Morphometric abnormalities in the spleen of the progeny of mice fed epigallocatechin during gestation and nursing.

It is very difficult to cure pregnant females suffering from infections, because of the risk which might occur during treatment by several, even herbal, medications. Many of these substances, among them extracts from plants, have antimicrobial, anti-inflammatory and immunostimulatory properties owing to their polyphenols content, but also may reveal unwanted effects on the fetal development because of their anti-angiogenic properties. The aim of the present study was to elucidate whether daily feeding pregnant and nursing mice 0.2 mg/kg epigallocatechin (EGC), previously recognized as angiogenesis inhibitor, may lead to abnormalities in morphology of spleen and in some parameters of immune function of their adult, 6-week old progeny. Morphometry of EGC offspring spleens revealed lower number of lymphatic nodules and their larger diameter than those found in the control offspring. Cellularity of spleens was lower in EGC offspring than in the controls. Cytometric analysis showed that this decline concerns lymphocytes with CD335 (p<0.001), CD19 (p<0.01) and CD4 (p<0.05) markers. No differences were observed in the humoral response to the immunization with SRBC, and in the proliferative response of splenocytes to mitogens PHA, ConA and LPS.

Morphometric abnormalities in spleen and kidney of the progeny of mice fed American cranberry extract (Vaccinium macrocarpon) during pregnancy and lactation.

Cranberries and cranberry-derived diet supplements are often recommended for the treatment of urinary tract infections, also during pregnancy. These products contain strongly anti-angiogenic chemical compounds which could not be indifferent to the developing fetus. In the present work we evaluated the effect of feeding pregnant and lactating mice American cranberry extract (daily dose 0.88 mg) on the morphology and some parameters of spleen and kidney function of their adult progeny. Six weeks after delivery the morphometry of spleen and kidney, cytometric analysis of spleen lymphocytes, evaluation of humoral response to SRBC (Sheep Red Blood Cells), and examination of serum creatinine/urea concentration, were performed in the offspring. Spleens of progeny from experimental (E) group differed from the spleens of progeny of control mice in the lower number of lymphatic nodules and their larger diameter. Cytometry of spleen cells from progeny of E mothers revealed more CD19+ and CD8+ lymphocytes than in the control group. No difference was seen in the response to immunization by red blood cells of sheep (SRBC) between control and E offspring. An increase in the diameter of glomeruli was observed in the kidneys of the experimental group in comparison with the control group. No abnormalities in creatinine and urea serum level were observed. A higher concentration of VEGF and bFGF in E offspring sera in comparison to the controls was seen. CONCLUSION: Although the observed differences between the control and experimental group were not large, caution is recommended in using cranberries and their extracts during pregnancy until more research will be done on this topic.

Spleen content of selected polyphenols, splenocytes morphology and function in mice fed Rhodiola kirilowii extracts during pregnancy and lactation.

UNLABELLED: The genus Rhodiola (Crassulaceae) consists of many species, growing mainly in Asia and traditionally used as adaptogens and anti-inflammatory drugs. In order to elaborate herbal immunostimulator which could be safely given to pregnant women, we performed a study on immunotropic effects of feeding pregnant and lactating mice Rhodiola kirilowii extracts. This paper presents the results of the first part of our study - spleen content of selected polyphenols, spleen cellularity, splenocytes phenotype and their response to mitogens. Experiments were performed on adult inbred females of Balb/c strain, mated with adult males. Females, since copulatory plug was noted, up to the 28-th day after delivery were fed daily with 20 mg/kg b.m. water (RKW) or hydro-alcoholic (RKW-A) extracts of Rhodiola kirilowii. RESULTS: 1. Significantly lower proportion of pregnant mice in experimental groups than in the control. 2. Cellularity of spleen and flavonol quercetin spleen concentration were significantly lower in experimental groups in comparison to the controls. 3. Flavanols ((+)-catechin and epicatechin) levels were significantly higher in the spleens of experimental mice than in the controls. 4. Positive correlation between spleen cellularity and quercetin, and negative correlation between spleen cellularity and epicatechin content were observed. 5. Spleen mass and spleen lymphocytes phenotype and proliferation in RKW and RKW-A fed mice did not differ from the control. These results, together with suspicion of some embryo-toxicity, are worrying and eliminate the possibility of use Rhodiola kirilowii extracts for long-term treatment in pregnant females.

