High serum AMH inhibits pathological growth of the low biomass endometrial microbiome.

OBJECTIVE: Although host microbiome play a role in both hormonal status and fertility, this issue has not yet been clarified. Since the endometrium is a sterile tissue, it is accepted that microbiota does not grow under normal conditions. The aim of the study was to reveal the characteristics of endometrial microbiota according to serum AMH levels in women with implantation failure. PATIENTS AND METHODS: Forty-five women aged 20-30 years with two or more implantation failures were included in the study. They were divided into 3 groups according to their serum AMH values: Group 1 -AMH <1.3 ng/ml; Group 2 - AMH between 1.3-2.6 ng/ml; Group 3 - AMH >2.6 ng/ml. Twenty-two healthy fertile women who were the same age as the infertile group and applied for cervical smear screening were accepted as the control group. Following the embryo transfer, the tip of the catheter was inserted into the transport medium under sterile conditions. Sowing was carried out by touching the tips of the catheter to the blood agar medium. After the evaluation of the petri dishes at the end of 48 hours of incubation, colonies were stained with Gram stain. Microorganisms in the colonies were identified with the Vitek-2 device according to their gram-staining characteristics and their antibiograms were made. RESULTS: A negative correlation was detected between low AMH values and the microbiome detection rates in endometrial cultures. In patients with low serum AMH levels, the chance of endometrial microbiota growth was higher in the endometrial culture medium. The most common bacteria were found to be MSSA, MRKNS and lactobacillus. Clinical pregnancy rates were found to be significantly higher in the group with high AMH levels. As AMH levels increased, positive flora detection rates decreased, while clinical pregnancy rates increased. CONCLUSIONS: Low serum AMH level increases the rate of positive endometrial microbiome in culture and decreases clinical pregnancy rates.

Adrenal cortical carcinoma metastatic to the breast.

A 56 year old woman was diagnosed with adrenal cortical carcinoma in May 2003, for which she underwent left radical adrenalectomy. Eight months later, in January 2004, she presented with a solitary, well delineated, left breast mass with central pleomorphic calcifications on mammographic examination. A diagnosis of metastatic adrenal cortical carcinoma was made on core biopsy. Subsequently, the patient underwent a lumpectomy of the mass, which confirmed the diagnosis. To our knowledge, this is the first case report of adrenal cortical carcinoma metastatic to the breast.

Bradykinin metabolism in the isolated perfused rabbit heart.

BACKGROUND: Bradykinin is a potent cardioprotective hormone, the beneficial role of which in vivo appears to be limited by its rapid metabolism. Inhibitors of peptidases that degrade endogenously formed bradykinin are themselves cardioprotective, presumably by increasing local bradykinin concentrations. As bradykinin-degrading peptidases are potential therapeutic targets, it is important to identify these enzymes in different animal models of cardiac function. OBJECTIVE: To determine the mechanism of bradykinin degradation in the coronary circulation of the rabbit, using an isolated perfused heart preparation. DESIGN AND METHODS: [3H]Bradykinin (16 nmol/l) was perfused as a bolus through the isolated rabbit heart in the presence and absence of specific peptidase inhibitors. The effluent was collected and the radiolabeled metabolites of [3H]bradykinin were separated by high performance liquid chromatography, identified, and quantified. RESULTS: [3H]Bradykinin was metabolized to the extent of 62 +/- 3% in a single passage through the rabbit coronary circulation at a physiological flow rate. The metabolites were identified as [3H]bradykinin(1-5) and [3H]bradykinin(1-7),accounting for 50 +/- 4 and 12 +/- 2% of the radioactivity, respectively. Co-perfusion with the angiotensin converting enzyme inhibitor, ramiprilat, completely blocked formation of these metabolites. CONCLUSIONS: Angiotensin-converting enzyme fully accounts for the metabolism of [3H]bradykinin in the rabbit coronary circulation. This result contrasts with data obtained using rat heart, which demonstrated a prominent role for aminopeptidase P in bradykinin metabolism in this species.

Cardioprotective effects of the aminopeptidase P inhibitor apstatin: studies on ischemia/reperfusion injury in the isolated rat heart.

Aminopeptidase P and angiotensin-converting enzyme (ACE) are responsible for the metabolism of exogenously administered bradykinin in the coronary circulation of the rat. It has been shown that ACE inhibitors decrease cytosolic enzyme release from the ischemic rat heart and reduce reperfusion-induced ventricular arrhythmias by increasing endogenous levels of bradykinin. It was hypothesized that the aminopeptidase P inhibitor apstatin could do the same. In an isolated perfused rat heart preparation subjected to global ischemia and reperfusion, both apstatin and ramiprilat (an ACE inhibitor) significantly decreased creatine kinase (CK) and lactate dehydrogenase (LDH) release. The difference between the postischemia and preischemia levels of released CK was reduced 68% by apstatin and 68% by ramiprilat compared with control. The corresponding reductions in LDH release were 74% for apstatin and 81% for ramiprilat. A combination of the inhibitors was not significantly better than either one alone. Apstatin and ramiprilat also significantly reduced the duration of reperfusion-induced ventricular fibrillation by 69 and 61%, respectively. The antiarrhythmic effect of apstatin was reversed by HOE140, a bradykinin B2-receptor antagonist, suggesting that apstatin is acting by potentiating endogenously formed bradykinin. The results demonstrate that the aminopeptidase P inhibitor apstatin is cardioprotective in this model of cardiac ischemia/ reperfusion injury.

Inhibition of both aminopeptidase P and angiotensin-converting enzyme prevents bradykinin degradation in the rat coronary circulation.

