Interferon-alpha 2b modulation of doxorubicin sensitivity in a multidrug resistant cell line.

We evaluated the effect of interferon-alpha2b as a chemosensitiser on HCT-15 cell line in treatment with doxorubicin. Chemosensitivity was determined by [3H]-thymidine incorporation and tetrazolium assays. The levels of expression of P-glycoprotein, Bcl-2 oncoprotein and HLA-ABC complex, and cell cycle/apoptosis analysis were determined by flow cytometry. Dox 50 ng/ml - IFN alpha 2b 500 IU/ml treatment inhibited cell proliferation (47.2 +/- 1.4%, p < 0.0001; MTT assay: 40.6 +/- 1.2%, p < 0.0001) and augmented the expression of P-170, Bcl-2 and HLA-ABC, while it didn't exert apoptosis, producing a slight G2/M arrest. A concentration of IFN-alpha2b, that by itself is not cytotoxic, can potentiate the efficacy of the anticancer drug. This effect is not due to a down-modulation of P-170. The absence of apoptosis and augmented levels of Bcl-2 expression suggests that this could be one of the mechanisms of drug resistance exerted by these cells.

Bone marrow fibroblasts in patients with advanced lung cancer

In a previous study we demonstrated that the incidence of fibroblast colony-forming units (CFU-F) was very low in bone marrow primary cultures from the majority of untreated advanced non-small lung cancer patients (LCP) compared to normal controls (NC). For this reason, we studied the ability of bone marrow stromal cells to achieve confluence in primary cultures and their proliferative capacity following four continuous subcultures in consecutive untreated LCP and NC. We also evaluated the production of interleukin-1ß (IL-1ß) and prostaglandin E2 (PGE2) by pure fibroblasts. Bone marrow was obtained from 20 LCP and 20 NC. A CFU-F assay was used to investigate the proliferative and confluence capacity. Levels of IL-1ß and PGE2 in conditioned medium (CM) of pure fibroblast cultures were measured with an ELISA kit and RIA kit, respectively. Only fibroblasts from 6/13 (46 percent) LCP confluent primary cultures had the capacity to proliferate following four subcultures (NC = 100 percent). Levels of spontaneously released IL-1ß were below 10 pg/ml in the CM of LCP, while NC had a mean value of 1,217 + or - 74 pg/ml. In contrast, levels of PGE2 in these CM of LCP were higher (77.5 + or - 23.6 pg/ml) compared to NC (18.5 + or - 0.9 pg/ml). In conclusion, bone marrow fibroblasts from LCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1ß and PGE2 production

Bone marrow fibroblasts in patients with advanced lung cancer.

In a previous study we demonstrated that the incidence of fibroblast colony-forming units (CFU-F) was very low in bone marrow primary cultures from the majority of untreated advanced non-small lung cancer patients (LCP) compared to normal controls (NC). For this reason, we studied the ability of bone marrow stromal cells to achieve confluence in primary cultures and their proliferative capacity following four continuous subcultures in consecutive untreated LCP and NC. We also evaluated the production of interleukin-1beta (IL-1beta) and prostaglandin E2 (PGE2) by pure fibroblasts. Bone marrow was obtained from 20 LCP and 20 NC. A CFU-F assay was used to investigate the proliferative and confluence capacity. Levels of IL-1beta and PGE2 in conditioned medium (CM) of pure fibroblast cultures were measured with an ELISA kit and RIA kit, respectively. Only fibroblasts from 6/13 (46%) LCP confluent primary cultures had the capacity to proliferate following four subcultures (NC = 100%). Levels of spontaneously released IL-1beta were below 10 pg/ml in the CM of LCP, while NC had a mean value of 1,217 +/- 74 pg/ml. In contrast, levels of PGE2 in these CM of LCP were higher (77.5 +/- 23.6 pg/ml) compared to NC (18.5 +/- 0.9 pg/ml). In conclusion, bone marrow fibroblasts from LCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1beta and PGE2 production.