In vivo inhibitory effect of Aloe vera gel on the ability of mouse parental splenic lymphocytes to induce cutaneous angiogenesis in recipient F1 mice.

UNLABELLED: Lymphocyte-induced angiogenesis test (LIA) is a model of local graft-versus-host (GVH) reaction, marker of the earliest events resulting from activation of donor lymphocytes after contact with host semi-allogeneic histocompatibility antigens. The effect of in vivo oral administration of Aloe vera gel for 21 days to maternal strain (Balb/c) donor mice on the ability of their splenic lymphocytes to induce cutaneous angiogenesis (LIA test) in F1 Balb/c x C3H recipients, was studied. RESULTS: Neovascular reaction evaluated 72 hours after cells grafting was significantly lower in F1 mice grafted with lymphocytes collected from Aloe- fed donors, than in recipients of lymphocytes collected from respective controls. CONCLUSIONS: This observation opens the promise of safe and ethically acceptable possibility of use of Aloe vera gel in human donors in prevention of GVHD in recipients of bone marrow grafts.

Inhibitory effect of herbal remedy PERVIVO and anti-inflammatory drug sulindac on L-1 sarcoma tumor growth and tumor angiogenesis in Balb/c mice.

Anticancer activity of many herbs was observed for hundreds of years. They act as modifiers of biologic response, and their effectiveness may be increased by combining multiple herbal extracts . PERVIVO, traditional digestive herbal remedy, contains some of them, and we previously described its antiangiogenic activity. Numerous studies documented anticancer effects of nonsteroidal anti-inflammatory drugs. We were the first to show that sulindac and its metabolites inhibit angiogenesis. In the present paper the combined in vivo effect of multicomponent herbal remedy PERVIVO and nonsteroidal anti-inflammatory drug sulindac on tumor growth, tumor angiogenesis, and tumor volume in Balb/c mice was studied. These effects were checked after grafting cells collected from syngeneic sarcoma L-1 tumors into mice skin. The strongest inhibitory effect was observed in experimental groups treated with PERVIVO and sulindac together. The results of our investigation showed that combined effect of examined drugs may be the best way to get the strongest antiangiogenic and antitumor effect.

In vivo stimulatory effect of multi-component herbal remedy PADMA 28 on mitogen-induced proliferation of mice splenic lymphocytes and their chemokinetic activity.

UNLABELLED: PADMA 28, a natural herbal multi-compound remedy originates from traditional Tibetan medicine and possesses a variety of beneficial effects on experimental and clinical models of inflammation and atherosclerosis, as well as angioprotecive, antioxidative and wound-healing properties. The aim of the present study was to evaluate the in vivo influence of this remedy on the in vitro mitogen-induced proliferation of murine splenic lymphocytes and their chemokinetic activity in cell culture.The study was performed on 6-8 weeks old inbred Balb/c mice. PADMA28 was administered to mice per os in daily doses 5.8 mg (calculated from the highest dose recommended for human) or 0.085 mg (dose from the range of active doses of other herbal extracts containing polyphenolic substances used previously by us in experiments with mice), for 7 days. Control groups received water. RESULTS: No substantial differences were observed between groups of mice fed with low and high PADMA doses. In both of them, response of splenic lymphocztes to mitogen PHA (p < 0.001) and their in vitro chemokinetic activity (p < 0.001 for low dose and p < 0.01 for high dose) were highly significantly increased as compared to the controls. CONCLUSION: The results of our investigations suggest that PADMA 28 can stimulate cell-mediated immunity in mice and might be used for this purpose in the wide spectrum of doses.

The effects of prenatal exposure to methylxanthines.

