Lesion kinetics in a non-human primate model of endometriosis.

BACKGROUND: Endometriosis is a common cause of pelvic pain and infertility in women of reproductive age. It is characterized by the presence of endometrial tissue outside the normal location, predominantly in the pelvic peritoneum causing severe abdominal pain. However, the severity of the symptoms of endometriosis does not always correlate with the anatomic severity of the disease. This lack of correlation may be due to morphological lesion variation during disease progression. This study examined lesion kinetics in a non-human primate model of endometriosis to better understand lesion dynamics. METHODS: Endometriosis was experimentally induced in nine normal cycling female adult olive baboons (Papio anubis) by i.p. inoculation of autologous menstrual endometrium on Day 2 of menses for two consecutive menstrual cycles. Diagnostic laparoscopies were performed between Day 8-12 post-ovulation at 1, 3, 6, 9 and 12 months, followed by a necropsy at 15 months, after the second inoculation. In two animals, lesions were excised/ablated at 6 months and they were monitored for lesion recurrence and morphological changes by serial laparoscopy. Furthermore, five control animals underwent surgeries conducted at the same time points but without inoculation. RESULTS: A total of 542 endometriotic lesions were observed. The location, macroscopic (different colours) and microscopic appearance confirmed distinct endometriosis pathology in line with human disease. The majority of the lesions found 1 month after tissue inoculation were red lesions, which frequently changed colour during the disease progression. In contrast, blue lesions remained consistently blue while white lesions were evident at the later stages of the disease process and often regressed. There were significantly lower numbers of powder burn, blister and multicoloured lesions observed per animal in comparison to black and blue lesions (P-value≤0.05). New lesions were continually arising and persisted up to 15 months post-inoculation. Lesions reoccurred as early as 3 months after removal and 69% of lesions excised/ablated had reoccurred 9 months later. Interestingly, endometriotic lesions were also found in the non-inoculated animals, starting at the 6-month time point following multiple surgeries. CONCLUSIONS: Documentation of lesion turnover in baboons indicated that lesions changed their colour from red to white over time. Different lesion types underwent metamorphosis at different rates. A classification of lesions based on morphological appearance may help disease prognosis and examination of the effect of the lesion on disease symptoms, and provide new opportunities for targeted therapies in order to prevent or treat endometriosis. Surgical removal of endometriotic lesions resulted in a high incidence of recurrence. Spontaneous endometriosis developed in control baboons in the absence of inoculation suggesting that repetitive surgical procedures alone can induce the spontaneous evolution of the chronic disease. Although lesion excision/ablation may have short-term benefits (e.g. prior to an IVF cycle in subfertile women), for long-term relief of symptoms perhaps medical therapy is more effective than surgical therapy.

The pluripotency transcription factor Krüppel-like factor 4 is strongly expressed in intratubular germ cell neoplasia unclassified and seminoma.

Germ cell tumors of the testis are the most frequent tumors in men between 20 and 40 years. Their most common subtype is the seminoma, which arises like the embryonal carcinoma from an intratubular germ cell neoplasia unclassified (IGCNU), i.e. fetal germ cells that escaped from the control of the developing testicular stem cell niche, eventually leading to a fully developed seminoma (or embryonal carcinoma). The molecular causes for the development of an IGCNU are still unknown. However, IGCNU cells share the expression of several factors with primordial germ cells and gonocytes and, interestingly, also with pluripotent embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. One factor playing important roles in both iPS and ES cells is the transcription factor Krüppel-like factor 4 (KLF4). This study examined KLF4 expression data from 179 human testicular samples including normal controls and seminoma, deposited in Gene Expression Omnibus repository for microarray data at the National Centre for Biotechnology Information. Immunohistochemistry was used to detect KLF4 protein expression in IGCNU (n = 6), seminoma (n = 14) and fetal human testes (n = 14). Microarray data from three independent sources suggest higher mRNA expression in seminoma than in normal testis. Normal spermatogonia, which are the stem cells of spermatogenesis, controlled by their stem cell niche, do not express KLF4. In contrast, IGCNU and seminoma cells strongly express KLF4. In conclusion, this finding suggests that KLF4 may be an important factor for the maintenance of the developmental and the tumorigenic potential of IGCNU as well as for the malignancy of seminoma.

c-KIT is frequently mutated in bilateral germ cell tumours and down-regulated during progression from intratubular germ cell neoplasia to seminoma.