Tumor necrosis factor-alfa production by monocytes from lung and colorectal cancer patients.

Tumour necrosis factor-alpha (TNF-alpha) is a monocyte (MO)-derived cytokine that plays an essential role in the immunological system. In the present study our aim was to evaluate the levels of TNF-alpha secreted by MO from cancer patients. Blood MO were obtained from 10 lung cancer patients (LCP), 10 colorectal cancer patients (CCP) and 10 healthy donors (HD). TNF-alpha levels in MO culture supernatants spontaneously (sp) secreted or after stimulation with LPS treatment were evaluated using a commercial ELISA kit (sensibility: 10-1000 pg/ml). Mean values, expressed as pg/ml were: LCP: sp= 452.6+/-107.2, LPS= 589.5+/-126.7); CCP: sp= 84.1+/-25.0, LPS= 437.3+/-93.2; HD: sp= 74.2+/-21.5, LPS= 573.5+/-87.1. We concluded that MO from LCP secrete high levels of TNF-alpha spontaneously (p< 0.003 versus HD) and it was also observed an absence of response to LPS treatment in the 33% of the cases in these patients.

In vitro secretion of cytokines and prostaglandin-E2 by monocytes from lung cancer patients.

Monocytes (MO) from cancer patients present functional abnormalities, such as an altered secretion of soluble factors. In the present study, our aim was to evaluate the levels of interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and prostaglandin-E2 (PGE2) secreted in vitro by MO from lung cancer patients (LCP), spontaneously or after stimulation with lipopolysaccharide (LPS). Results showed that cytokine secretion was higher for MO from LCP than for MO from healthy controls, while in the 25% of the patients analysed, an absence of response to LPS treatment was found.

Apoptosis and expression of Bcl-2 and Bax in eutopic endometrium from women with endometriosis.

OBJECTIVE: To evaluate and compare spontaneous apoptosis and Bcl-2 and Bax expression in eutopic endometrium from women with and without endometriosis. DESIGN: Apoptosis and Bcl-2 and Bax expression were examined in eutopic endometrium from women with and without endometriosis. SETTING: Instituto de Biología y Medicina Experimental-CONICET, Department of Gynecology and Department of Gynecological Pathology, Clínicas University Hospital, Buenos Aires, Argentina. PATIENT(S): Women with untreated endometriosis (n = 14) and controls (n = 16). INTERVENTION(S): Collection of endometrial samples during diagnostic or therapeutic laparoscopy. MAIN OUTCOME MEASURE(S): Apoptotic cells were detected with use of the dUTP nick-end labeling (TUNEL) assay; Bcl-2 and Bax expressions were assessed with use of immunohistochemical techniques. RESULT(S): Spontaneous apoptosis was significantly lower in eutopic endometrium from patients with endometriosis, compared with healthy controls (2.26 +/- 0.53 and 9.37 +/- 1.69 apoptotic cells/field, respectively) and was independent of cycle phase. An increased expression of Bcl-2 protein was found in proliferative eutopic endometrium from patients with endometriosis. Bax expression was absent in proliferative endometrium, whereas there was an increase in its expression in secretory endometrium from both patients and controls. CONCLUSION(S): Women with endometriosis show decreased number of apoptotic cells in eutopic endometrium. The abnormal survival of endometrial cells may result in their continuing growth into ectopic locations.

Regulation of HLA-DR antigen in monocytes from colorectal cancer patients by in vitro treatment with human recombinant interferon-gamma.