This review discusses epidemiology and laboratory studies on the effects of prenatal methylxanthine administration on some systems developing organisms. They are mainly absorbed from coffee, tea and cocoa products such as cola beverages and chocolate bars. Prenatal methylxanthine exposure can induce several unfavourables changes in the developing organism, which are persistent even in later phases of life. Based on results obtained from animal studies, the effect on embryogenesis is not only poorly understood but also controversial. It is therefore important to study interspecies differences as results may differ depending on animals used and administration methods.

In vitro angiomodulatory activity of sera from type 2 diabetic patients with background retinopathy.

Diabetic retinopathy is the leading cause of adult vision loss and blindness. The most important contributors to the development of diabetic retinopathy are hyperglycemia and hypoxemia that lead to increased vasopermeability, endothelial cell proliferation, and pathological neovascularization. In our previous studies, close relationship between proangiogenic activity of sera from type 2 diabetes mellitus patients (DM2) with background retinopathy, assessed in the in vivo serum-induced mouse cutaneous test (SIA), and VEGF and IL-18 serum concentration were observed. Moreover, it was clearly shown that IGF-1 might play an important role in the negative regulation of neoangiogenesis induced by DM2 patients' sera by diminishing the VEGF stimulatory effect. To confirm the observed phenomenon we evaluated the effect of DM2 patients' sera on the in vitro proliferative activity of human endothelial cells, which is critical for the sprouting and generation of new blood capillaries. Endothelial proliferative activity was significantly higher in the presence of sera from DM2 patients than from healthy controls (P<0.001), as estimated by the MTT test. Moreover, the examined sera from DM2 patients were characterized by increased IL-18 (P<0.05), diminished IGF-1 (P<0.02), and unchanged VEGF levels compared with those in controls. In conclusion, the present study showed a strong stimulatory effect of DM2 patients' sera on the proliferation of endothelial cells, which, along with the findings of our previous studies, proves that the described phenomenon is universal and valid for both animal and human endothelium.

Immunohistochemical and microscopic studies on giant cells in tuberous sclerosis.

Tuberous sclerosis (TSC) is an autosomal dominant disease, caused by mutations in TSC1 or TSC2 genes, encoding hamartin and tuberin, respectively. The clinical picture of the disease is connected with the formation of hamartomas, mainly in the heart, kidneys and the brain. In three types of brain lesions: cortical tubers, subependymal nodules and subependymal giant-cell astrocytoma (SEGA) characteristic, so-called "giant cells" are found. In the present review we summarise immunohistochemical findings of two types of studies performed on giant cells aiming at establishing the expression of hamartin and tuberin level and determining the presence of neuron- or astrocyte-specific markers. Moreover, we support our argument with the summary of ultrastructural research done with the purpose of demonstrating structures characteristic of neural and/or glial cells. We conclude that giant cells in cortical tubers and SEGAs are the same undifferentiated cells that, depending on individual determination, can show neural or glial features.

Multiple effects of theobromine on fetus development and postnatal status of the immune system.

