Embryologic origin of endometriosis: analysis of 101 human female fetuses.

The etiology of endometriosis, a gynecological disease characterized by the presence of endometrial glands and stroma outside the uterine cavity, is still unknown. Our research group has recently demonstrated the presence of ectopic endometrium in human female fetuses at different gestational ages. In this manuscript we describe four new cases of fetal endometriosis found among a series of 52 female fetuses analyzed at autopsy. The anatomical localization of this ectopic endometrium, and its histological and immunohistochemical characteristics are depicted. We suggest that endometriosis is caused by dislocation of primitive endometrial tissue outside the uterine cavity during organogenesis. The clinical and pathological implications of these findings are discussed.

Endocrine disruptors in utero cause ovarian damages linked to endometriosis.

Timed pregnant Balb-C mice were treated from day 1 of gestation to 7 days after delivery with the endocrine disruptor bisphenol a (BPA) (100, or 1,000 microg/kg/day). After delivery, pups were hold for three months; then, ovaries were analyzed in their entirety. We found that in the ovaries of BPA-treated animals the number of primordial follicles and of developing follicles was significantly lower than in the untreated animals. Moreover, the number of atretic follicles was significantly higher in the treated animals. Finally, we found that the animals displaying endometriosis-like phenotype had a more severe impairment of the ovaries in term of number of primordial and developing follicles in comparison with the other mice exposed to BPA. In conclusion, we describe for the first time a complex phenotype in mice, elicited by pre-natal exposition to BPA, that includes ovarian lesions and endometriosis. Considering the high incidence of endometriosis and of the premature ovarian failure associated to infertility in these patients, the data showed prompt a thoroughly reconsideration of the pathological framing of these lesions.

New evidence of the presence of endometriosis in the human fetus.

The aetiology of endometriosis, a gynaecological disease defined by the histological presence of endometrial glands and stroma outside the uterine cavity, is still open to debate. Research has recently found evidence for endometriosis in human female fetuses at different gestational ages. This paper reports a new case of fetal endometriosis in a 25-week female fetus, deceased due to placental pathology, from a series of 13 female fetuses analysed at autopsy. The exact anatomical localization of this misplaced endometrium, as well as its histopathological and immunohistochemical characteristics are illustrated. The case suggests that endometriosis can be caused by dislocation of primitive endometrial tissue outside the uterine cavity during organogenesis.

Pre-natal exposure of mice to bisphenol A elicits an endometriosis-like phenotype in female offspring.

Endometriosis is a chronic gynecological disease characterized by the growth of endometrial tissue outside the uterine cavity. Exposure to endocrine disruptors during critical period of development causes long-lasting effects, being the genital system one of the targets. This study describes the effects on female genital system caused by developmental exposure to the endocrine-disrupting chemical bisphenol A (BPA) during pre- and peri-natal development in mice. To this end, timed pregnant Balb-C mice were treated from day 1 of gestation to 7 days after delivery with BPA (100, or 1000 microg/kg/day). After delivery, pups were held for 3 months; then, pelvic organs were analyzed in their entirety and livers of both pups and moms were studied for the presence of BPA. We found in the adipose tissue surrounding the genital tracts of a consistent number of treated animals, endometriosis-like structure with the presence of both glands and stroma and expressing both estrogen receptor and HOXA-10. Moreover, cystic ovaries, adenomatous hyperplasia with cystic endometrial hyperplasia and atypical hyperplasia were significantly more frequent in treated animals respect to the controls. Finally, BPA was found in the livers of exposed moms and female offspring. In conclusion, we describe for the first time an endometriosis-like phenotype in mice, elicited by pre-natal exposition to BPA. This observation may induce to thoroughly reconsider the pathogenesis and treatment of endometriosis, considering the high incidence of endometriosis and the problems caused by associated infertility.

Ectopic endometrium in human foetuses is a common event and sustains the theory of müllerianosis in the pathogenesis of endometriosis, a disease that predisposes to cancer.