During the immune response, a great number of cytokines that modulate the function of mononuclear phagocytes are produced. Since interferons are one of the most important cytokines, the aim of this study was to evaluate the expression of HLA-DR antigen after an 18-h culture with human recombinant interferon-gamma (hrIFN-gamma) on freshly isolated peripheral blood monocytes from 16 colorectal cancer patients and 16 healthy donors, using an indirect immunofluorescence method. The results obtained showed that there was a decreased percentage of HLA-DR+ monocytes in the colorectal cancer patents (51 +/- 3%, p <0.01) compared with the healthy donors (77 +/- 2%). Treatment for 18 h with hrIFN-gamma increased the percentages of monocytes expressing HLA-DR: 71 +/- 3% for the cancer patients and 84 +/- 2% for the healthy donors (p <0.01 and <0.001, respectively). Our results demonstrate that after in vitro treatment with hrIFN-gamma, functionally altered monocytes from colorectal cancer patients can reach major histocompatibility complex II antigen expression values similar to those of healthy donors, thus improving the host's cellular immune response against the tumor.

Apoptosis y expresión de Bcl-2 y Bax en el endometrio eutópico de pacientes con endometriosis / Apoptosis and Bcl-2 and Bax in normal endometrium of patients with endometriosis

El objetivo de este trabajo es investigar una posible predisposición de las células endometriales de pacientes con endometriosis (EDT), a ser resistentes a la muerte celular programada. Se evaluó apoptosis y expresión de las proteínas Bcl-2 y Bax en 30 cortes de tejido de endometrio eutópico, 14 de mujeres con EDT y 16 de controles (C). Para la determinación de apoptosis, se utilizó el kit Apoptag-Plus basado en la localización y tinción de los extremos 3'-OH de los fragmentos de ADN. Los resultados se expresan como nº de células apoptóticas (CA)/campo a 630x de aumento. Se detectaron CA sólo en el epitelio glandular. Se observó menor cantidad de CA en tejido endometrial proveniente de pacientes con EDT: 2,26 ñ 0,53 vs 9,37 ñ 1,69 en los C (p<0,001). Esta disminución se conservó a lo largo del ciclo menstrual. En la fase proliferativa tardía, los resultados fueron de: 7,08 ñ 0,92 vs 1,9 ñ 0,73 (p<0,05), para las muestras provenientes de mujeres C y pacientes con EDT respectivamente; mientras que en el endometrio secretorio los niveles de apoptosis detectados fueron de 11,6 ñ 1,74 en los C vs 2,7 ñ 0,82 en EDT (p<0,001). No se observaron diferencias significativas de apoptosis en endometrio de acuerdo al grado de la enfermedad. La evolución de los productos de protooncógenes, bcl-2 y bax se realizó utilizando metodologías de inmunohistoquímica. Se halló incrementada la expresión de la proteína antiapoptótica Bcl-2 en el endometrio proliferativo proveniente de pacientes con EDT comparado con el C. La expresión del antagonista de Bcl-2, Bax, aumentó durante la fase secretoria tanto en muestras provenientes de pacientes como de C, y se halló ausente durante la fase proliferativa. De los resultados se desprende que las células endometriales de pacientes con EDT poseen características apoptóticas alteradas que las harían más susceptibles a crecer en un sitio ectópico. Estas no se ven modificadas a lo largo del ciclo menstrual. La proteína Bcl-2 estaría implicada en la protección de la apoptosis de las células endometriales eutópicas de pacientes con EDT durante la fase proliferativa del ciclo menstrual. La proteína Bax estaría involucrada en la regulación de la muerte celular programada que se produce en el endometrio eutópico previo a la menstruación. La resistencia a la muerte celular programada que posee el endometrio eutópico de pacientes con EDT, estaría relacionada con la etiología y/o fisiopatología de la enfermedad

Cisplatin containing chemotherapy influences HLA-DR expression on monocytes from cancer patients.

The effect of cisplatin-containing chemotherapy regimen was evaluated on the expression of HLA-DR antigen in peripheral blood monocytes from patients with lung (LCP) and colorectal (CCP) cancer. Chemotherapeutic schedules employed in patients were etoposide and cisplatin for LCP and 5-fluorouracil and cisplatin for CCP. The results obtained showed a diminished percentage of monocytes expressing HLA-DR antigen in LCP (52.4 +/- 2.6, p < 0.004) and CCP (50.1 +/- 2.1, p < 0.001) respectively versus healthy donors (71.0 +/- 1.1%), and that their values increased during chemotherapy, raising them up to control level after the second cycle of treatment, independently of the course of the cancer growth. We conclude that both modalities of treatment allowed an increase of monocytes expressing HLA-DR antigen, suggesting that this effect may be due to cisplatin action.

