The ellagic acid metabolites urolithin A and B differentially affect growth, adhesion, motility, and invasion of endometriotic cells in vitro.

STUDY QUESTION: What are the effects of plant-derived antioxidant compounds urolithin A (UA) and B (UB) on the growth and pathogenetic properties of an in vitro endometriosis model? SUMMARY ANSWER: Both urolithins showed inhibitory effects on cell behavior related to the development of endometriosis by differentially affecting growth, adhesion, motility, and invasion of endometriotic cells in vitro. WHAT IS KNOWN ALREADY: Endometriosis is one of the most common benign gynecological diseases in women of reproductive age and is defined by the presence of endometrial tissue outside the uterine cavity. As current pharmacological therapies are associated with side effects interfering with fertility, we aimed at finding alternative therapeutics using natural compounds that can be administered for prolonged periods with a favorable side effects profile. STUDY DESIGN, SIZE, DURATION: In vitro cultures of primary endometriotic stromal cells from 6 patients subjected to laparoscopy for benign pathologies with histologically confirmed endometriosis; and immortalized endometrial stromal (St-T1b) and endometriotic epithelial cells (12Z) were utilized to assess the effects of UA and UB on endometriotic cell properties. Results were validated in three-dimensional (3D) in vitro co-culture spheroids of 12Z and primary endometriotic stroma cells of one patient, and organoids from 3 independent donors with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects on cell growth were measured by non-radioactive colorimetric assay to measure cellular metabolic activity as an indicator of cell viability (MTT assay) and flow cytometric cell cycle assay on primary cultures, St-T1b, and 12Z. Apoptosis analyses, the impact on in vitro adhesion, migration, and invasion were evaluated in the cell lines. Moreover, Real-Time Quantitative Reverse Transcription polymerase chain reaction (RT-qPCR) assays were performed on primary cultures, St- T1b and 12Z to evaluate a plausible mechanistic contribution by factors related to proteolysis (matrix metalloproteinase 2, 3 and 9 -MMP2, MMP3, MMP9-, and tissue inhibitor of metalloproteinases -TIMP-1-), cytoskeletal regulators (Ras-related C3 botulinum toxin substrate 1 -RAC1-, Rho-associated coiled-coil containing protein kinase 2 -ROCK2-), and cell adhesion molecules (Syndecan 1 -SDC1-, Integrin alpha V-ITGAV-). Finally, the urolithins effects were evaluated on spheroids and organoids by formation, viability, and drug screen assays. MAIN RESULTS AND THE ROLE OF CHANCE: 40 µM UA and 20 µM UB produced a significant decrease in cell proliferation in the primary endometriotic cell cultures (P < 0.001 and P < 0.01, respectively) and in the St-T1b cell line (P < 0.001 and P < 0.05, respectively). In St-T1b, UA exhibited a mean half-maximum inhibitory concentration (IC50) of 39.88 µM, while UB exhibited a mean IC50 of 79.92 µM. Both 40 µM UA and 20 µM UB produced an increase in cells in the S phase of the cell cycle (P < 0.01 and P < 0.05, respectively). The same concentration of UA also increased the percentage of apoptotic ST-t1b cells (P < 0.05), while both urolithins decreased cell migration after 24 h (P < 0.001 both). Only the addition of 5 µM UB decreased the number of St-T1b adherent cells. TIMP-1 expression was upregulated in response to treating the cells with 40 µM UA (P < 0.05). Regarding the 12Z endometriotic cell line, only 40 µM UA decreased proliferation (P < 0.01); while both 40 µM UA and 20 µM UB produced an increase in cells in the G2/M phase (P < 0.05 and P < 0.01, respectively). In this cell line, UA exhibited a mean IC50 of 40.46 µM, while UB exhibited a mean IC50 of 54.79 µM. UB decreased cell migration (P < 0.05), and decreased the number of adherent cells (P < 0.05). Both 40 µM UA and 20 µM UB significantly decreased the cellular invasion of these cells; and several genes were altered when treating the cells with 40 µM UA and 10 µM UB. The expression of MMP2 was downregulated by UA (P < 0.001), and expression of MMP3 (UA P < 0.001 and UB P < 0.05) and MMP9 (P < 0.05, both) were downregulated by both urolithins. Moreover, UA significantly downregulated ROCK2 (P < 0.05), whereas UB treatment was associated with RAC1 downregulation (P < 0.05). Finally, the matrix adhesion receptors and signaling (co)receptors SDC1 and ITGAV were downregulated upon treatment with either UA or UB (P < 0.01 and P < 0.05, respectively in both cases). Regarding the effects of urolithins on 3D models, we have seen that they significantly decrease the viability of endometriosis spheroids (80 µM UA and UB: P < 0.05 both) as well as affecting their area (40 µM UA: P < 0.05, and 80 µM UA: P < 0.01) and integrity (40 µM UA and UB: P < 0.05, 80 µM UA and UB: P < 0.01). On the other hand, UA and UB significantly inhibited organoid development/outgrowth (40 and 80 µM UA: P < 0.0001 both; 40 µM UB: P < ns-0.05-0.001, and 80 µM UB: P < 0.01-0.001-0.001), and all organoid lines show urolithins sensitivity resulting in decreasing viability (UA exhibited a mean IC50 of 33.93 µM, while UB exhibited a mean IC50 of 52.60 µM). LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was performed on in vitro endometriosis models. WIDER IMPLICATIONS OF THE FINDINGS: These in vitro results provide new insights into the pathogenetic pathways affected by these compounds and mark their use as a potential new therapeutic strategy for the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was funded EU MSCA-RISE-2015 project MOMENDO (691058). The authors have no conflicts of interest to declare.

