Imre Földesi

Fecal MMP-9: A new noninvasive differential diagnostic and activity marker in ulcerative colitis

Background:Ulcerative colitis (UC) is characterized by frequent relapses, with the presence of colorectal inflammation and mucosal lesions. Matrix-metalloprotease 9 (MMP-9) is elevated in colonic biopsies, urine, and blood plasma of UC patients. MMP-9 has been suggested as a predictor of UC in the urine of children; however, 20% of the controls tested positive. So far, fecal MMP-9 levels have never been measured. Our aims were: 1) to compare fecal MMP-9 levels in UC patients to control subjects and a functional gastrointestinal disorder characterized by diarrhea (IBS-D); 2) to test the correlation between UC disease activity and fecal levels of MMP-9; and 3) to correlate fecal MMP-9 levels with a known fecal marker of UC activity, calprotectin. Methods:UC (n = 47), IBS-D (n = 23) patients, and control subjects (n = 24) provided fecal samples for MMP-9 analysis. In UC patients, disease severity was evaluated by the Mayo score. Fecal MMP-9 and calprotectin levels were measured by enzyme-linked immunosorbent assay and lateral flow assay, respectively. Results:MMP-9 was undetectable or ⩽0.22 ng/mL in the feces of all controls and IBS-D patients. In UC patients, fecal MMP-9 levels significantly correlated with the overall Mayo score (P < 0.001), the endoscopic score (P < 0.001), and the serum C-reactive protein levels (P = 0.002). Additionally, in UC patients fecal MMP-9 levels showed a significant correlation with a known disease activity marker, fecal calprotectin (P = 0.014). Conclusions:These results highlight fecal MMP-9 as a useful tool in the differential diagnosis of diarrheic disorders and in the noninvasive evaluation of disease activity and mucosal healing in UC.

Endocrine and behavioral effects of neuromedin S.

The present experiments focused on the effects of neuromedin S on hypothalamic-pituitary-adrenal (HPA) activation and behavior. The peptide (0.25-1 nmol) was administered intracerebroventricularly to rats, the behavior of which was monitored by means of telemetry, open field observations and an elevated plus-maze (EPM) test. Autonomic functions such as the temperature and the heart rate were recorded by telemetry. The action on the HPA axis was assessed via measurements of the plasma corticosterone and ACTH levels. To reveal the transmission of the endocrine responses, animals were pretreated with corticotrophin releasing hormone receptor (CRHR) antagonists (1 nmol). In the open field test, the animals were pretreated with either a CRHR(1) antagonist (antalarmin) or haloperidol (10 microg/kg), while in the EPM test they were pretreated with antalarmin or diazepam (1 mg/kg). The dopamine release from striatal and amygdala slices after peptide treatment was measured with a superfusion apparatus. Neuromedin S exerted dose-dependent effects on the HPA system, which were inhibited by antalarmin. It also activated grooming and decreased the entries to and time spent in the open arms during the EPM test. The grooming response was abolished by haloperidol and antalarmin pretreatment, while diazepam and antalarmin showed a tendency to attenuate the response evoked in the EPM test. In the superfusion studies, neuromedin S enhanced the dopamine release from the amygdala slices. These results demonstrate that neuromedin S stimulates the HPA axis through the CRHR(1) pathway and evokes stereotyped behavior and anxiety through mesolimbic dopamine and corticotrophin releasing hormone release.

Determination of RU486 (mifepristone) in blood by radioreceptorassay; A pharmacokinetic study

A human progesterone receptor assay has been developed for the measurement of the biologically active molecular fraction of RU486 (RU486 binding equivalent) for studying its pharmacokinetic properties. Thirty-nine healthy pregnant volunteers with amenorrhoea of 49 days or less receiving a single oral dose of 200 mg, 400 mg or 600 mg RU486 orally in a single dose were involved in this study. Blood samples were collected within 48 hours for the analysis. It was found that the pharmacokinetics of the RU486 binding equivalent followed an open two-compartment model. The dose was rapidly absorbed and peak serum concentrations were measured within 1-2 hours after ingestion of the drug. The distribution was also rapid, but the elimination was slow, the elimination half-life ranging between 83 and 90 hours. Significant differences were found between the peak plasma values for the 200 mg and 600 mg doses (p < 0.05) and between the AUCs for the 200 mg and 600 mg doses (p < 0.01) and the 400 mg and 600 mg doses (p < 0.05). It can be concluded that this newly developed radioreceptor assay satisfies the requirements of radioligand binding techniques and can be used to determine the serum levels of RU486 and its metabolites, which are able to bind to human myometrial progesterone receptors. The pharmacokinetics for the RU486 binding equivalent is similar to that for RU486, with the exception of very slow elimination, which may originate from the measurement of the biologically active metabolites together with the parent compound.

Endocrine, behavioral and autonomic effects of neuropeptide AF.

