Biomedical subjects

F Kohen

Publications and source records attributed to F Kohen.

At least 19 recordsLinked to original sources

PubMed2005

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A.

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.

Binding Sites↗

PubMed2004

Role of putative membrane receptors in the effect of androgens on human vascular cell growth.

We have reported previously that dihydrotestosterone (DHT) induces a biphasic effect on DNA synthesis in human vascular smooth muscle cells (VSMC), i.e. stimulation at low concentrations and inhibition at high concentrations. In contrast, DHT dose-dependently stimulated [(3)H]thymidine incorporation in a human endothelial cell line (ECV304). Additionally, DHT increased the specific activity of creatine kinase (CK) in both vascular cell types. In the present study, we have determined whether some of these effects are exerted via membrane-binding sites. We measured changes in DNA synthesis and CK after treatment with DHT and the membrane-impermeant testosterone-3-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (T-BSA). High concentrations of either DHT or T-BSA inhibited VSMC proliferation (by 52+22% and 51+25% respectively). DHT as well as T-BSA increased DNA synthesis in ECV304 cells dose-dependently. In contrast, T-BSA did not affect CK in either cell type. In both cell types, DHT as well as T-BSA increased mitogen-activated protein kinase (MAPK) kinase activity as measured by total phosphorylated MAPK. Further, the inhibitory effect of either the free or protein-bound androgens on DNA synthesis was blocked by UO126, an inhibitor of MAPK kinase activity. T-BSA conjugate labeled with Europium showed binding to whole VSMC, which could be displaced by excess T-BSA, but not by estradiol-BSA or the free hormones. Finally, using T-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for androgen in VSMC. Hence, the inhibitory effects of testosterone on DNA synthesis in VSMC are apparently exerted by membrane-binding sites for androgen, do not require intracellular entry of the hormone and its binding to the classical nuclear receptors and are linked to MAPK activation.

Butadienes↗

PubMed2003

Preliminary evaluation of a lateral flow immunoassay device for screening urine samples for the presence of sulphamethazine.

A lateral flow immunoassay (LFIA) device was developed and applied to testing urine samples for residues of the antimicrobial sulphamethazine (SMZ). This report describes the preparation of a rat monoclonal antibody to SMZ and its characterisation in an ELISA format. Apart from SMZ, the antibody showed high (> or =50%) cross-reactivity to N4-acetyl-sulphamethazine (55%), sulphamerazine (59%) and sulphisoxazole (50%) and lower cross-reactivity of 18% to sulphachlorpyridazine and sulphadiazine. The LFIA device consisted of a nitrocellulose membrane spotted with SMZ-ovalbumin and goat anti-mouse antibody as capture line and control line, respectively. Mouse anti-rat IgG F(ab')2 fragment specific antibody, adsorbed to colloidal carbon, was used as the detection ligand in the LFIA. The LFIA device had a cut-off value of 6.3 ng/ml in diluted (1/10) urine. Urine samples from SMZ-treated pigs, and bovine and porcine urine samples fortified with SMZ were used for a blind, four-laboratory evaluation of the performance of the LFIA device. Concentrations of SMZ in the test samples (n=29), as determined by LC-MS/MS, ranged from 0 (<3) to 1174 ng/ml. The evaluation of the LFIA device showed an overall sensitivity of 100%, a specificity of 71%, and positive and negative prediction values of 73% and 100%, respectively. The LFIA device has been fabricated as a test kit for determining SMZ residues in animals produced for slaughter.

Animals↗

PubMed2003

Carboxy derivatives of isoflavones as affinity carriers for cytotoxic drug targeting in adrenocortical H295R carcinoma cells.