BACKGROUND: Endometriosis is a gynecological disease defined by the histological presence of endometrial glands and stroma outside the uterine cavity. Women with endometriosis have an increased risk of different types of malignancies, especially ovarian cancer and non-Hodgkin's lymphoma. Though there are several theories, researchers remain unsure as to the definitive cause of endometriosis. Our objective was to test the validity of the theory of müllerianosis for endometriosis, that is the misplacing of primitive endometrial tissue along the migratory pathway of foetal organogenesis METHODS: We have collected at autopsy 36 human female foetuses at different gestational age. We have performed a morphological and immunohistochemical study (expression of oestrogen receptor and CA125) on the pelvic organs of the 36 foetuses included en-block and totally analyzed. RESULTS: In 4 out of 36 foetuses we found presence of misplaced endometrium in five different ectopic sites: in the recto-vaginal septum, in the proximity of the Douglas pouch, in the mesenchimal tissue close to the posterior wall of the uterus, in the rectal tube at the level of muscularis propria, and in the wall of the uterus. All these sites are common location of endometriosis in women. CONCLUSION: We propose that a cause of endometriosis is the dislocation of primitive endometrial tissue outside the uterine cavity during organogenesis.

Right ventricular cardiomyocyte apoptosis in patients with acute myocardial infarction of the left ventricular wall.

Cardiac remodeling after acute myocardial infarction (AMI) is characterized by molecular and cellular mechanisms involving both the left (LV) and right ventricular (RV) walls. Cardiomyoycte apoptosis in the peri-infarct and remote LV myocardium has a central role in cardiac remodeling. Whether apoptosis also occurs in the right ventricle of patients with ischemic heart disease has not been investigated. The aim of the present study was to investigate the presence of cardiomyocyte apoptosis in the right ventricle in patients with AMI. We assessed the number of apoptotic cardiomyocytes using multiple samplings in the LV and RV walls of 12 patients selected at autopsy who died 4 to 42 days after AMI. Five patients without cardiac disease were also selected at autopsy as controls. Apoptotic rates were calculated from the number of cardiomyocytes showing double positive staining for in situ end-labeling of DNA fragmentation (TUNEL) and for activated caspase-3. Potentially false-positive results (DNA synthesis and RNA splicing) were excluded from cell counts. The apoptotic rate in the right ventricle in patients with AMI was significantly higher than in control hearts (median 0.8%, interquartile range 0.3 to 1.0 vs median 0.01%, interquartile range 0.01 to 0.03, p <0.001). RV apoptosis significantly correlated with such parameters of global adverse remodeling as cardiac diameter to LV free wall thickness (R = +0.57, p = 0.050). RV apoptosis was significantly higher in five cases (42%) with infarct involving the ventricular septum and an adjacent small area of the RV walls (median 1.0%, interquartile range 0.8 to 2.2 vs median 0.5%, interquartile range 0.2 to 1.0, p = 0.048, p <0.001 vs controls). The association between apoptotic rate in the right ventricle and cardiac remodeling was apparent even after exclusion of cases with RV AMI involvement (R = +0.82, p = 0.023 for diameter to LV wall thickness ratio and R = -0.91, p = 0.002 for RV free wall thickness). In conclusion, patients with cardiac remodeling after AMI had a significant increase in RV apoptosis even when ischemic involvement of the RV wall was not apparent.

Patterns of tumor response in canine and feline cancer patients treated with electrochemotherapy: preclinical data for the standardization of this treatment in pets and humans.

Electrochemotherapy (ECT) is a novel anticancer therapy that is currently being evaluated in human and pet cancer patients. ECT associates the administration of an anti-tumor agent to the delivery of trains of appropriate waveforms. The increased uptake of chemotherapy leads to apoptotic death of the neoplasm thus resulting in prolonged local control and extended survival. In this paper we describe the histological features of a broad array of spontaneous tumors of companion animals receiving pulse-mediated chemotherapy. Multivariate statistical analysis of the percentage of necrosis and apoptosis in the tumors before and after ECT treatment, shows that only a high percentage of necrosis and apoptosis after the ECT treatment were significantly correlated with longer survivals of the patients (p < 0.0001 and p = 0.004, respectively). Further studies on this topic are warranted in companion animals with spontaneous tumors to identify new molecular targets for electrochemotherapy and to the develop new therapeutical protocols to be translated to humans.

Identification of protein disulfide isomerase as a cardiomyocyte survival factor in ischemic cardiomyopathy.