Potential use of ellagic acid for endometriosis treatment: its effect on a human endometrial cell cycle, adhesion and migration.

Endometriosis is a common and challenging condition of reproductive-aged women that is defined as the presence of endometrial-like tissue outside the uterine cavity. Despite its prevalence, there is still no effective therapeutics; so we aim to evaluate the ellagic acid (EA) effect on the most relevant aspects that are known to be altered in endometriosis. Endometrial primary cultures from women with and without endometriosis and endometrial cell lines were incubated with EA (50 and 100 µM) for 24 and 48 h. The results demonstrated that EA arrests an endometrial stromal cell cycle on the G2/M phase, after 48 h. In addition, 100 µM EA treatment significantly decreased ECC-1 cell migration at 20 h and T-HESC cell migration at 10 h and 20 h, while 50 µM EA significantly decreased T-HESC cell migration at 20 h. On the other hand, we proved that the treatment with EA for 24 h reduces T-HESC and ECC-1 adhesion to plastic. However, we did not find an effect of EA on cell proliferation. EA has an inhibitory effect on endometrial cell adhesion, migration and cell cycle progression in vitro. These highlight the idea to investigate natural compounds as novel and promising candidates for therapeutic treatment of endometriosis.

Natural therapies assessment for the treatment of endometriosis.

STUDY QUESTION: Can resveratrol and epigallocatechin-3-gallate (EGCG) inhibit the growth and survival of endometriotic-like lesions in vivo in a BALB/c model of endometriosis, and in vitro in primary cultures of human endometrial epithelial cells (EECs)? SUMMARY ANSWER: Resveratrol and EGCG exerted a potent inhibitory effect on the development of endometriosis in a BALB/c murine model and on the survival of EECs. WHAT IS KNOWN ALREADY: Endometriosis is a common condition associated with infertility and pelvic pain in women of reproductive age. Resveratrol and EGCG are two polyphenols with anticarcinogenic and antioxidant properties that have been proposed as natural therapies to treat endometriosis. STUDY DESIGN, SIZE, DURATION: Fifty-six 2-month-old female BALB/c mice underwent surgical induction of endometriosis. Treatments with resveratrol or EGCG started 15 days post-surgery and continued for 4 weeks. Human biopsies were taken with a metal Novak curette from the posterior uterine wall from 16 patients with untreated endometriosis and 15 controls who underwent diagnostic laparoscopy for infertility. MATERIALS, SETTING, METHODS: After the treatments, animals were sacrificed and lesions were counted, measured, excised and fixed. Immunohistochemistry for proliferating cell nuclear antigen and CD34 was performed for cell proliferation and vascularization assessment in the lesions. The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) technique was performed for apoptosis evaluation. Peritoneal fluid was collected to analyze vascular endothelial growth factor levels. Human EECs were purified from proliferative-phase endometrial biopsies and cultured. The effect of both polyphenols on cell proliferation was determined by a colorimetric assay using the CellTiter 96®AQueous One Solution Cell Proliferation Assay kit and on apoptosis by the TUNEL technique, using an In Situ Cell Death Detection Kit with Fluorescein. MAIN RESULTS: In the mouse model, both treatments significantly reduced the mean number (P < 0.05 versus control) and the volume of established lesions (P < 0.05 versus control). Treatments consistently statistically significantly diminished cell proliferation (resveratrol P < 0.01 and EGCG P < 0.05, versus control), reduced vascular density (resveratrol P < 0.01 and EGCG P < 0.001, versus control) and increased apoptosis within the lesions (resveratrol P < 0.01 and EGCG P < 0.05, versus control). Both compounds induced reduction in human EEC proliferation (P < 0.05 versus basal) and increased apoptosis (P < 0.05 versus basal) in primary cultures. LIMITATIONS: In vitro studies were only carried out in epithelial cells from human eutopic endometrium. WIDER IMPLICATIONS OF THE FINDINGS: The present findings are promising and will assist the development of novel natural treatments for endometriosis. STUDY FUNDING: This study was supported by ANPCYT (PICT 6384 BID 1201 OC-AR) and CONICET (PIP 5471), Argentina. None of the authors has any conflict of interest to declare.