Caffeine and its active derivative, theobromine, are probably the most frequently ingested pharmacologically active substances. Considering their uninhibited transport via the placental barrier as well as immature enzymatic activities and metabolic pathways in embryos and infants resulting in the longer half-life of methyloxanthines and their accumulation, unrestrained uptake of these substances might result in noticeably more pronounced biological effects during pregnancy and the postnatal period. Our previous studies have shown that methyloxanthines are significant inhibitors of angiogenic growth factors production and angiogenesis itself. We have hypothesized that increased uptake of these substances might affect embryonal angiogenesis and, later in the postnatal period, maturation and functional activity of the offspring's immune system. The study was performed on 2-month-old Balb/c mice fed theobromine 2 or 6 mg/day during pregnancy and lactation. On day 18 of pregnancy the number and weight of embryos were assessed as was their tissue angiogenic activity, using the cutaneous angiogenesis assay. In the group of 4-week-old sucklings, body and spleen were weighed together with the trunk, and tail and limb length were measured. Six weeks after birth the splenocytes' mitogen-induced activity and their ability to induce graft-versus-host reaction as well as the humoral response to SRBC antigen were evaluated. Content of theobromine in the embryos' tissue was estimated by high liquid performance chromatography (HPLC). Theobromine feeding resulted in significant inhibition of embryo growth as assessed by their weight and decreased angiogenic activity of their tissue. The theobromine content in embryo tissue from treated groups was higher than in the controls, and the difference was close to significant. In the postnatal period the discrepancies in the treated 4-week-old group's development were also observed in the significantly shorter limbs in comparison to the controls. Moreover in the treated group of 6-week-old sucklings, considerable variations in the immune system's functional activity were registered as far as cellular and immune response were concerned. Respectively, the splenocytes' mitogen-induced proliferative activity was significantly suppressed while the graft-versus-host reaction was up-regulated, and the serum antibodies titer was elevated in correspondence to the observed spleen enlargement. We concluded that a theobromine-enriched diet affects progeny development in both prenatal and postnatal periods. Consequently, particular attention should be paid to the reduction of theobromine consumption, and most probably that of other methyloxanthines, during pregnancy and lactation.

Chocolate feeding of pregnant mice influences length of limbs of their progeny.

UNLABELLED: We previously reported the inhibitory effect of various methyloxantines and phenolic compounds on tumor-induced angiogenesis and the production of angiogenic growth factors. The aim of the present work was to evaluate the effect of chocolate (CH), food containing substantial amounts of methyloxantine theobromine and polyphenols (mainly catechins), given to mice during pregnancy and the lactation period, on weight of organs, length of limbs, and bone vascular endothelial growth factor (VEGF) concentration (tested by ELISA), in 4-week old offspring. The study was performed on 2-month old Balb/c mice fed during pregnancy and lactation 400 mg of CH daily. Content of polyphenols (catechines) and theobromine in the chocolate was estimated by high liquid perforance chromatography (HPLC). Concentration of VEGF was tested by ELISA. Feeding pregnant mice chocolate produced the following effects: decrease of relative length of limbs and thigh bones in 4-week old progeny and decrease in VEGF content of offspring femoral bones. CONCLUSION: attention should be paid to possible unwanted effects of catechine- and methyloxantine-rich food and beverages during pregnancy and lactation. 200 mg of chocolate per mouse corresponds to 100 g per person.

Pentoxifylline inhibits perforin-dependent natural cytotoxicity in vitro.

Pentoxifylline (PTX) is commonly used in peripheral blood vessel diseases, however it has also been found to decrease the level of proinflammatory cytokines such as IL-12, TNF-alpha and IFN-gamma. Moreover, some authors reported that PTX suppresses spontaneous cytotoxicity of peripheral blood mononuclear cells (PBMC) in vitro. It could influence the mechanism of killing target cells by PBMC. For this reason we evaluated the influence of PTX on spontaneous cytotoxicity of PBMC against K562 and CaSki cell lines. Subsequently, we compared this effect to that evoked by dexamethasone, one of the most effective anti-inflammatory drugs. Our study revealed that PTX inhibits natural cytotoxicity preferentially through inhibition of perforin-mediated cell membrane damage, without a statistically significant influence on apoptosis induction. Furthermore, pentoxifylline inhibits natural cytotoxicity as effectively as dexamethasone. However, the result of PTX inhibitory influence is observed much earlier than that of dexamethasone. Currently PTX is commonly used in diseases that occur more frequently in elderly patients. We suggest that PTX, inhibiting perforin-dependent PBMC cytotoxic activity, could weaken anti-cancer action of immune system thus accelerating the progress of neoplasm formation in these patients.

Clinical aspects of disrupted Hedgehog signaling (Review).