Carboxy derivatives of isoflavones that exhibit oestrogenic/anti-oestrogenic properties were used as carriers for affinity drug targeting to H295R adrenocortical carcinoma cells that express transcripts of oestrogen receptor (ER) alpha and beta. These derivatives were prepared by introducing a carboxymethyl group at the 6-position of genistein and of biochanin A, yielding 6CG and 6CB respectively. In transactivation assays, 6CG displayed mixed agonist/antagonist activity for ERalpha, whereas 6CB displayed only weak antagonist activity. Low concentrations of oestrogen, 6CG and 6CB were capable of inducing proliferation in H295R cells and of stimulating creatine kinase (CK) specific activity, suggesting that these cells were sensitive to oestrogenic compounds. In in vivo experiments, both 6CG and 6CB were capable of inhibiting oestrogen-induced CK specific activity in rat tIssues. For affinity drug targeting, the cytotoxic drug daunomycin was coupled to 6CB and 6CG, yielding 6CB-Dau and 6CG-Dau respectively. These conjugates were tested for their antiproliferative ability to inhibit DNA synthesis as assessed by incorporation of [(3)H]thymidine in H295R cells. A dose-dependent cytoxicity was observed with both conjugates. At 0.3-3 nM, both conjugates were 10 to 30 times more potent than daunomycin. At 30 nM these conjugates were two to three times more potent than daunomycin. At concentrations ranging between 300 and 3000 nM, no difference in cytotoxicity was observed between the conjugates and daunomycin. When the cells were treated over a wide range of concentrations with a combination of 6CG plus daunomycin, the observed cytotoxicity was less than with daunomycin alone. When non-transformed rat enterocytes, which do not express ER, were treated with 6CG-Dau or daunomycin, the antiproliferative effect of 6CG-Dau was the same as that of daunomycin over the concentration range tested. These pilot studies suggest that the ready availability of oestrogenic binding sites in H295R cells can be exploited for site-directed chemotherapy.

Adrenal Cortex Neoplasms↗

PubMed2002

6-Carboxymethyl genistein: a novel selective oestrogen receptor modulator (SERM) with unique, differential effects on the vasculature, bone and uterus.

The novel genistein (G) derivative, 6-carboxymethyl genistein (CG) was evaluated for its biological properties in comparison with G. Both compounds showed oestrogenic activity in vitro and in vivo. On the other hand G and CG differed in the following parameters: (i) only CG displayed mixed agonist-antagonist activity for oestrogen receptor (ER) alpha in transactivation assays and (ii) only CG was capable of attenuating oestrogen (E(2))-induced proliferation in vascular smooth muscle cells and of inhibiting oestrogen-induced creatine kinase (CK) specific activity in rat tissues. On the other hand only G enhanced the stimulatory effect on CK specific activity in the uterus. In comparison to the selective oestrogen receptor modulator (SERM) raloxifene (RAL), CG showed the same selectivity profile as RAL in blocking the CK response to E(2) in tissues derived from both immature and ovariectomized female rats. Molecular modelling of CG bound to the ligand binding domain (LBD) of ERbeta predicts that the 6-carboxymethyl group of CG almost fits the binding cavity. On the other hand, molecular modelling of CG bound to the LBD of ERalpha suggests that the carboxyl group of CG may perturb the end of Helix 11, eliciting a severe backbone change for Leu 525, and consequently induces a conformational change which could position Helix 12 in an antagonist conformation. This model supports the experimental findings that CG can act as a mixed agonist-antagonist when E(2) is bound to its receptors. Collectively, our findings suggest that CG can be considered a novel SERM with unique effects on the vasculature, bone and uterus.

Analysis of Variance↗

PubMed2001

Effects of phytoestrogens on DNA synthesis and creatine kinase activity in vascular cells.

BACKGROUND: The aim of this study was to assess the effect of phytoestrogens on the human vascular wall in vitro. METHODS: We compared the effects of E2 to those of genistein (G), daidzein (D), biochanin A (BA), equol (EQ), and quecertin (Qu) on 3[H] thymidine incorporation and creatine phosphokinase (CK) activity in human vascular smooth muscle cells (VSMC) and in a human endothelial cell line (E304). RESULTS: In VSMC, E2, the estrogen antagonist raloxifene (RAL), G, and D stimulated DNA synthesis at low concentrations and suppressed 3[H] thymidine incorporation at higher concentrations. In contrast, BA and EQ had a monophasic stimulatory effect on 3[H] thymidine incorporation (87% +/- 9% and 54% +/- 17%, respectively) whereas Qu had only an inhibitory effect (-36 +/- 16% at 30 nmol/L). In E304 cells, all phytoestrogens stimulated DNA synthesis in a dose-related manner. In both cell types E2, RAL as well as all phytoestrogens increased CK-specific activity. The administration of phytoestrogens to immature female rats resulted in increased CK in the aorta (Ao) (60% to 220%) and in the left ventricle of the heart (Lv) (45% to 160%). Similar increases in Ao and Lv CK were also induced by E2 and all five phytoestrogens in ovariectomized (OVX) female rats. RAL antagonized phytoestrogen-induced CK activity in human vascular cells and in the rat Ao and Lv tissue but did not block phytoestrogen effects on DNA synthesis in human VSMC. CONCLUSIONS: Although phytoestrogens have estrogen-mimetic effects on cell growth and CK in cultured human vascular cells and on CK in rat vascular tissues in vivo, the effects on human VSMC replication are highly dependent on the concentration and the particular phytoestrogen under investigation.