Effect of GnRH analogues on apoptosis and expression of Bcl-2, Bax, Fas and FasL proteins in endometrial epithelial cell cultures from patients with endometriosis and controls.

BACKGROUND: Our purpose was to evaluate the effect of the GnRH agonist (GnRHa), leuprolide acetate (LA), and the GnRH antagonist (GnRHant), Antide, on apoptosis and expression of apoptosis-related proteins in endometrial epithelial cell (EEC) cultures from patients with endometriosis and controls (infertile women without endometriosis). METHODS: Biopsy specimens of eutopic endometrium were obtained from 22 patients with endometriosis and from 14 women that served as controls. Apoptosis was examined in EEC after incubation with LA and Antide. Bax, Bcl-2, Fas and FasL expression was evaluated after exposure to LA, Antide or a combination of both. The percentage of apoptotic cells (%ApC) was assessed by the acridine orange-ethidium bromide technique, and protein expression was evaluated by western blot and immunocytochemistry. RESULTS: LA 100 and 1000 ng/ml increased the %ApC in EEC from patients with endometriosis (both P < 0.05) and controls (p < 0.05 and P < 0.01, respectively). Antide 10(-5) M increased the %ApC in EEC from patients with endometriosis and controls (P < 0.01). In EEC from women with endometriosis, Bax expression increased after treatment with LA, Antide and LA + Antide (P < 0.05, P < 0.001 and P < 0.001), whereas Bcl-2 expression decreased after exposure to LA and Antide (P < 0.001 and P < 0.01). FasL expression increased after LA, Antide and LA + Antide treatments (P < 0.01, P < 0.001 and P < 0.01). No significant changes were observed on Fas expression. CONCLUSIONS: GnRH analogues enhanced apoptosis in EEC, and this was accompanied by an increase in expression of the pro-apoptotic proteins Bax and FasL and a decrease in expression of the anti-apoptotic protein Bcl-2.

Interleukin-1 beta, transforming growth factor beta 1, prostaglandin E2, and fibronectin levels in the conditioned mediums of bone marrow fibroblast cultures from lung and breast cancer patients.

We analyzed the ability of the bone marrow (BM) stromal cells to achieve confluence and their proliferative capacity in BM primary cultures from 30 untreated lung cancer patients (LCP), 27 breast cancer patients (BCP), and 30 normal controls (NC) when these confluent cells were induced to proliferate following four continuous subcultures. Moreover, we evaluated the production of interleukin-1 beta (IL-1beta), transforming growth factor beta 1 (TGF-beta1), fibronectin, and prostaglandin E2 (PGE2) by pure fibroblasts (fourth passage). A fibroblast colony-forming units (CFU-F) assay was used to investigate the proliferative and confluence capacity. Levels of IL-1beta, TGF-beta1, and fibronectin in conditioned mediums (CM) of fibroblast cultures were measured by enzyme-linked immunosorbent assay (ELISA) kit and PGE(2) by radioimmunoassay (RIA) kit. Confluence was achieved in the 60% of LCP and 78% of BCP primary cultures compared with 100% of NC, and only fibroblasts from seven LCP and six BCP cultures had the capacity to proliferate following four subcultures. Levels of IL-1beta were below 10 pg/ml in both patient groups, while NC had a mean value of 5882.57+/-221.61 pg/ml. Levels of TGF-beta1 in BCP were lower than NC values ( P<0.05). LCP and BCP had significantly decreased levels of fibronectin when compared to NC values ( P<0.05 and P<0.01, respectively). Levels of PGE2 in LCP were higher compared to NC ( P<0.01). In conclusion, BM fibroblasts from LCP and BCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1beta, TGF-beta1, fibronectin, and PGE2 production.

