Expression of mRNA and protein of IL-18 and its receptor in human follicular granulosa cells.

PURPOSE: There is no information available about the IL-18 receptor in ovarian follicles, so the present study attempts to demonstrate the expression of IL-18 and its receptor in human granulosa cells (GCs). METHODS: To evaluate the concentration of IL-18 in serum and follicular fluid (FF), we collected serum and FF from 102 women undergoing oocyte retrieval. Also, to detect expression of IL-18 and its receptor by luteinized GCs, these cells were pooled six times from a total of twenty individual patients with 5-16 follicles each. The IL-18 concentration was determined by ELISA and the expression of IL-18 and its receptor by immunocytochemistry and reverse transcription polymerase chain reaction. RESULTS: Our results showed that the median IL-18 concentration in serum, 159.27 pg/ml (IQR 121.41-210.1), was significantly higher than in FF, 142.1 pg/ml (IQR 95.7-176.5), p < 0.001. Moreover, we found that IL-18 and its receptor are expressed by GCs. CONCLUSION: The presence of IL-18 in FF and the expression of IL-18 and its receptor by GCs suggest an important role for this cytokine in ovarian function.

Ovulation detection in saliva, is it possible.

BACKGROUND/AIMS: The new mini-microscope Geratherm® ovu control was evaluated for its recognition of saliva ferning in a collective of 47 patients taking part in an artificial reproductive technology program on the day of follicular puncture. METHODS: The ferning phenomenon was evaluated by patients and laboratory staff according to the criteria: no ferning, slight ferning and good ferning. RESULTS: Geratherm® ovu control showed a specificity of 78% and a sensitivity of 80% in relation to rising E2 levels under follicle-stimulating hormone/human chorionic gonadotrophin. A comparison of the evaluations of the saliva test carried out by patients and by laboratory staff resulted in a high and substantial agreement of 89.4% (κ). CONCLUSION: Evaluations performed by ovu control were similar to those achieved with a highly sophisticated inverted microscope.

Comparison of c-DNA microarray analysis of gene expression between eutopic endometrium and ectopic endometrium (endometriosis).

PROBLEM: As recent studies have suggested abnormalities in the regulation of specific genes in the development of endometriosis, we investigated differentially expressed genes in endometriosis compared to endometrium. METHOD OF STUDY: Gene expression profiles using the Atlas microarray were performed in endometriotic tissue and endometrium. Nine of the 13 genes of endometriotic tissue showed an up-regulation in relation to endometrium and four of the 13 genes a down-regulation. RESULTS: Of the 1176 genes on the Atlas Human 1,2 array, only 13 differentially expressed identical genes were detected after repeating the gene analysis three times. CONCLUSION: According to our c-DNA analysis some differentially expressed genes may be involved in the pathogenesis of endometriosis. An imbalance in the genes responsible for the reproductive process may lead to a decrease in embryo implantation in patients with endometriosis.

Is granulocyte colony-stimulating factor level predictive for human IVF outcome?

BACKGROUND: We investigated granulocyte colony-stimulating factor (G-CSF) in human reproduction. METHODS: From a total sample of 93 patients, we analysed in group 1 (n = 82) the level of G-CSF and estradiol (E(2)) in serum and follicular fluid (FF) on day of follicular puncture (FP). Furthermore, in response to ovarian stimulation, G-CSF levels in serum were compared between low (n = 11), moderate (n = 53) and high (n = 18) response patients. In group 2 (n = 23) serum for G-CSF assessment was collected throughout menstrual cycle until gestation. Group 3 (n = 11) patients with endometriosis were assessed for G-CSF in serum and FF on day of FP without further differentiation. RESULTS: G-CSF in FF was higher than in serum (P < 0.01). G-CSF in serum increased from low through moderate to high response (P < 0.001); pregnancy rates were 0, 24.5 and 33.5% respectively. G-CSF in serum increased throughout stimulation, reached a peak with ovulation induction (P = 0.01) and decreased until embryo transfer (P=0.001). G-CSF level only in pregnant patients (n = 11) increased from embryo transfer to implantation to gestation (P = 0.005). In endometriosis patients G-CSF in serum and FF was lower than in non-endometriosis patients (P < or = 0.03) and corresponded with low response patients. CONCLUSIONS: G-CSF is involved in follicle development and may be a predictor of IVF outcome.