Bradykinin (Bk), which is produced locally in the heart, exhibits potent cardioprotective effects. However, these effects appear to be limited by rapid degradation of the peptide. To determine the mechanism of Bk metabolism in the coronary circulation, [3H]Bk was perfused through the isolated rat heart via the aorta in the presence and absence of specific peptidase inhibitors. The radiolabeled metabolites were collected from the pulmonary artery and then separated, identified, and quantified by reversed-phase high-performance liquid chromatography (HPLC) by using a radioactive flow detector. In the absence of inhibitors, only 45 +/- 2% of the radioactivity eluted from the coronary circulation as intact [3H]Bk. The chromatograms suggested that Bk was being hydrolyzed at the Arg1-Pro2 bond by aminopeptidase P and at the Pro7-Phe8 bond by angiotensin-converting enzyme. When the aminopeptidase P inhibitor, apstatin (200 microM), was coperfused with [3H]Bk, cleavage at the Arg1-Pro2 bond was blocked and the amount of intact [3H]Bk in the perfusate increased to 57 +/- 5% (p < 0.05 vs. control). Coperfusion with the angiotensin-converting enzyme inhibitor, ramiprilat (0.5 microM), alone blocked cleavage at the Pro7-Phe8 bond and increased intact [3H]Bk to 75 +/- 3% (p < 0.001 vs. control). When both apstatin and ramiprilat were present, almost all of the radioactivity (96 +/- 1%) eluted as intact [3H]Bk (p < 0.01 vs. ramiprilat alone). The results indicate that the degradation of Bk in the rat coronary circulation can be fully accounted for by aminopeptidase P (approximately 30%) and angiotensin-converting enzyme (approximately 70%).

Involvement of free radicals in the cardioprotective effect of defibrotide.

Ischemia followed by reperfusion has deleterious effects on myocardial tissue and a wide range of drugs have been investigated to modulate these changes. Defibrotide (polydeoxyribonucleotides from bovine lung), a drug with antithrombotic and fibrinolytic activities, has also proven to be cardioprotective against myocardial ischemia/reperfusion damage. However, the mechanism of this protective effect has not been clarified yet. The aim of this study was to determine whether this effect is due to protection against free radical induced changes. The experimental model in rabbits includes coronary artery ligation for 60 min followed by a reperfusion period of 45 min. In this model, free radical damage was estimated by different parameters of lipid peroxidation such as diene conjugation, carbonyl content, and thiobarbituric acid reactive substances, together with protein oxidation determinations. The results demonstrate that defibrotide prevents free radical induced changes after myocardial ischemia/reperfusion.

Protective effects of cilazapril against free radical injury in myocardial ischaemia-reperfusion.

Cilazapril is a prodrug which is rapidly hydrolysed to the pharmacologically active cilazaprilat following absorption to the bloodstream. In clinical pharmacological studies, administration of cilazapril resulted in potent, reversible, selective and competitive angiotensin converting enzyme inhibition. In this study, we have examined the protective effect of cilazapril on a myocardial ischaemia-reperfusion model by using different parameters of lipid peroxidation and protein oxidation. We have observed increased levels of diene conjugates, carbonyls and malondialdehyde as well as protein carbonyls after ischaemia-reperfusion, whereas protein sulphydryl groups were decreased. Our results clearly demonstrate that cilazapril, a non-sulphydryl, long-acting angiotensin converting enzyme inhibitor, has free-radical-scavenging potential in a model comparable to the clinical situation observed in humans.

Morphological changes in carotid arteries of rabbits induced by defibrotide infusion.

Defibrotide is an antithrombotic and profibrinolytic drug which modulates endothelial function. The drug increases prostacyclin and tissue plasminogen activator while it decreases plasminogen activator inhibitor synthesis by endothelial cells. In this study, in vivo effects of defibrotide on the morphology of endothelial cells and vessel wall of the healthy rabbits were investigated by light and electron microscopy. The examination of the carotid arteries of healthy rabbits after infusion of saline or defibrotide (10 mg/kg/hr) in saline solution for three hours revealed that the drug had induced dramatic morphological changes in all the test animals while no change was observed in control group. The changes observed after defibrotide administration, such as the decrease in hill and valley-like appearance of endothelial surface, and thinning of the intimal layer provides evidence for the vasorelaxant effect of the drug, while the decrease in the number of blood cells adhering to the endothelial surface confirms the antithrombotic effect of defibrotide. Finally the decrease in the number of crater-like structures may be due to the cytoprotective effect of the drug.

Subcellular localization of radioactively labelled defibrotide in cultured endothelial cells.

Defibrotide is a new antithrombotic and fibrinolytic drug which is obtained by controlled depolymerization of mammalian DNA. In various models of arterial and venous thrombosis, it has been shown that it induces tissue plasminogen activator [tPA] and prostacyclin [PGI2] release from the vessel wall. We have previously shown the presence of specific binding sites with a Kd of 4.2 micrograms/ml for radioactively labelled defibrotide. The present study was undertaken to identify the location of the binding site. Confluent cultures of endothelial cells from human umbilical vein were incubated with media containing 3H-acetyl-defibrotide for various intervals of time. Cells were then washed and harvested nonenzymatically. Subcellular location of 3H-defibrotide was investigated by fractionating cells on discontinuous sucrose gradient and measuring the distribution of radioactivity. 5'-nucleotidase enzyme activity was also measured to ensure the location of membrane fraction. Our results suggest that the major location of 3H-defibrotide in endothelial cells is the plasma membrane. On the other hand, nuclei also contain a considerable amount of the drug which suggests a mechanism where binding to a membrane protein is followed by internalization.