The Hedgehog (HH) signaling pathway is involved in patterning and development of a variety of organ systems, including the nervous system, the skeletal system, the craniofacial structures, and the gastrointestinal tract. Recent evidence also implicates this signaling pathway in the postembryonic regulation of stem-cell number in epithelia and blood. The family of HH proteins consists of at least three different members, i.e., sonic HH (SHH), Indian HH (IHH), and desert HH (DHH). SHH is the most broadly expressed member of this family and is probably responsible for the major effects of this signaling pathway. The HH signal is received and transduced via a specific receptor complex composed of patched (PTCH) and smoothened (SMOH) transmembrane proteins. Abnormalities in this signaling cascade have been found in various developmental pathologies and neoplasms such as basal cell carcinoma. The abnormalities are associated with congenital or sporadic genetic alteration affecting function of different components of the HH signaling pathway, including SHH, PTCH, SMOH and GLI proteins.

Potential application of cytokine level measurement in corneal epithelium.

The aim of the work was to evaluate whether rat corneal epithelial (RtCE) cell line, spontaneously established from rat corneal epithelium in our laboratory, could be used for the evaluation of cornea inflammatory state. Production of proinflammatory cytokines IL-1beta, IL-6 and TNF-alpha by RtCE line in response to a non-specific irritating agent (Triton) was tested. Supernatants from RtCE cells treated for 1 h with 20 microM, 50 microM and 100 microM Triton, were collected after 1 and 24 h, and tested with enzyme-linked immunosorbent assay (ELISA) for IL-1beta, IL-6 and TNF-alpha. The control groups did not produce significant levels of any of the cytokines. However, after stimulation with Triton, the cells did not produce TNF-alpha, while the concentration of IL-1beta and IL-6 increased over 10 times. These results show that in response to a proinflammatory agent RtCE line produces cytokines that could be used for measuring the effect of irritants on the cornea.

Characterisation of epithelial cell line from rat cornea.

PURPOSE: Characterisation of RtCE-1 cells, newly established rat corneal epithelial cell line. METHODS: Morphology of RtCE-1 cells was characterised by light and electron microscopy. Expression of cytokeratins was studied by polyacrylamide electrophoresis and Western blotting. Characterisation of RtCE-1 cells also included karyotype analysis and in vitro study of growth kinetics. RESULTS: The line shows morphological similarities to normal corneal epithelium. The cells express cornea-specific cytokeratins. Karyotype analysis revealed that the cells are polyploid with modal number of chromosomes at passage 30 equalling 72 +/- 14. Growth of the line is partially dependent on EGF. CONCLUSION: RtCE-1 cells may constitute a model for the evaluation of proliferation, function and differentiation of corneal epithelium in vitro.

Angiotensin-converting enzyme activity and angiomodulatory effects of sera in patients with diabetic retinopathy.

Aberrant neovascularization plays a crucial role in ocular complications in diabetic patients. Sera from these patients contain high levels of angiostimulatory factors, the most important of which is vascular endothelial growth factor (VEGF). Many authors have described elevation of angiotensin-converting enzyme (ACE) activity in the sera of diabetic patients. It is important to determine the possible relationship between these two phenomena. We studied ACE serum activity and VEGF concentrations in patients with type 1 and type 2 diabetes and retinopathy We also investigated the effect of their sera on cutaneous angiogenesis induced in mice by grafting healthy human mononuclear blood leukocytes. We found a negative correlation between the angiostimulatory effect and ACE level in the sera of patients with type 1 diabetes and no correlation between these two parameters in patients with type 2 diabetes. VEGF concentrations were lower and ACE activity was significantly higher in the sera of patients with type 1 diabetes than in the sera of those with type 2 diabetes.

[Studies on conditions for the culture of rabbit corneal epithelium]. / Badania nad ustaleniem optymalnych warunków hodowli nablonka rogówki królika.