OBJECTIVES: The aim of the study was to analyze the molecular mechanisms activated during postinfarction remodeling in human hearts. BACKGROUND: The molecular mechanisms of initial response to ischemic insult in the heart and the pathways involved in compensation and remodeling are still largely unknown. METHODS: Up-regulation or down-regulation of gene expression in the human viable peri-infarct (vs. remote) myocardial region was investigated by complementary deoxyribonucleic acid array technology and confirmed at a single-gene/protein level with reverse transcriptase polymerase chain reaction and immunohistochemistry. An in vitro model of cardiomyocyte hypoxia in HL1 cells was used to validate anti-apoptotic effects of the candidate gene/protein and to assess the associated downstream cascade. Finally, a mouse model of myocardial infarction was used to test the in vivo effects of exogenous transfection with the candidate gene/protein. RESULTS: Protein disulfide isomerase (PDI), a member of the unfolded protein response, is 3-fold up-regulated in the viable peri-infarct myocardial region, and in a postmortem model, its expression is significantly inversely correlated with apoptotic rate and with presence of heart failure (HF) and biventricular dilatation. Induced PDI expression in HL1 cells conferred protection from hypoxia-induced apoptosis. Adenoviral-mediated PDI gene transfer to the mouse heart resulted in 2.5-fold smaller infarct size, significantly reduced cardiomyocyte apoptosis in the peri-infarct region, and smaller left ventricular end-diastolic diameter versus mice treated with a transgene-null adenoviral vector. CONCLUSIONS: These results suggest that PDI promotes survival after ischemic damage and that zinc-superoxide dismutase is one of the PDI molecular targets. Pharmacological modulation of this pathway might prove useful for future prevention and treatment of HF.

Ischemia and apoptosis in an animal model of permanent infarct-related artery occlusion.

Apoptosis is a pathologic feature of cardiomyocytes in acute myocardial infarction (AMI) and heart failure. The temporal course of apoptosis in the peri-infarct area in the weeks following an AMI is still uncompletely defined. In order to study the time course of apoptosis after AMI, 16 rabbits underwent left coronary artery ligation and were sacrificed at 16, 26, 35, and 56 days after surgery. Increased apoptotic rate (AR) was observed at in the peri-infarct region than in remote myocardium (5.4% [2.5-9.6] vs 0.4% [0.1-0.9], respectively, P<0.001) and than in sham-operated cases (0.01% [0-0.02], P<0.001). A gradual decrease of AR in the peri-infarct region was observed over time with a 90% reduction at 8 weeks after coronary ligation.

Analysis of HtrA1 serine protease expression in human lung cancer.

HtrA1 is a serine protease homologue to the bacterial serine-protease HtrA, also known as DegP, a heat shock-induced envelope-associated serine protease. It has been shown that over-expression of HtrA1 in human cancer cells inhibits cell growth and proliferation in vitro and in vivo, thus, suggesting a possible role as a tumor suppressor. The expression of HtrA1 was investigated in depth by means of immunohistochemistry in a large group of human lung cancer specimens and corresponding lymph node metastases. Univariate analysis showed, that the only statistically significant correlation was found between the HtrA1 expression level detected in the primary tumors and in the lymph node metastases. This result was also confirmed when the analysis was restricted only to the cases where both the primary tumor and the autologous lymph node metastasis were available. Our data suggest that HtrA1 may be involved in lung cancer progression by targeting several molecular pathways.

The ubiquitin-proteasome system and inflammatory activity in diabetic atherosclerotic plaques: effects of rosiglitazone treatment.

The role of ubiquitin-proteasome system in the accelerated atherosclerotic progression of diabetic patients is unclear. We evaluated ubiquitin-proteasome activity in carotid plaques of asymptomatic diabetic and nondiabetic patients, as well as the effect of rosiglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma activator, in diabetic plaques. Plaques were obtained from 46 type 2 diabetic and 30 nondiabetic patients undergoing carotid endarterectomy. Diabetic patients received 8 mg rosiglitazone (n = 23) or placebo (n = 23) for 4 months before scheduled endarterectomy. Plaques were analyzed for macrophages (CD68), T-cells (CD3), inflammatory cells (HLA-DR), ubiquitin, proteasome 20S activity, nuclear factor (NF)-kappaB, inhibitor of kappaB (IkappaB)-beta, tumor necrosis factor (TNF)-alpha, nitrotyrosine, matrix metalloproteinase (MMP)-9, and collagen content (immunohistochemistry and enzyme-linked immunosorbent assay). Compared with nondiabetic plaques, diabetic plaques had more macrophages, T-cells, and HLA-DR+ cells (P < 0.001); more ubiquitin, proteasome 20S activity (TNF-alpha), and NF-kappaB (P < 0.001); and more markers of oxidative stress (nitrotyrosine and O2(-) production) and MMP-9 (P < 0.01), along with a lesser collagen content and IkappaB-beta levels (P < 0.001). Compared with placebo-treated plaques, rosiglitazone-treated diabetic plaques presented less inflammatory cells (P < 0.01); less ubiquitin, proteasome 20S, TNF-alpha, and NF-kappaB (P < 0.01); less nitrotyrosine and superoxide anion production (P < 0.01); and greater collagen content (P < 0.01), indicating a more stable plaque phenotype. Similar findings were obtained in circulating monocytes obtained from the two groups of diabetic patients and cultured in the presence or absence of rosiglitazone (7.0 micromol/l). Ubiquitin-proteasome over-activity is associated with enhanced inflammatory reaction and NF-kappaB expression in diabetic plaques. The inhibition of ubiquitin-proteasome activity in atherosclerotic lesions of diabetic patients by rosiglitazone is associated with morphological and compositional characteristics of a potential stable plaque phenotype, possibly by downregulating NF-kappaB-mediated inflammatory pathways.