Functional and phenotypic alterations in peritoneal macrophages from patients with early and advanced endometriosis.

The aim of this study was to elucidate whether peritoneal macrophage (pMO) alterations are a generalized feature in all stages of endometriosis and the effect of hormonal treatment on this leukocyte population. For this purpose we quantified the number of pMO, the expression of HLA-DR antigen (pMO DR+), percentages of pMO that reduced nitro-blue tetrazolium (pMO NBT+), and interleukin-1 (IL-1) and prostaglandin E2 (PGE2) production by pMO from patients with early (stages I/II) and advanced (stages III/IV) endometriosis, we also analyzed some of these properties in pMO from patients which had been treated for 6 months with 800 mg/day of Danazol or gonadotropin releasing hormone agonist (GnRHa). We found that there were a significant increase of the pMO number in both types of patients, though the highest values were obtained in early endometriosis (p < 0.001). Percentages of pMO DR+ were decreased in all patients (p < 0.01) while percentages of pMO NBT+ were significantly increased. Production of IL-1 by early and advanced endometriosis pMO were considerably enhanced. PGE2 release was not altered in early endometriosis pMO but, in advanced endometriosis, pMO PGE2 levels were 100-fold higher than control values. In posttreatment patients, the number of pMO and percentage of pMO NBT+ were similar to early endometriosis patients, though the percentage of pMO DR+ was within the normal range. We conclude that the pMO population, as well as IL-1 and PGE2 production, were altered in all stages of endometriosis, and that these changes could be involved in the pathogenesis of endometriosis and associated infertility. Hormonal treatments do not reverse the pMO changes.

Bone marrow fibroblastic progenitors in patients with advanced breast cancer.

Bone marrow fibroblast colony-forming cells (CFU-F) were studied in fifteen consecutive untreated breast cancer patients (BCP) with clinical stages III and IV, and in sixteen normal controls (NC). A decreased number of CFU-F was observed in BCP compared to NC (p < 0.004). Confluence of the adherent cell layer was observed in all normal bone marrow mononuclear cells (MC) cultures, while a lower proportion of cultures from BCP (11/15) showed confluent adherent cell layers. When MC cultures of BCP were treated with indomethacin (Indo, 10(-6)M) 50% of them increased the number of CFU-F compared to the value obtained without treatment. In addition, a significant increase in the release of PGE2 in BCP cultures was observed before Indo treatment. Moreover, after MC were fractionated into adherent and non-adherent progenitors, the CFU-F decreased in both types of fractions of BCP compared to NC value (p < 0.02 and < 0.05, respectively). The number of light density MC per 10 ml of bone marrow aspirate and the number of trypsin-sensitive adherent progenitors were lower than NC in BCP (p < 0.02 and 0.013, respectively). Total MC and fibroblasts (fourth passage) were cultivated to evaluate the production of interleukin-1 beta (IL-1 beta) by ELISA methodology. Results indicated no difference of IL 1 beta spontaneous release when total MC cultures of both groups were compared. However, the levels of this cytokine were lower (< 10 pg/ml) in fibroblast culture supernatants of BCP compared to NC (1,217 +/- 74 pg/ml). Fibroblast cultures from BCP showed low or no release of IL-1 beta after muramyl-dipeptide (MDP. 1 microgram/ml) stimulation. In conclusion, the defective proliferative and confluence capacity of BCP fibroblastic progenitors may be related to the decrease in the production of IL-1 beta by these precursors.