Relationship of ovarian stimulation response with vascular endothelial growth factor and degree of granulosa cell apoptosis.

BACKGROUND: The aim of this study was to evaluate the concentration of vascular endothelial growth factor (VEGF) in follicular fluid and in granulosa cell cultures in relation to the degree of apoptosis in granulosa cells from patients with different types of ovarian response to controlled ovarian hyperstimulation. METHODS: We studied 30 women who underwent controlled ovarian hyperstimulation and oocyte retrieval. Group A comprised patients with 1-4 follicles (n = 10), group B patients with 5-14 follicles (n = 10) and group C patients with >15 follicles (n = 10). RESULTS: Mean (+/-SD) VEGF concentrations in follicular fluid were 1232 +/- 209, 813 +/- 198 and 396 +/- 103 pg/ml for groups A, B and C respectively (P > 0.01). Concentrations of VEGF in granulosa cell supernatant were 684 +/- 316, 1101 +/- 295 and 1596 +/- 227 pg/ml respectively (P < 0.05). Percentages of apoptotic cells in granulosa cells culture was 55.02 +/- 7.5, 23.98 +/- 4.4 and 14.2 +/- 2.3% respectively (A versus B, P < 0.01, A versus C, P < 0.006, B versus C, NS). CONCLUSIONS: Our findings showed that in patients with decreased ovarian response to controlled ovarian hyperstimulation, follicular fluid VEGF concentration is elevated, the concentration from granulosa cells culture supernatant is decreased and the percentage of apoptotic granulosa cells is increased, while opposite findings occurred in patients with normal or hyper-responses.

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 s

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

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.

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.

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.

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.

Chemiluminescence production by neutrophils and immune complex levels in cancer patients.

We studied the production of chemiluminescence (CL) by peripheral blood neutrophils from 24 normal subjects, 13 lung cancer patients with clinical stages (CS) III and IV, and 27 breast cancer patients with CS II, III, and IV. Evaluations were made before chemo- and radiotherapy treatments. CL was expressed as: baseline of the record background activity; area under the curve (AUC) of opsonized zymosan response; maximum peak (MP) response; time to MP (TMP); and total time (TT). Simultaneously, circulating immune complexes (CIC) in sera were evaluated by polyethylene glycol precipitation technique. The results demonstrated that lung cancer patients with CS III had an increase of TMP and TT values, while breast cancer patients with CS IV showed the lowest values of AUC and MP compared with the control group. The CIC levels were increased in all the cancer patients. There was no correlation between the baseline CL activity and the levels of immune complexes.

Differential PGE2 and cAMP production by allogeneic and syngeneic pregnant mice uteri.

Prostaglandin E2 (PGE2) and cAMP production in allogeneic and syngeneic pregnant mice uteri, were measured in relation to the ratio of plasma estrogen/progesterone levels. PGE2 generation by allopregnant uteri varied with the days of pregnancy. The increment of the prostanoid coincided with the increase in plasma estrogen concentration, whereas the decrement of its production was in parallel with the increment of plasma progesterone. The syngeneic pregnant uterus was unable to increase the release of PGE2 above basal values during the whole pregnancy. The rise of PGE2 production by the allogeneic pregnant uterus was correlated with an increase in cAMP levels. It is proposed that the pregnant mouse uterus increases its ability to release PGE2 in response to an ovarian steroids.

Functional alterations of murine peritoneal macrophages during pregnancy.

We have studied some functional characteristics of murine peritoneal macrophages (MOp) related to hormonal changes produced during pregnancy. Maximal expression of Ia antigen and release of interleukin 1 (IL1) were observed during the first week of pregnancy (implantation), when the highest peak of estradiol was produced. Both Ia antigen expression and IL1 levels progressively decreased as gestation advanced. Inversely, MOp capability to phagocyte and reduce nitro blue tetrazolium (NBT) was diminished at the beginning of pregnancy but returned to normal values in the last week. Sexual steroid levels (estradiol and progesterone) were inversely related, and the release of prostaglandin E2 (PGE2) by MOp decreased when progesterone levels increased. Although PGE2 production had no evident relation with Ia antigen expression and IL1 activity during the first and second weeks of pregnancy, the increment in PGE2 values and percentages of NBT+ cells could indicate a different stage of macrophage activation. These results suggest a possible hormonal regulation of macrophage functionality.