PURPOSE: Research was aimed at comparison of isolation methods as well as determination of growth and differentiation dynamics of rabbit corneal epithelium (CE) in vitro. Adhesion, growth and differentiation of CE cells growing on collagen membranes were evaluated. MATERIAL AND METHODS: Research was performed on the cells of rabbit corneal epithelium isolated mechanically or enzymatically (Dispase II) from comeas excised at the edge of limbus. CE cells were cultured in media with high or low contents of calcium, with addition of FCS, insulin, cholera toxin and EGF. RESULTS: In comparison with mechanical isolation, enzymatic isolation yielded 4-5 times more living undifferentiated (CE) cells. The highest dynamics of in vitro growth was observed in primary cultures in low-calcium medium supplemented with the above substances. After 20 population doublings cells were differentiated and died. Only few cells on collagen membranes adhered to the collagen but did not enter division. CONCLUSIONS: Current research allowed for determination of methodology for CE excision and isolation. Optimal conditions for in vitro growth have been established. Growth dynamics and proliferation of CE in vitro have been evaluated. Growth of CE on standard collagen membrane has not been observed.

Production of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha by a rat corneal epithelial cell line.

Production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by rat corneal epithelial cells in response to lipopolysaccharide and phorbol-12-myristate-13-acetate (PMA) was tested. Supernatants from rat corneal epithelial cells treated with lipopolysaccharide and PMA were collected after 6, 24 and 48 h and tested with enzyme-linked immunosorbent assay for IL-1 beta, IL-6 and TNF-alpha. The activity of TNF-alpha was additionally confirmed with bioassay on L929 cells. It was found that control groups did not produce significant levels of either cytokine. However, after stimulation with lipopolysaccharide, cells produced mainly IL-6, whereas after PMA they produced mainly TNF-alpha. IL-6 levels 24 and 48 h after PMA stimulation were also elevated, which could have been caused by the presence of TNF-alpha. Production of IL-1 beta in all groups was very low and remained within the test sensitivity range. These results show that the rat corneal epithelial cell line produces inflammatory cytokines in response to proinflammatory mediators. For this reason, it could be used for measuring the effects of irritants on the cornea.

The effect of salbutamol treatment on the cellular immunity of the offspring of pregnant mice: spleen cell activity.

Preterm delivery is one of the greatest problems in obstetric care. One of the most commonly used treatments for high risk cases is salbutamol, a beta-2-adrenoceptor agonist. The aim of the present study was to determine if such treatment causes any changes in the neonatal immune system which should therefore be a concern in the care of the newborn. The experiments were performed on 4 to 5 or 6 to 7-week old female and male offspring of salbutamol-treated C3H/W inbred mice. In the first part of the study, the number of spleen cells, phenotypes and activity (phytohemagglutinin-induced proliferation, ability to induce local graft versus host reaction) were determined. We observed lowering of cell number and lowered proportions of cluster of differentiation (CD)3, CD4 and CD8 positive lymphocytes in spleens of progeny of salbutamol-treated mice. However, CD4+ to CD8+ ration was higher in the progeny of salbutamol-treated mothers than in the corresponding controls. In addition, reactivity to phytohemagglutinin and ability to induce local graft vs. host reaction were higher (popliteal lymph node test) or undisturbed (lymphocyte-induced angiogenesis test) in this group of mice.

[Morphological characteristics of epithelial cell line from rat cornea]. / Charakterystyka morfologiczna linii komórkowej RtCE wyprowadzonej z przedniego nablonka rogówki szczura.

PURPOSE: Morphological characteristics of established cell line (RtCE) derived from rat anterior corneal epithelium. MATERIAL AND METHODS: Morphology of cultured RtCE cells was evaluated by light and electron microscopy. Anterior corneal epithelium of rat served as control. RESULTS: Both in light and electron microscope we observed morphological similarity with anterior corneal epithelium (several layers of cells, numerous desmosomal connections). Moreover, similar morphological changes were observed in relation to the control material, i.e. degeneration and flattening of cells in superficial layers. CONCLUSIONS: The morphological analysis of RtCE cell line proves its usefulness for further cytomorphological, cytogenetical and functional analyses, as well as for evaluation of influence of different physico-chemical and biological factors on growth and regeneration of corneal epithelium in vitro.