Animals↗

PubMed2001

The role of the Fas/Fas ligand system in estrogen-induced thymic alteration.

PROBLEM: Estrogen induces atrophy in the thymus by an unknown mechanism. Since the Fas/FasL system is one of the main pathways in T cell apoptosis, we tested the hypothesis that estrogen-induced thymic atrophy is mediated by the Fas/FasL system. METHODS OF STUDY: In vivo experiments were done using ovariectomized female rats treated with estrogen or saline. In vitro experiments were performed using isolated thymocytes. Estrogen receptor (ER) alpha and beta expression was characterized using flow cytometry, RT-PCR and immunofluorescence. Fas and FasL mRNA and protein expression was evaluated using RT-PCR and Western blot analysis respectively. RESULTS: ERalpha and ERbeta are present in thymocytes and stromal cells. ER expression is mainly localized in the Double Positive CD4+CD8+ thymocytes. Estrogen treatment decreases thymus size and increase FasL expression. CONCLUSION: CD4+CD8+ thymocytes and thymic stroma cells express ERalpha and ERbeta. In vivo and in vitro we showed that estrogen treatment increases FasL expression while decreasing thymus cell number. These findings support the hypothesis that estrogen-induced thymic atrophy occurs as a result of apoptosis and is mediated by estrogen-induced FasL expression.

Animals↗

PubMed2001

A novel form of platelet-type 12-lipoxygenase mRNA in human vascular smooth muscle cells.

The lipoxygenase pathway has been implicated in the growth, migration, and contraction of vascular smooth muscle cells (VSMCs). However, the precise type of lipoxygenase present in the vascular wall has not been characterized. In this study, we used a specific reverse-transcriptase polymerase chain reaction method with 2 sets of specific primers on total RNA and polyA (+)RNA of normal human VSMCs prepared from umbilical artery. Two forms of platelet-type 12-lipoxygenase mRNA were present in human VSMCs: the already published form cloned from human erythroleukemia cells and a variant form of platelet-type 12-lipoxygenase, which includes 2 additional sequences consistent with the 2 introns (D and E). This novel form of 12-lipoxygenase poly A (+)RNA was downregulated by lipopolysaccharide (10 ug/ml) and upregulated by epidermal growth factor (100 ng/ml) but was not affected by angiotensin II (10(-7) mol/l). We developed a rabbit anti-human platelet-type 12-lipoxygenase polyclonal antibody directed against a 24-amino acid peptide encoded within exon 4. Western immunoblotting of protein extracted from VSMCs and umbilical artery and platelet extract with this antibody showed a coordinate 110-kDa protein and the already-described 70-kDa band detected in platelets and cord homogenate. Another 120-kDa protein was consistently detected in cord extracts but not in platelet or VSMC homogenates. The immunohistochemistry study performed with the same antibody showed extensive cytoplasmic staining of VSMCs. The specific role of these different forms of platelet-type 12-lipoxygenase is subject to further investigation.

Alternative Splicing↗

PubMed2001

Antivascular treatment of solid melanoma tumors with bacteriochlorophyll-serine-based photodynamic therapy.