Testicular germ cell tumours (TGCTs) are the most frequent cancer type in young men; 5% of these patients develop a second TGCT in the contralateral testis. The pathogenesis of TGCT is closely linked to primordial germ cells (PGCs) or gonocytes. The receptor tyrosine kinase (c-KIT) is necessary for migration and survival of PGCs and is expressed in intratubular neoplastic germ cells (IGCNUs) and seminomas. We studied the frequency of c-KIT exon 11 and 17 mutations in 155 unilateral (108 seminomas and 47 non-seminomas) and 22 bilateral (18 seminomas, two embryonal carcinomas, two IGCNU) cases. While no mutations were detected in exon 11, the mutation frequency in exon 17 was significantly higher in bilateral (14/22, 63.6%) compared to unilateral TGCT (10/155, 6.4%) (p < 0.001). Different activating mutations (Y823D, D816V, D816H and N822K) were detected in bilateral TGCT. Y823D mutation was identical in both testes in three cases and quantitative pyrosequencing showed that up to 76% of the cells analysed in tumour samples carried this mutation. One bilateral synchronous seminoma revealed a S821F mutation in one testis and a Y823D mutation contralaterally. To study the role of c-KIT in TGCT progression, we compared its expression in 41 seminomas and adjacent IGCNUs. Immunohistochemical analysis revealed that c-KIT expression was significantly reduced in seminomas compared to IGCNUs (p < 0.006) and that there were no significant changes in c-KIT mRNA copy numbers in progressed compared to low-stage seminomas. In summary, our study shows that patients with c-KIT mutations are more prone to develop a bilateral TGCT and suggests that in a portion of bilateral TGCTs, c-KIT mutations occur early during embryonal development, prior to the arrival of PGCs at the genital ridge. Furthermore, our findings show that c-KIT down-regulation occurs during the progression of IGCNU to seminoma.

Gene signatures of testicular seminoma with emphasis on expression of ets variant gene 4.

Gene expression patterns of testicular seminoma were analysed applying oligonucleotide microarrays in 40 specimens of different tumour stages (pT1, pT2, pT3) and in normal testes. Transcripts of maternally expressed 3 transcripts were expressed in seminoma without correlation with delta-like 1 homologue expression indicating an impaired imprinting status in seminoma. Interestingly, the transcripts of bromodomain-containing 2 and nuclear autoantigenic sperm protein associated with spermatogenesis were significantly upregulated in progressing tumour stages. Transcription factors TEA domain family member 4 and ETS variant gene 4 (ETV4), weakly expressed in normal testis, were strongly augmented during tumourigenesis. For ETV4 expression, a significant correlation with the increased expression of matrix metalloproteinase 2 and a disintegrin and metalloproteinase domain 15 was determined. The ETV4 protein was localised to nuclei of spermatogonia and revealed an intense staining in seminoma cells. Taken together, we characterised additional transcription factors and spermatogenesis-associated genes involved in the progression of seminoma.

Quantitative determination of MDR1 mRNA expression in peripheral blood lymphocytes: a possible role of genetic polymorphisms in the MDR1 gene.

BACKGROUND: P-glycoprotein is a transmembrane efflux pump that extrudes a wide variety of drugs, thereby reducing their intracellular access. In humans, P-glycoprotein is encoded by the MDR1 gene. Recently, several single nucleotide polymorphisms in the MDR1 gene were identified. Moreover, it was postulated that, in addition to the full-length P-glycoprotein, a 'mini' P-glycoprotein was also present in lymphocytes. MATERIALS AND METHODS: We investigated the effect of the genetic polymorphisms G2677T and C3435T in the MDR1 gene on MDR1 mRNA expression in FACS-sorted peripheral blood CD4+, CD8+, CD19+, and CD56+ cells. MDR1 mRNA expression was determined in 45 healthy individuals using a real-time quantitative RT-PCR. RESULTS: We detected the highest expression of MDR1 mRNA in CD56+ cells, followed by CD8+ > CD4+ > CD19+ cells. However, genetic polymorphisms of the MDR1 gene failed to affect (P > 0.05) MDR1 mRNA levels in the peripheral blood lymphocytes. Furthermore, the transcript levels for the MDR1 N-terminal half were almost two-fold lower than that of the MDR1 C-terminal half in all cell populations investigated (P < 0.0001). CONCLUSIONS: An almost two-fold difference in MDR1 C- and N-terminal half expressions supports the presence of mini-P-glycoprotein, an alternatively spliced form of the full-length molecule, in peripheral blood lymphocytes.