Fibroblastic colony-forming units and levels of tumor necrosis factor and prostaglandin E2 in bone marrow cultures from patients with advanced lung carcinoma.

BACKGROUND: Although alterations of the bone marrow (BM) fibroblast colony-forming cells are involved in the development of diverse hematologic disorders, these progenitors still have not been well characterized in patients with solid tumors. METHODS: The incidence of fibroblast colony-forming units (CFU-F) was evaluated in the cultures of unseparated and fractionated light density BM mononuclear cells (MC) from 25 consecutive untreated lung carcinoma patients (LCP) and 16 normal controls (NC). Unseparated MC also were cultured in the presence of indomethacin (10[-6] M). Finally, the authors evaluated the spontaneous production of prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-alpha) in culture conditioned mediums of unseparated MC by radioimmunoassay and enzyme-linked immunoadsorbent assay methodology, respectively. RESULTS: A decreased number of CFU-F was observed in unseparated and fractionated (adherent and nonadherent) light density MC cultures from LCP compared with NC. When unseparated MC of LCP were treated with indomethacin, a slightly increase in the number of CFU-F was found. Adherent MC (stromal cells) achieved confluence only in 44% of LCP primary cultures compared with 100% of NC. Overproduction of PGE2 and TNF-alpha was found in the conditioned mediums of LCP compared with the mean values obtained in NC (P < 0.05 and P < 0.02, respectively). CONCLUSIONS: The lack of confluence and suppression of CFU-F in BM of LCP may be related to the increase production of PGE2 and TNF-alpha. Future investigation will allow the determination of how these modifications influence tumor cell growth and will prove if more alterations of the hematopoietic microenvironment imply a worse prognosis.

Interleukin-1 beta levels in human embryo culture supernatants and their predictive value for pregnancy.

Interleukin 1 (IL-1) is possibly one factor produced by the embryo that might have a role in the maternal immunological recognition of pregnancy. The purpose of this study was to identify an embryo-related factor suitable for prediction of pregnancy during IVF procedures. For this purpose, IL-1 beta levels were measured in 21 samples of human embryo culture-conditioned media. The average number of embryos per sample was 5 +/- 1. Simultaneously, 16 cell culture media containing 10% autologous serum but no embryos were tested as controls. IL-1 beta levels were measured using the ELISA technique, and the biological activity of IL-1 was measured by means of a C3H/HeJ mice thymocyte proliferation assay. The average IL-1 beta level +/- S.E.M. was 49 +/- 7 pg/ml in embryo culture-conditioned media and 12 +/- 2 pg/ml in controls (P < 0.001). The average IL-1 beta level in embryo culture-conditioned media from viable pregnancy cycles was 82 +/- 6 pg/ml (n = 8), while in those cases that did not result in viable pregnancies the IL-1 beta level was significantly lower (28 +/- 4 pg/ml, n = 13, P < 0.001). The IL-1 activity of embryo culture-conditioned media, measured by [3H]thymidine incorporation in thymocytes was increased, compared with control media (442 +/- 51 counts/min vs. 337 +/- 13 counts/min, P < 0.05), and the highest values corresponded to media containing those embryos that resulted in pregnancies (589 +/- 41 counts/min, P < 0.01 vs. controls). We conclude that the determination of the levels of this cytokine in embryo culture-conditioned media might be a predictive parameter for pregnancies in patients undergoing IVF-ET.

Determination of IL-1 and IL-6 levels in human embryo culture-conditioned media.