Perioperative levels of interleukin-1beta and interleukin-6 in women with breast cancer.

OBJECTIVE: Proinflammatory cytokines, such as Interleukin-1beta and Interleukin-6, are known to play an important biological role in trauma, sepsis and malignant disease. Surgery can modulate the immune system, especially in patients with malignant diseases, by influencing the serum levels of Interleukin-1beta and Interleukin-6. It is known that cytokine levels depend on the grade of tissue injury. We have investigated the serum levels of Interleukin-1beta and Interleukin-6 during the perioperative period in women with breast cancer undergoing simple mastectomy or segmental resection. The aim of the study was to analyse whether breast cancer surgery influences cytokine expression in the peripheral blood and whether the concentrations of the measured interleukines differ according to the surgical method. METHODS: Blood samples of 45 women with breast cancer (Stage I and II) undergoing simple mastectomy (n = 16) or segmental resection (n = 29) were collected at six different times: before (T1), during (T2), three hours after (T3), one day after (T4), three days (T5) and five days after (T6) surgery. The serum levels of Interleukin-1beta and Interleukin-6 were measured by ELISA. RESULTS: In both groups Interleukin-1beta serum concentrations (p < 0.05) increased significantly on the first day after surgery. On the third day after surgery the concentrations of Interleukin-1b decreased to the preoperative level. A significant difference in Interleukin-1beta concentrations as a consequence of the surgical method was not detectable. Changes in the serum levels of Interleukin-6 within the measurement period were not observed. CONCLUSIONS: Surgery in patients with breast cancer leads to increased Interleukin-1beta serum levels on the first postoperative day. It has been shown that elevated Interleukin-1beta and Interleukin-6 levels are correlated with a high rate of recurrence. Therefore, this may be of consequence to patients with malignant diseases. The method of surgery for both types, however, had no influence on the peripheral cytokine expression. Therefore, a nearly equal influence on the immune system can be stated.

[Effect of estradiol and progesterone on production of macrophage colony-stimulating factor by human granulosa cells in vitro].

OBJECTIVE: To investigate the effect of estradiol (E(2)) and progesterone (P) on macrophage colony-stimulating factor (M-CSF) production by human luteinized granulosa cells in vitro. METHODS: Human luteinized granulosa cells were isolated from follicular fluid of superovulated infertile patients undergoing intracytoplasmatic sperm injection (ICSI). Luteinized granulosa cells were cultured with HAM's F-10 medium with various concentrations of E(2) or P (0, 1 x 10(-7), 1 x 10(-6), 1 x 10(-5), 1 x 10(-4), 1 x 10(-3) mol/L). Media were removed at 72 h after culture. M-CSF in media was measured by a solid phase enzyme-linked immunosorbant assay (ELISA) and estradiol and progesterone in media were measured by enzyme immunoassays (EIA). RESULTS: The basic concentration of M-CSF in cultivated granulosa cells without E(2) stimulation was low (47 +/- 15 ng/L). However, E(2) at concentrations of 1 x 10(-6) - 1 x 10(-3) mol/L caused a significant increase of M-CSF secretion by luteinized granulosa cells in a dose-dependent mode (2.3, 4.5, 6.9, 7.9 times higher than the basic level, respectively) (P < 0.05). E(2) at a concentration of 1 x 10(-7) mol/L, however, did not stimulate the production of M-CSF (P > 0.05). P at concentrations of 1 x 10(-7) - 1 x 10(-3) mol/L showed no effect on M-CSF production by luteinized granulosa cells (P > 0.05). CONCLUSIONS: Estradiol can stimulate M-CSF production by luteinized granulosa cells in vitro in a dose-dependent mode, on the other hand P cannot induce M-CSF production by luteinized granulosa cells. The mechanism of estradiol regulating follicular development may partially via M-CSF/receptor pathway.

Interaction of interleukin-6 on human granulosa cell steroid secretion.