We describe here a strategy for photodynamic eradication of solid melanoma tumors that is based on photo-induced vascular destruction. The suggested protocol relies on synchronizing illumination with maximal circulating drug concentration in the tumor vasculature attained within the first minute after administrating the sensitizer. This differs from conventional photodynamic therapy (PDT) of tumors where illumination coincides with a maximal concentration differential of sensitizer in favor of the tumor, relative to the normal surrounding tissue. This time window is often achieved after a delay (3-48 h) following sensitizer administration. We used a novel photosensitizer, bacteriochlorophyll-serine (Bchl-Ser), which is water soluble, highly toxic upon illumination in the near-infrared (lambda max 765-780 nm) and clears from the circulation in less than 24 h. Nude CD1 mice bearing malignant M2R melanotic melanoma xenografts (76-212 mm3) received a single complete treatment session. Massive vascular damage was already apparent 1 h after treatment. Changes in vascular permeability were observed in vivo using contrast-enhanced magnetic resonance imaging (MRI), with the contrast reagent Gd-DTPA, by shortening spin-spin relaxation time because of hemorrhage formation and by determination of vascular macromolecular leakage. Twenty-four hours after treatment a complete arrest of vascular perfusion was observed by Gd-DTPA-enhanced MRI. Histopathology performed at the same time confirmed primary vascular damage with occlusive thrombi, hemorrhage and tumor necrosis. The success rate of cure of over 80% with Bchl-Ser indicates the benefits of the short and effective treatment protocol. Combining the sensitizer administration and illumination steps into one treatment session (30 min) suggests a clear advantage for future PDT of solid tumors.

Animals↗

PubMed2000

Measurement of estrogen receptors in intact cells by flow cytometry.

BACKGROUND: Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D(5). The stained cells were analyzed by flow cytometry. RESULTS: The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80-99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5. CONCLUSIONS: Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis.

Antibodies, Anti-Idiotypic↗

PubMed2000

Decreased ovarian hormones during a soya diet: implications for breast cancer prevention.

Ovarian hormones are biomarkers for breast cancer risk. Soybean consumption may be responsible in part for lower levels of ovarian hormones and decreased rates of breast cancer in women in Asia compared with Western populations. Soybeans contain a significant amount of the isoflavones daidzein and genistein, which are weak estrogens. The purpose of this study was to determine whether soya feeding decreases circulating levels of ovarian hormones and gonadotropins. Ten healthy, regularly cycling women consumed a constant soya-containing diet on a metabolic unit, starting on day 2 of a menstrual cycle until day 2 of the next cycle. Blood and urine samples were obtained daily for one menstrual cycle before and during soy feeding. The diet was calculated to maintain constant body weight, included 400 kilocalories from a 36-ounce portion of soymilk, and provided 113-207 mg/day (154.0+/-8.4 mg/day, mean +/- SE) of total isoflavones. For the group, the soya diet provided more carbohydrate and less protein than the home diets. Daily consumption of the soya diet reduced circulating levels of 17beta-estradiol by 25% (P<0.01, Wilcoxon signed rank test, two-tailed) and of progesterone by 45% (P<0.0001) compared with levels during the home diet period but had no effect on luteinizing hormone or follicle-stimulating hormone. Mean menstrual cycle length did not change during the soya diet; a slight decrease in mean luteal cycle length was marginally statistically significant (P = 0.06). Urinary excretion of isoflavones was 33.8+/-5.3 mg/day (mean +/- SE) and when expressed as percentage of intake, varied substantially (21.9+/-3.3% of intake; range, 9.1-36.7%) among the subjects. Mean daily serum levels of daidzein and genistein (free and conjugated forms) 15 h after soymilk were 2.89+/-0.53 microg/ml and 0.85+/-0.22 microg/ml, respectively, indicating systemic bioavailability of these substances. Secondary analyses by multiple regression showed that decreases in follicular and luteal phase 17beta-estradiol levels were positively associated with urinary isoflavone excretion, an association affected by age, and were inversely associated with decreases in protein intake. Decreases in progesterone levels during the soya diet were inversely associated with increases in intakes of genistein and were affected by the interaction of the intakes of daidzein with energy or with fiber. Consumption of an isoflavone-containing soya diet reduced levels of ovarian steroids in normal women over the entire menstrual cycle without affecting gonadotropins. This suggests that at least under the conditions of this study, soya-induced reductions of circulating ovarian steroids are not mediated by gonadotropins. Decreases in ovarian hormones are related to isoflavones contained in soy and also to energy intake and other components such as protein and fiber but not fat. Our results may explain decreased ovarian hormone levels and decreased risk of breast cancer in populations consuming soya diets and have implications for reducing breast cancer risk by dietary intervention.

Adult↗

PubMed2000

Vitamin D analogs modulate the action of gonadal steroids in human vascular cells in vitro.