Adjuvant electrochemotherapy for the treatment of incompletely resected canine mast cell tumors.

BACKGROUND: Electrochemotherapy (ECT) is a novel anticancer therapy that combines the delivery of trains of appropriate waveforms with the local administration of chemotherapy agents. The purpose of this investigation was to assess the adjuvant potentials of ECT for the treatment of incompletely excised mast cell tumors (MCT). MATERIALS AND METHODS: Twenty-eight privately-owned dogs with incompletely removed MCT were treated with intralesional bleomycin (1.5 IU/cm2) followed by the application of trains of biphasic pulses (8 pulses, 1300 V/cm, 50 + 50 micros duration, 1 Hz frequency). RESULTS: The overall response rate was 85% with a mean estimated time to recurrence of 52.76 +/- 6.5 months (range: 39.99 to 65.54 months, 95% CI). At the time of writing this report, the median survival time was not reached. Three dogs died of metastatic disease that they developed at the same time of local recurrence, one developed multiple cutaneous nodules at different locations and one with recurrence was re-treated and is currently disease-free after 22 months. No major local or systemic toxicities were noted for the duration of the study. CONCLUSION: ECT is a safe and effective therapy for incompletely excised MCTs in companion animals. Its ease of administration, lack of toxicities and low cost make it an attractive alternative to standard treatments and warrants further investigation.

Infarct-related artery occlusion, tissue markers of ischaemia, and increased apoptosis in the peri-infarct viable myocardium.

AIMS: Unfavourable cardiac remodelling often complicates acute myocardial infarction (AMI) as a result of increased cardiomyocyte apoptosis. It is currently unclear whether ongoing or recurrent ischaemia is an independent determinant for increased apoptosis in peri-infarct viable myocardium. METHODS AND RESULTS: In order to assess the link between infarct-related artery (IRA) occlusion, ischaemia, and apoptosis, 30 subjects dying 7-120 days after AMI (16 with IRA occlusion and 14 with patent IRA) and five control subjects were selected at autopsy. Cardiomyocytes were defined as apoptotic if co-expressing TUNEL and activated caspase-3. Expression of both hypoxia-inducible factor-1 and cyclo-oxygenase-2 was assessed in the peri-infarct myocardium and considered as tissue markers of ischaemia. Evidence of ischaemia was significantly more frequent in cases with IRA occlusion (53%) than in cases with patent IRA (15%) or control hearts (0%, P=0.026). The finding of IRA occlusion and markers of ischaemia identified cases with higher apoptotic rates (ARs) in the peri-infarct viable myocardium [12.2% (8.2-14.0), P<0.001 vs. others], whereas IRA occlusion without ischaemia was associated with lower AR, not significantly different from patent IRA [3.0% (1.0-7.9) vs. 2.2% (1.0-5.8), respectively, P=0.42] CONCLUSION: Ischaemia in the peri-infarct viable myocardium is present in over 50% of subjects dying late after AMI with IRA occlusion, and it is associated with increased apoptosis. Relief of ischaemia after AMI may prove of benefit in preventing apoptosis and its consequences.

Ferritin contributes to melanoma progression by modulating cell growth and sensitivity to oxidative stress.