Effects of sexual steroid hormones on the functionality of murine peritoneal macrophages.

In the present work, we studied the possible effect of steroid hormones, estradiol, progesterone, and 5 alpha-dihydrotestosterone, on different phenotypic and functional characteristics of peritoneal adherent mononuclear cells. We used female and male mice of Balb/c strain, normal, gonadectomized, and gonadectomized with hormonal replacement. We found that gonadectomy in both sexes produced a significant decrease in the functionality of membrane receptors for the complement and in phagocytic activity of Candida albicans-anti-C albicans system. In addition, the percentages of cells that reduced nitroblue tetrazolium were diminished in castrated animals. Ovariectomized females injected with estradiol presented normal levels of phagocytic and metabolic capacities, but the expression of membrane receptors for complement remained decreased. In contrast, progesterone treatment of ovariectomized animals had the opposite effect. Simultaneous treatment with estradiol plus progesterone gave results similar to those observed with estradiol only. Dihydrotestosterone per se did not affect any of the parameters measured in the conditions used here. These results suggest that female steroids affect macrophage functionality, probably by regulating surface receptors that are involved in phagocytic activity.

[Evaluation of the interleukin 1 production by adherent cells in patients with diabetes type II]. / Evaluación de la producción de interluequina 1 por células adherentes en pacientes con diabetes tipo II.

We have studied the comitogenic activity of IL 1 produced by cultures of mononuclear adherent cells obtained from Diabetes Mellitus (DM) type II or non insulin dependent diabetic patients. This activity was measured by the incorporation of 3H Thymidine into cultures of C3H/HeJ mice thymocytes in the presence of phytohemagglutinin (PHA). We have observed that IL 1 from patients with DM type II did not produce a synergistic effect with PHA, since the lymphoproliferation levels were similar to those obtained in the absence of this lectin. This lack of comitogenic activity (P less than 0.001) in relation to the response obtained with IL 1 from normal subjects plus PHA, could be due to the release of one or several soluble substances capable of blocking glycosylated receptors to mitogens or of impairing the cellular activation process.

Evaluación de la producción de interluequina 1 por células adherentes en pacientes con diabetes tipo II. / [Evaluation of the interleukin 1 production by adherent cells in patients with diabetes type II]

We have studied the comitogenic activity of IL 1 produced by cultures of mononuclear adherent cells obtained from Diabetes Mellitus (DM) type II or non insulin dependent diabetic patients. This activity was measured by the incorporation of 3H Thymidine into cultures of C3H/HeJ mice thymocytes in the presence of phytohemagglutinin (PHA). We have observed that IL 1 from patients with DM type II did not produce a synergistic effect with PHA, since the lymphoproliferation levels were similar to those obtained in the absence of this lectin. This lack of comitogenic activity (P less than 0.001) in relation to the response obtained with IL 1 from normal subjects plus PHA, could be due to the release of one or several soluble substances capable of blocking glycosylated receptors to mitogens or of impairing the cellular activation process.

Some characteristics of neutrophils from diabetic patients and their relation to the levels of circulating immune complexes.

In the present work we studied different characteristics of neutrophils from diabetic patients and their relation to the levels of circulating immune complexes (CIC). Twenty-five insulin-dependent (IDD) and 25 non-insulin-dependent diabetic (NIDD) patients were evaluated. Each group was then subdivided according to the presence or absence of microvascular complications (MC). We found that the chemiluminescence (CL) emitted by opsonized zymosan (Zop) stimulated neutrophils in IDD and NIDD patients was significantly increased when compared to healthy subjects (p less than 0.01 and p less than 0.02, respectively). The CL values were correlated to CIC levels and both parameters were related to the presence of MC. On the other hand, the percentage of neutrophils capable of reducing nitroblue tetrazolium was diminished in the two groups of diabetic patients (p less than 0.05 for IDD and p less than 0.01 for NIDD). The percentage of neutrophils with functional C3b receptors was normal in diabetic patients; however, the proportion of phagocytic cells through Fc receptors was significantly decreased in both types of patients (p less than 0.05 and p less than 0.01 for IDD and NIDD, respectively). Furthermore, the number of granulocytes with immune complexes (IC) bound to their cell surface was increased in diabetics. We suggest that the increase of CIC level may produce an increase in IC binding to the neutrophil membrane. These IC could block the Fc receptors, diminish phagocytic capacity and, simultaneously, stimulate the release of toxic oxygen products, thus contributing to produce tissue damage.