We have previously reported that estradiol (E2) and dihydrotestosterone (DHT) regulate cell growth in human umbilical arterial smooth muscle cells (SMC) and in an endothelial cell line (E304). In SMC both gonadal steroids stimulated DNA synthesis at low concentrations and suppressed 3[H] thymidine incorporation at high concentrations, whereas in E304 cells E2 and DHT dose dependently enhanced DNA synthesis. In both cell types gonadal steroids also induced the specific activity of creatine kinase BB (CK). Previous evidence suggets that the in vitro and in vivo CK responses to gonadal steroids in bone cells are upregulated by pretreatment with vitamin D analogs due to increased level of cellular estrogen receptors (ER). Here we analyzed the interaction of the vitamin D analogs hexafluorovitamin D (FL), JK-1624 F2-2 (JKF), and CB 1093 (CB) with gonadal steroids in regulating DNA synthesis and CK activity in human vascular cells in vitro. In E304 cells, daily treatment with FL, JKF, or CB (1 nmol/L for 3 days) increased DNA synthesis by 110 +/- 11%, 65 +/- 16%, and 88 +/- 23% respectively. In contrast, the same analogs inhibited 3[H] thymidine incorporation by 52 +/- 21%, 46 +/- 19%, and 50 +/- 10%, respectively, in SMC. In both cell types all three analogs increased CK by 25% to 75% and amplified the CK response to E2 and to DHT by twofold to threefold. In E304 cells the vitamin D analogs also increased DNA response to gonadal steroids from 50% to 60% to 200% to 280%. In SMC these analogs did not modify the DNA synthetic response to a low E2 concentration, but prevented the suppression of DNA synthesis exerted by high concentrations of E2 and DHT. Vitamin D inhibitors known to block cellular calcium mobilization, had no effect on the proliferative activity induced by vitamin D analogs. However, the inhibitor of the nuclear effects of vitamin D, ZK 159222, blocked the stimulatory effects of CB on DNA synthesis in E304 cells. Finally, both 1,25(OH)2 D3, and JKF decreased the expression of ERbeta proteins in SMC and increased the ERalpha isoform in E304 cells by 40% to 75%. The results indicate that vascular cells are targets for both vitamin D and gonadal steroid action and suggest a possible interaction between these hormones in the regulation of cell proliferation via modulation of vascular ER or interaction with proteins associated with ER.

Androgens↗

PubMed2000

Influence of the hapten conjugation site on the characteristics of antibodies generated against metabolites of clostebol acetate.

4-chloro-androst-4-ene-3,17-dione (CLAD) and 4-chlorotestosterone (clostebol, beta-CLT or CLT) were made immunogenic by coupling to protein carriers via the 3 and 17 positions, respectively. These immunogens were used to elicit polyclonal and monoclonal antibodies to CLAD and to clostebol. The antibodies were characterized in an enzyme immunoassay for sensitivity and specificity. Polyclonal antisera generated through position 17 reacted preferentially with 4-chlorotestosterone-17-acetate (clostebol acetate, CLTA), 4-chloro-epitestosterone (epi-clostebol, 17alpha-clostebol, 17alpha-CLT), and clostebol, whereas polyclonal antisera generated through the 3 position almost did not react with these derivatives. Interestingly, the monoclonal antibody generated through the 3 position recognized (35%) epi-clostebol. These results suggest that polyclonal antisera generated through the 17 position have a broad specificity profile and can be used to analyze by immunoassay methods urinary metabolites of clostebol acetate and thereby detect the illegal use of clostebol acetate in livestock farming.

Antibodies, Monoclonal↗

PubMed2000

Apoptosis and apoptosis-related proteins (Fas, Fas ligand, Blc-2, p53) in lymphoid elements of human ovarian tumors.

Different types of lymphocytes have different roles in tumor suppression. Thus, their expression of apoptosis-related proteins (ARP - Fas and Fas ligand, bcl-2, p53) in lymphocytes and their apoptosis were analyzed immunohistochemically in ovarian tumors of different grades. Ovaries without oncologic disorders had few lymphocytes, mainly T cells, and no ARP. Benign cysts presented features of weak immune reaction: small lymphoid infiltration and few lymphocytes. The ARP were present in 13.7% to 23.5% of the lymphocytes, and apoptosis was rare. In borderline tumors, expansion of lymphoid infiltrates and increased density of lymphocytes resulted in a tenfold rise in total lymphocytes, reflecting intensification of the immune response. Most lymphocytes were T cells (92%) predominated by CD8+ cells that were in direct contact with tumor epithelial cells. ARP species were found in 47% to 65% of the lymphocytes, and apoptosis in 2.2%. In carcinomas with ligh lymphoid infiltration, lymphocytes were 2.5 times more abundant, and the apoptotic index as well as the number of CD20+ and CD25+ lymphocytes rose sharply, whereas bcl-2 positive lymphocytes decreased to 8% of their number in borderline tumors. In carcinomas with low lymphoid infiltration, the total lymphocyte count decreased eightfold compared to carcinomas with high lymphoid infiltration, reflecting the deep subcompensation of the lymphoid system. Few p53-positive lymphocytes were found in the carcinomas. In conclusion, we found a positive correlation between apoptosis and the numbers of CD4+ or CD8+ lymphocytes in epithelial ovarian tumors. This correlation could reflect the antitumor activity of T cells. However, the high expression of ARP studied by immune cells at the vicinity of the tumor ARP reveals the lymphoid vulnerability to apoptosis, resulting in devastation of the lymphoid tissue, and consequently in tumor progression.