PROBLEM: The aim of this study was to evaluate the presence of interleukin-1 (IL-1) and IL-6 in 24-hour human embryo culture-conditioned media (HECCM) and to find any embryo-related factor(s) to predict pregnancy during IVF procedure. METHOD: IL-1 beta and IL-6 levels were measured in 36 samples of HECCM and 17 cell culture medial that had not been exposed to embryos, which were used as controls. Both cytokine levels were measured by the ELISA technique, using commercially available kits. RESULTS AND CONCLUSIONS: We found an average IL-1 beta level +/- SEM of 82 +/- 4 pg/ml (n = 11) in HECCM from viable pregnancies and a significantly lower value, 14 +/- 2 pg/ ml (n = 25, P < 0.001), in HECCM from embryos that did not lead to viable pregnancies. In control medial the average IL-1 beta level was 11 +/- 1 pg/ml (n = 17), (P < 0.001 vs. HECCM from viable pregnancies). In contrast IL-6 levels were undetectable in all samples analyzed. Judging by our results we suggest that measurement of IL-1 level in HECCM could be a useful parameter for predicting implantation in techniques of assisted reproduction.

Effect of peritoneal fluid from patients with mild and severe endometriosis on endometrial stromal cell proliferation.

The aim of this study was to evaluate and compare the mitogenic effect of peritoneal fluid (PF) from women with mild and severe endometriosis on the endometrial stromal cell proliferation. Increasing concentrations of PF from women with and without mild or severe endometriosis were added to primary endometrial stromal cell cultures and 3H-thymidine incorporation was used to assess DNA synthesis in these cultures. PF from women with mild endometriosis induced a statistically significant dose-dependent increase in stromal cell thymidine uptake ranged from 5.8 to 14.5 fold, whereas PF from women with severe endometriosis produced an average 51% inhibition of stromal cell proliferation of compared with cells exposed to non-endometriosis PF or exposed to nutrient medium supplemented with 2.5% calf serum alone. PF samples from patients with stage I endometriosis induced a statistically dose-dependent increase in stromal cell proliferation, whereas PF from patients with stage IV endometriosis caused a significant inhibition.

Intracytoplasmatic and extracellular interleukin-1 production by monocytes of lung and colorectal cancer patients.

We studied the production of interleukin-1 (IL-1) by peripheral blood monocytes (Mo) from twelve normal subjects (NS) and eight and nine untreated lung and colorectal cancer patients (CP), respectively. No significant changes of extracellular IL-1 biological activity was observed between CP and NS by thymocyte proliferation assay. This result was independent that the cells were treated or not with lipopolisaccharide from E. coli (LPS, 10 micrograms/ml). Moreover, CP present normal amount of antigenic IL-1 beta in LPS treated Mo culture supernatants by enzyme-linked immunosorbent assay (ELISA). The biological activity of IL-1 released was not significant modified after indomethacin (Indo, 10(-6)M) and LPS + Indo treatments. Furthermore, patients showed a low percentage of LPS activated Mo with intracytoplasmatic IL-1 (alpha + beta) compared to normal values. These results were obtained by immuno-alkaline phosphatase staining using monoclonal antibody anti IL-1 (alpha + beta). In conclusion, CP had a reduced number of Mo with intracytoplasmatic IL-1 (alpha + beta) and the difference observed may depend on degradation or in the rate of synthesis of this cytokine.

Murine peritoneal macrophages in syngeneic and allogeneic pregnancies.

We have previously observed that some functional characteristics of peritoneal macrophages (MOp) are altered during syngeneic murine pregnancy. To determine if these alterations are related to the immunological stimulation that the embryo produces on the mother, we evaluated MOp activity in allogeneic pregnancy. We also compared expression of the la antigen, ability to phagocyte and reduce nitro blue tetrazolium (NBT) and to produce interleukin-1 (IL-1) in allogeneic and syngeneic pregnancies. We observed that at Day 7 of pregnancy the increment in MOpIa(+) percentages was more evident in allogeneic (P < 0.05) than syngeneic pregnancies, and that these values remained high during the second week of gestation. We also observed a significant decrease in the macrophages that reduced NAT during the first week both in allogeneic and syngeneic pregnancies. Yet, in the former, the percentages of MOpNBT(+) were still low in the last week of pregnancy (P < 0.05). No differences were found in IL-1 production or in estradiol and progesterone levels between the 2 types of pregnancies. Thus, it is possible to postulate that during the first week of pregnancy the strong antigenic challenge that the embryo represents may activate MOp and that this activation could be augmented when major antigenic differences between mother and embryo are present.