PURPOSE: Employing an in vitro model system of human melanoma progression, we previously reported ferritin light chain (L-ferritin) gene overexpression in the metastatic phenotype. Here, we attempted to characterize the role of ferritin in the biology of human melanoma and in the progression of this disease. EXPERIMENTAL DESIGN: Starting from the LM human metastatic melanoma cell line, we engineered cell clones in which L-ferritin gene expression was down-regulated by the stable expression of a specific antisense construct. These cells were then assayed for their growth capabilities, chemoinvasive properties, and sensitivity to oxidative stress. Additionally, ferritin protein content in primary and metastatic human melanomas was determined by immunohistochemistry. RESULTS: Artificial L-ferritin down-regulation in the LM cells strongly inhibited proliferation and chemoinvasion in vitro and cell growth in vivo. In addition, L-ferritin down-regulated cells displayed enhanced sensitivity to oxidative stress and to apoptosis. Concurrently, immunohistochemical analysis of a human melanoma tissue array revealed that ferritin expression level in metastatic lesions was significantly higher (P < 0.0001) than in primary melanomas. Furthermore, ferritin expression was constantly up-regulated in autologous lymph node melanoma metastases when compared with the respective primary tumors in a cohort of 11 patients. CONCLUSIONS: These data suggest that high ferritin expression can enhance cell growth and improve resistance to oxidative stress in metastatic melanoma cells by interfering with their cellular antioxidant system. The potential significance of these findings deserves to be validated in a clinical setting.

A case of granulomatous skin lesions caused by Mycobacterium marinum in the Campania region.

The diagnosis of cutaneous Mycobacterium marinum infection is frequently presumptive, as detection by conventional methods is difficult. We describe a patient with granulomatous skin lesions on the right dorsal hand and forearm. Histological examinations were presumptive for mycobacterium lesions. We identified Mycobacterium marinum directly in the patient's lesional skin biopsy combining polymerase chain reaction (PCR) amplification using Mycobacterium genus-specific primers, and subsequent restriction enzyme analysis enabling identification to the species level. The symptoms were no longer present after specific therapy, thereby confirming the initial diagnosis.

Pattern of expression of HtrA1 during mouse development.

The human HtrA family of proteases consists of four members: HtrA1, HtrA2, HtrA3, and HtrA4. In humans the four HtrA homologues appear to be involved in several important functions such as cell growth, apoptosis, and inflammatory reactions, and they control cell fate via regulated protein metabolism. In previous studies it was shown that the expression of HtrA1 was ubiquitous in normal adult human tissues. Here we examined the expression of HtrA1 protein and its corresponding mRNA during mouse embryogenesis using Northern blotting hybridization, RT-PCR, and immunohistochemical staining analyses. Our results indicate that HtrA1 is expressed in a variety of tissues in mouse embryos. Furthermore, this expression is regulated in a spatial and temporal manner. Relatively low levels of HtrA1 mRNA are detected in embryos at the beginning of organogenesis (E8), and the levels of expression increase during late organogenesis (E14-E19). Our results show that HtrA1 was expressed during embryonic development in specific areas where signaling by TGFbeta family proteins plays important regulatory roles. The expression of HtrA1, documented both at mRNA and protein levels by RT-PCR and immunohistochemistry in the developing nervous system, is consistent with a possible role of this protein both in dividing and postmitotic neurons, possibly via its documented inhibitory effects on TGFbeta proteins. An exhaustive knowledge of the different cell- and tissue-specific patterns of expression of HtrA1 in normal mouse embryos is essential for a critical evaluation of the exact role played by this protein during development.

Expression of angiogenic factors during acute coronary syndromes in human type 2 diabetes.

Inadequate angiogenic response to ischemia in diabetic myocardium could result in poor collateral formation. Because hypoxia-inducible factor (HIF)-1alpha is a transcriptional activator of vascular endothelial growth factor (VEGF) and is critical for initiating angiogenic responses to hypoxia, we investigated the expression of HIF-1alpha and VEGF in specimens of human heart tissue to elucidate the molecular responses to myocardial ischemia in diabetic patients during unstable angina. Moreover, accumulation of a marker of protein nitration nitrotyrosine, as well as the superoxide anion (O(2)(-)) levels and inducible nitric oxide synthase (iNOS), were evaluated. Ventricular biopsy specimens from 15 type 2 diabetic and 14 nondiabetic patients presenting with unstable angina (ischemic group) and from 20 patients (11 type 2 diabetic and 9 nondiabetic patients) who underwent coronary bypass surgery without angina within the preceding 10 days (control group) were collected during coronary bypass surgery. Nondiabetic patients had higher HIF-1alpha and VEGF expressions compared with diabetic patients (P < 0.001). As compared with nondiabetic specimens, diabetic specimens showed higher levels of both iNOS mRNA and protein levels (P < 0.001) associated with the highest tissue levels of nitrotyrosine and O(2)(-) (P < 0.001). Diabetes is associated with increased myocardial tissue levels of iNOS, O(2)(-), and nitrotyrosine and reduced expression of myocardial angiogenesis factors during ischemia.