Adenocarcinoma↗

PubMed1999

TRH is a tonic secretagogue in growth hormone secreting but not in nonfunctioning pituitary tumors.

In most patients with growth hormone (GH) secreting pituitary adenomas and clinically nonfunctioning pituitary tumors (NFPT) the intravenous injection of thyrotropin releasing hormone (TRH) augments the secretion of GH and subunits of gonadotropin hormones respectively. Similar hormone responses to TRH have been detected in rat pituitary cell lines and in primary human pituitary tumor cultures in vitro. Nevertheless the TRH effect on tumor hormonal secretion has not been well characterized. In the present study we examined TRH-induced hormone secretion in GH secreting tumors and in NFPT in vitro. Cultured cells secreted betaLH and betaFSH (NFPT) or GH (GH secreting adenomas) up to 14 days in culture. In NFPT TRH (10(-8) mol/l) elicited peak betaLH and betaFSH secretion at 60 to 90 min, with no further increase at 24 h. TRH-stimulated GH secretion peaked at 90-120 min, and decreased after 3 h, but a secondary rise occurred after 24 h of incubation. Chronic daily exposure to TRH followed by an acute TRH challenge resulted in a further increase of GH secretion after one hour. In contrast, acute TRH administration following chronic exposure did not elicit increased P-subunits secretion in NFPT. Coadministration of cycloheximide did not change TRH induced beta-subunits secretion in NFPT. However, when it was administered 24 h prior to TRH, it blocked both basal and TRH induced beta-subunits levels in NFPT. Cycloheximide had no effect on basal or stimulated GH secretion when administered concomitantly or 24 h before TRH. Incubation of cultured GH secreting tumors with cycloheximide during 5 days blocked both basal and TRH stimulated GH secretion, thus indicating dependency on protein synthesis during the chronic, secondary phase. Since the acute secretion was not affected by coadministration of cycloheximide, these early increases in hormone levels apparently reflect the release of stored hormone. In summary, GH secreting adenomas and NFPT differ significantly in their hormonal response to continuous exposure to TRH. The mechanisms underlying the sustained effect of TRH on GH secretion in vitro remain to be investigated. If endogenous TRH exerts a similar continuous effect it may contribute to the disregulated GH secretion in acromegaly.

Adenoma↗

PubMed1999

Platelet-derived endothelial cell growth factor inhibits DNA synthesis in vascular smooth muscle cells.