Numerous studies have shown that a variety of cytokines are involved in the regulation of ovarian function. Interleukin-6 (IL-6), has been found in follicular fluid. In this report we show by immunocytochemical methods the localization of the extracellular domain of the IL-6-receptor and its associated signal transducer glycoprotein gp 130 on the surface of granulosa cells (GCs). The possibility that IL-6 may also influence the basal and FSH-stimulated production of estradiol (E2) and progesterone (Prog) by GCs in vitro was also investigated. GCs were obtained from infertile patients undergoing in vitro fertilization and embryo transfer treatment and cultured for 72 h or were given increasing concentrations of human recombinant IL-6 (8--128 pg/ml) in the absence or presence of FSH (96 U/ml). For the time-course studies, FSH-stimulated GCs were treated in the absence or presence of IL-6 (128 pg/ml) and supernatants were assayed at 24 h intervals (24-96 h) for E2 and Prog productions. The results show that increasing amounts of IL-6 significantly inhibit E2 production in the absence or presence of FSH vs untreated controls (P=0.025 at IL-6=128 pg/ml and P=0.016 at IL-6=16 pg/ml respectively). IL-6 also inhibited FSH-stimulated but not unstimulated Prog release (P=0.038 at IL-6=8 pg/ml). These findings suggest that IL-6 may be one of the factors that plays a local regulatory role in the course of FSH-stimulated E2 and Prog release. The time-course studies of the effect of the absence or presence of IL-6 demonstrated a significant inhibitory effect on both E2 and Prog secretion (P<0.001) by FSH-stimulated GCs. As infections of the female reproductive tract are often accompanied by elevated local IL-6 levels, this factor may be one of the links leading to endocrine reproductive dysfunction during genital infections.

Characterization of a MAb to ovarian cancer and its possible diagnostic application.

We report the distribution pattern of the target antigen of an IgG1 monoclonal antibody, Ki-OC III raised in BALB/C mice against solubilised ovarian adenocarcinoma, in normal tissue and in a collection of human tumour types. Special reference was made to benign and malignant ovarian tumours. The reactive antigen protein purified to homogeneity for a planned amino acid analysis showed three bands between 37-40 kDa. Immunohistochemical analysis revealed a specificity of 98% and a sensitivity of 77%. The tracer kinetics of the radiolabelled antibody were tested on a human ovarian carcinoma cell line in athymic mice. The results were compared to cell lines derived from breast and stomach carcinomas as well as to a human glioblastoma cell line. The results show the preferential uptake by ovarian cancer cells followed by breast cancer cell lines. In animal models, scintigraphic monitoring and direct measurement of the radio-labelled monoclonal antibody showed a preferential accumulation in tumours, in addition to high level signals in the liver, kidney, spleen and heart which were related to degradation, excretion and high circulation. The Ki-OC III reactive antigen could be a potential candidate for immunomonitoring of ovarian and possibly also of breast cancer, for in vivo tumour imaging as well as for histopathologic examinations.

Isolation of monocytic serine esterase and evaluation of its proteolytic activity.

Monocytes are characterized by high activity of alpha-naphthyl acetate esterase (ANAE), distinguishing them from all other blood cells. The physiological function of this monocyte marker enzyme has not yet been elucidated. In this study ANAE's potential proteolytic activity was analyzed because serine esterases/proteases can function as effector molecules in cell-mediated cytotoxicity and because monocytes-macrophages are known to exert cytotoxic effects on tumor cells. This enzyme was purified from the monocytic cell line U-937 by preparative isoelectric focusing and a three-step high-performance liquid chromatography that conserved its catalytic activity. It has a molecular mass of 60 kd, and partial amino acid sequence revealed that the enzyme is not identical to known serine esterases/proteases. The purified enzyme failed to digest a couple of peptides, indicating lack of protease activity. In addition, the esterolytic activity of ANAE was not inhibited by protease inhibitors. The isolation and purification of ANAE enable further studies concerning its function in monocytes-macrophages and its relation to monocytic cytotoxicity.

Heterogenous expression and putative structure of human monocyte/macrophage serine esterase 1.