Citoquinas embrionarias y su posible uso como parámetro predictivo en técnicas de IVF / Embryonic cytokines and its possible use as prediction parameter in IVF techniques

El objetivo del presente trabajo fue evaluar los niveles de IL-1 e IL-6 en sobrenadantes de distinto número de embriones humanos de 24 horas en relación con la viabilidad de los embarazos, con el fin de determinar su posible utilidad como test predictivo en las prácticas de fertilización asistida. Para ello se analizaron 36 muestras de medios condicionados de cultivo de embriones humanos (MCCEH) de 24 horas y 17 medios controles (medios de cultivo igualmente adicionados con 10 por ciento de suero autólogo pero sin contacto con embriones). Se observó que los MCCEH de embriones que luego de ser transferidos produjeron embarazos viables tuvieron los valores más altos de IL-1b (82 ñ 4 pg/ml, n=11) con respecto a aquellos que no produjeron embarazos viables (14 ñ 2 pg/ml, n=25) p<0,001 y significativamente mayores a los medio controles (11 ñ 1 pg/ml, n=17). Estas diferencias en los niveles de IL-1ß fueron igualmente significativas al analizar los resultados obtenidos de acuerdo al número de embriones por muestra. La evaluación de los niveles de IL-6 sin embargo no dio resultados comparables a los obtenidos en la determinación de IL-1ß, puesto que los valores hallados fueron extremadamente bajos (= 20 pg/ml). Estos resultados indican que la determinación de los niveles de IL-1ß en MCCEH podría ser un buen parámetro predictivo en pacientes sometidas a técnicas de fertilización asistida

Human granulosa cells express HLA-DR antigen and are capable of synthesizing interleukin-1.

To study the origin of interleukin 1 (IL-1) present in human follicular fluid we determined the percentage of macrophages (MO) and cells with HLA-DR antigen (DR+) present in 22 samples of human follicular fluid (FF) from women undergoing in vitro fertilization, and examined the release of IL-1 beta by cultures of purified human granulosa cells (GC). The number of red blood cells (RBC) in the crude preparation was taken as a measure of possible contamination with peripheral blood monocytes (assuming a ratio of one monocyte or MO per 10(4) RBC). For the evaluation of MO and DR+ cells percentages we employed an indirect immunofluorescence technique using specific monoclonal antibodies. Total cells from FF were purified by Ficoll-Hypaque density gradient centrifugation (delta = 1.076 g/l and GC were purified using a gradient delta = 1.065 g/l. This method reduced the contamination with MO to 0-1%. The spontaneous release of IL-1 beta was measured by ELISA. We found that FF contained 9.81 +/- 1.47% of MO but only 7.85% were ovarian MO. In addition the total percentage of DR+ cells was 17.13 +/- 2.35% but only 9.81% corresponded to MO. Therefore about 7.32% of DR+ cells could be GC. Then purified GC (10(4)/0.2 ml/well) were cultured during 24 hours at 37 degrees C in serum free medium (DMEM:F12). IL-1 beta levels were 84 +/- 17 pg/ml and these values were increased by 44% when GC were stimulated with FSH (200 ng/ml). These results suggest that GC produced IL-1 beta and that the synthesis of this cytokine might be regulated by hormones.

Antitumor and cytotoxic screen of 5,6,7-trisubstituted flavones from Gomphrena martiana.

An in vivo antitumor screening of extracts of Gomphrena martiana indicated positive activity in the petroleum ether extract, and its further bioactivity-directed fractionation resulted in a lipophilic flavonoid fraction. Upon inoculation of various doses of 5,6,7-trisubstituted flavones on two murine tumor lines, Sarcoma 180 and Ehrlich's carcinoma, a decrease of tumor growth was observed. An in vitro KB cultured cell screen indicated cytotoxicity.