and macrophages. Positive immunostaining for SIS components in the precursors of endocrine organs was seen from 4 to 6 weeks of development, and was present thereafter in the pituitary body, thyroid, pancreatic islets and adrenals. J chain and immunoglobulins were found in all endocrine cells throughout intrauterine development, but the massive antigenic influence caused by chorioamnionitis decreased the latters immunoreactivity. The presence of SC in the precursors of adenohypophysis and pancreatic islet cells decreased significantly after their transformation into definitive endocrine organs. In the thyroidal follicular epithelium and the pars intermedia of the pituitary body cells, SC was present during the entire period of pregnancy. In adrenals, SC was not found. Maternal immunoglobulins, together with SC and J chain, are accumulated in endocrine gland cells from the early stages of intrauterine life. They are the major mechanism of endocrine cell defense during the early prenatal period when the common immune system is still structurally and functionally incompetent.

Our studies on the relationships among the lymphoid system, apoptosis and apoptosis-related proteins (ARP) in human ovarian benign cysts, borderline tumors, and carcinomas are reviewed and analyzed. Fas and Fas ligand are expressed in 50% to 80% of the epithelial cells in all studied tumors. Many bcl-2-positive tumor epithelial cells are seen in benign cysts and they disappear as tumorigenesis progresses, whereas p53 protein is found only in borderline tumors and in carcinomas. Many exceptions to the opinion that bcl-2 inhibits apoptosis and p53 promotes it are encountered. Bcl-2 is lacking in epithelial cells of mucoid tumors of all grades, and its absence does not stimulate their apoptosis. P53 protein is absent from most lymphocytes, macrophages and epithelial tumor cells, nevertheless, they undergo apoptosis. Indeed, in many tumors apoptosis is regulated without the participation of bcl-2 and p53. Different components of the immune system become active during different stages of tumor development. The weak reaction of T-cell killers and macrophages is typical in benign cysts. In borderline tumors, the activity of T-cell killers increases in the parenchyma, and that of T helpers and macrophages in the stroma. In carcinomas with high lymphoid infiltration, a strong reaction of macrophages and T cell killers in the tumoral parenchyma as well high reaction of T helpers and B lymphocytes in the stroma are typical. Apoptosis that should protect against tumor also stimulates apoptotic death of lymphocytes and macrophages, and this has catastrophic consequences, as seen in weakly infiltrated carcinomas. In conclusion, our studies indicate that during malignancy the major task of the immune system is curtailment and control of tumorigenesis.