Human monocyte serine esterase 1 (HMSE1) was purified from U937 cell extract. Since the N terminus of the enzyme was blocked, cleavage with trypsin was used to obtain several peptides accessible to amino acid sequencing. Based on partial amino acid sequence information, an oligonucleotide probe was synthesized and used to screen a U937 cDNA library. One clone was isolated and sequenced by us which contains an open reading frame of 503 amino acids that lacks about 50 amino acids at the N terminus relative to the protein. Computer analysis revealed an active site characteristic of known carboxylesterases with a catalytic active serine. Northern blot hybridization analysis revealed that the expression of HMSE1 is restricted to cells of the monocyte/macrophage system. In contrast to the moderate expression of HMSE1 in monocytes, alveolar macrophages showed very high amounts of the transcript. With the sequence features detected by computer analysis a structure model of HMSE1 as a dimeric, membrane-bound ectoenzyme was developed.

cDNA cloning and characterization of human monocyte/macrophage serine esterase-1.

Human monocyte/macrophage serine esterase (HMSE), commonly known as acid esterase or alpha-naphthylacetate esterase, comprises a group of five enzyme variants that can be distinguished by their isoelectric points from esterase variants of the other normal human blood cell populations. A cDNA for one of the monocytic enzyme variants (HMSE1) was cloned from a U-937 lambda gt11 cDNA library by screening with an oligonucleotide mixture designed according to amino acid sequence data of the purified enzyme. The cDNA contains 1,727 bp with an open reading frame of 1,512 bp coding for a protein of 503 amino acid residues. HMSE1 cDNA represents the first cloned monocyte/macrophage-specific serine esterase and its sequence shows up to 77% homology to other known serine esterases of different species. The amino acid composition of the putative active site of HMSE1 as deduced from the nucleotide sequence corresponds with the active sites of other serine esterases but not with the active sites of serine proteases. Hybridization of the cDNA with RNA of separated normal blood cell populations and hematopoietic cell lines shows restricted expression within the monocyte/macrophage lineage.

Six new monoclonal antibodies to serous, mucinous, and poorly differentiated ovarian adenocarcinomas.

Six monoclonal antibodies directed against ovarian adenocarcinoma were generated by use of 100,000 x g supernatants of Triton-X-100 solubilized extracts of ovarian serous adenocarcinoma as the antigen source. Immunoperoxidase preparation of frozen-sections and routinely processed paraffin section specimens revealed a highly restricted reactivity of these antibodies when tested with adult (n = 2) and fetal (n = 3) tissue types. Coreactivities were occasionally observed with epithelia of the kidney, mammary gland, and pancreas. One monoclonal antibody, Ki-OC I-6-2, cross-reacted only with epididymal epithelia. No coreaction occurred with normal tissue of the ovary, Fallopian tube, or uterus. All antibodies were additionally tested on 74 cases of nonovarian malignancies, 15 cases of ovarian metastases of nonovarian carcinomas, and 114 specimens of ovarian neoplasms other than carcinomas. Ki-OC I-6-2 had no cross-reactivity with these tumors except for one case of renal cell carcinoma. This monoclonal antibody recognized serous, mucinous, and poorly differentiated adenocarcinoma cell types. None of the six antibodies reacted with clear cell or endometrioid carcinoma. All were found to be of the IgG-1 subclass. The tumor antigen to which Ki-OC I-6-2 immunoreacted was estimated to have a molecular weight of 80 kilodaltons (KD).

[Monoclonal antibodies Ki-Sp II-13 and VI-2 react with surface membrane of human spermatozoa]. / Monoklonale Antikörper Ki-Sp II-13 und Ki-Sp VI-2 erkennen Oberflächen-Antigene humaner Spermatozoen.

Immunizing Balb-c mice with washed motile human spermatozoa enabled the production of 2 monoclonal spermatozoal antibodies designated Ki-Sp II-13 and Ki-Sp VI-2. Immunohistochemically Ki-Sp VI-2 reacts only with mature spermatozoa. The monoclonal antibody Ki-Sp II-13 recognizes besides spermatozoa also T- and B-lymphocytes. Electronmicroscopically both antibodies react with the surface membrane of spermatozoa in the area of the head, the neck and the proximal part of the tail. Functional tests show a high sperm-agglutinating and sperm-immobilizing activity, using different test systems for Ki-Sp II-13. Both antibodies proved to be IgG. On immunoprecipitation Ki-Sp II-13 was identified as IgG-2a and Ki-Sp VI-2 as IgG1. Immunoprecipitates of SDS-electrophoretically separated and radiolabeled sperm surface antigens revealed a molecular weight of 18 KD for Ki-Sp II-13 and of 24 KD for Ki-Sp VI-2.