Platelet-derived endothelial cell growth factor (PD-ECGF) is linked to angiogenesis in human cancer. Direct studies have demonstrated that PD-ECGF is a potent mitogen for endothelial cells in vivo. Because endothelial repair and smooth muscle cell proliferation are two processes that affect arterial wall structure and tone, we analyzed the effects of PD-ECGF on DNA synthesis and creatine kinase BB-specific activity (CK) in human umbilical artery smooth muscle cells (SMC) and in a human umbilical endothelial cell line (E304). In SMC, PD-ECGF (0.001 to 10 U/mL) inhibited DNA synthesis dose dependently (-24% + 6% to -63% + 15%) assessed by 3[H]thymidine incorporation into DNA, whereas in E304 it stimulated DNA synthesis dose dependently (30% + 4% to 100% + 4%). In both SMC and E304, however, PD-ECGF elicited an increase in CK-specific activity by 54% to 130% and 79% to 163%, respectively. These effects were reversed by a specific anti-PD-ECGF antibody. In E304 cells PD-ECGF enhanced 17beta-estradiol (E2) or dihydrotestosterone (DHT)-induced DNA synthesis from 56% to 122% and from 127% to 359%, and CK activity from 70% to 180% and from 90% to 190%, respectively. In SMC PD-ECGF, an inhibitor of 3[H]thymidine incorporation by itself, markedly enhanced the stimulatory effect of low concentrations of E2 and DHT on 3[H]thymidine incorporation. It also increased E2 and DHT CK induction from 40% to 140% and from 52% to 120%, respectively. In both E304 and SMC, PD-ECGF inhibited the proliferative and the CK-inducing effects of platelet-derived growth factor (PDGF) and immunoglobulin F1 (IGF1). Thus, PD-ECGF, an established growth promoter for endothelial cells, is a potent inhibitor of DNA synthesis in human arterial SMC. However, in both E304 endothelial cells and SMC, PD-ECGF enhances the stimulatory effect of low concentrations of gonadal steroids on 3[H]thymidine incorporation. PD-ECGF antagonizes PDGF- and IGF1-induced DNA synthesis in both E304 and SMC cells. By inhibiting arterial SMC proliferation and accelerating endothelial cell replication, PD-ECGF may buffer the effect of PDGF and favorably modulate arterial wall response to injury.

Cell Division↗

PubMed1999

Production and characterization of polyclonal antibodies to sulfamethazine and their potential use in immunoaffinity chromatography for urine sample pre-treatment.

An immunoaffinity chromatographic (IAC) method for isolating sulfamethazine (SMZ) from incurred urine samples was developed. This was achieved by (i) generating polyclonal antibodies that recognize equally well SMZ and its major urinary metabolites, (ii) evaluating in an ELISA procedure the influence of methanol, salt and pH on the antigen-antibody interaction in order to determine the optimum conditions for IAC and (iii) covalent coupling of the IgG fractions of anti-SMZ to CNBr activated Sepharose for the preparation of re-usable immunoaffinity columns, having a high capacity for SMZ (1900 ng SMZ mL-1 gel). For desorbing SMZ from the immunoaffinity column, different elution modes were evaluated, with 40% MeOH-0.1 mol L-1 HOAc-0.5 mol L-1 NaCl being the most efficient combination. Using the IAC column for processing SMZ spiked urine samples resulted in high recoveries, ranging from 92 to 100%. Because of the high cross-reactivity with the major metabolites of SMZ present in urine of treated animals, the antibodies show excellent properties for use in both IAC and ELISA. For the isolation and concentration of the parent drug and its major metabolites, the urine could be applied directly to the IAC column, without the time-consuming step of deconjugation. Moreover, the use of IAC prior to ELISA for the analysis of incurred urine samples showed good efficiency for the elimination of matrix interferences. Owing to the urine-tissue relationship, the urine concentrations can be used to predict the presence of the parent drug in tissues and so possible violations of the maximum residue limit (MRL) can be controlled.

Animals↗

PubMed1999

A nonisotopic enzyme-based immunoassay for assessing human exposure to genistein.

Phytoestrogenic isoflavones that are abundant in soybeans may be an important group of natural products that could play a critical role in preventing several chronic human diseases. To facilitate studying the relationship of soya exposure and chronic diseases, we report a simple method for measuring an isoflavone, genistein, in human urine and plasma. The method is a competitive enzyme-linked immunoassay that utilizes a conjugate of horseradish peroxidase (HRP) and genistein as tracer and a monoclonal antibody to genistein (clone 10D8) generated through the 6-position of genistein. Genistein, in diluted hydrolyzed urine or plasma of subjects who ingested soy milk, competes with HRP-genistein conjugate for the binding sites of anti-genistein antibody on rabbit anti-mouse IgG-coated plates. After a one-hour incubation, the wall-bound genistein-HRP activity, after reaction with a chromogen, is measured colorimetrically at 450 nm and is inversely correlated with concentrations of genistein over the range of 0.1-32 ng/well. The sensitivity limit of the method is 0.5 ng of genistein per well or 0.5 ng per 10 microliters of urine and plasma. Urine and plasma levels of genistein measured by this immunoassay correlated well (R2 = 0.92 for urine and 0.77 for plasma) with those determined by chromatographic techniques. This method can be used to assess soya exposure in humans and could facilitate epidemiological studies of the relationship of soya diets and chronic diseases, including cancer.

Adult↗