The actions of neuropeptide AF (NPAF), on the hypothalamic-pituitary-adrenal (HPA) axis, behavior and autonomic functions were investigated. NPAF (0.25, 0.5, 1, 2 nmol) was administered intracerebroventricularly to rats, the behavior of which was monitored by means of telemetry, open-field (OF) observations and elevated plus-maze (EPM) tests. The temperature and heart rate were recorded by telemetry, and the plasma ACTH and corticosterone levels were used as indices of the HPA activation. The dopamine release from striatal and amygdala slices after peptide treatment (100 nM and 1 microM) was measured with a superfusion apparatus. To establish the transmission of the HPA response, animals were pretreated with the corticotrophin-releasing hormone (CRH) receptor antagonist antalarmin or astressin 2B (0.5 nmol). In the OF test, the animals were pretreated with antalarmin or haloperidol (10 microg/kg), while in the EPM test they were pretreated with antalarmin or diazepam (1 mg/kg). NPAF stimulated ACTH and corticosterone release, which was inhibited by antalarmin. It activated exploratory locomotion (square crossings and rearings) and grooming in OF observations, and decreased the entries to and the time spent in the open arms during the EPM tests. The antagonists inhibited the locomotor responses, and also attenuated grooming and the EPM responses. NPAF also increased spontaneous locomotion, and tended to decrease the core temperature and the heart rate in telemetry, while it augmented the dopamine release from striatal and amygdala slices. These results demonstrate, that acute administration of exogenous NPAF stimulates the HPA axis and behavioral paradigms through CRH and dopamine release.

The Diagnostic Value of a New Fecal Marker, Matrix Metalloprotease-9, in Different Types of Inflammatory Bowel Diseases

BACKGROUND Only limited data are available regarding the diagnostic accuracy of fecal matrix metalloprotease-9 [MMP-9] for inflammatory bowel disease [IBD]. The aims of our study were to assess the diagnostic accuracy of fecal MMP-9 in patients with active Crohns disease [CD], ulcerative colitis [UC], and pouchitis, and to compare the diagnostic accuracy of fecal MMP-9 and fecal calprotectin [CP] in IBD. METHODS Stool and blood samples were collected in 50 CD, 54 UC, and 34 ileal pouch-anal anastomosis patients before control endoscopies were performed. Biopsies were taken for histologic purposes. The activities of CD, UC, and pouchitis were defined with the use of clinical, endoscopic, and histologic activity scores. Fecal CP and MMP-9 levels were quantified by enzyme-linked immunosorbent assay. RESULTS Active CD, UC, and pouchitis were detected in 38%, 54%, and 29% of the patients, respectively. A significant correlation was revealed between fecal CP and the clinical activities of CD and UC, and between fecal CP and the endoscopic activity of UC and pouchitis. Fecal MMP-9 did not correlate with any of the activity indices of CD; however, strong associations were shown between fecal MMP-9 and clinical, endoscopic, and histologic activities of both UC and pouchitis. CONCLUSIONS This is the first study assessing the diagnostic accuracy of MMP-9 in different types of IBD. Our results showed that fecal MMP-9 has high sensitivity in the detection of endoscopically active UC and pouchitis. These non-invasive methods help assess intestinal inflammation.

Autoimmune pancreatitis in Hungary: A multicenter nationwide study

Background: To date, most cases of autoimmune pancreatitis (AIP) have been reported from Japan. The aim of the present study was to assess the clinical features and management of AIP cases in Hungary. Methods: The demographics, clinical presentation, laboratory and imaging findings, extrapancreatic involvement, treatment response and recurrence were evaluated in the first 17 patients diagnosed with AIP in Hungary. Results: The mean age at presentation was 42.7 years (range: 16–74); 47% of the patients were women. New-onset mild abdominal pain (76%), weight loss (41%) and jaundice (41%) were the most common symptoms, with inflammatory bowel disease being the most frequent (36%) extrapancreatic manifestation. Diffuse pancreatic swelling was seen in 7 patients (41%) and a focal pancreatic mass in 8 (47%). Endoscopic retrograde cholangiopancreatography revealed pancreatic duct strictures in all study patients. The serum IgG4 level at presentation was elevated in 62% of the 8 patients in whom it was measured. All the percutaneous core biopsies (5 patients) and surgical specimens (2 patients), and 2 of the 4 biopsies of the papilla of Vater revealed the typical characteristic findings of AIP: a diffuse lymphoplasmacytic infiltration, marked interstitial fibrosis and obliterative phlebitis. Immunostaining indicated IgG4-positive plasma cells in 62% of the 8 patients in whom it was performed. Granulocytic epithelial lesions (GEL) were present in 3 patients. The patients without GELs were older (mean age 59 years), while those with GEL were younger (mean age 34 years), and 2 of 3 were female and had ulcerative colitis. A complete response to steroid treatment was achieved in all 15 patients. Because of the suspicion of a pancreatic tumor, 2 patients with focal AIP underwent partial pancreatectomy. One patient relapsed, but responded to azathioprine. Conclusions: This first Hungarian series has confirmed several previously reported findings on AIP. AIP with GEL was relatively frequent among our patients: these patients tended to be younger than in earlier studies and displayed a female preponderance with a high coincidence of ulcerative colitis. Performance of a percutaneous biopsy is strongly recommended. The response to immunosuppressive therapy was excellent.

The interaction of Urocortin II and Urocortin III with amygdalar and hypothalamic cotricotropin-releasing factor (CRF) – Reflections on the regulation of the hypothalamic–pituitary–adrenal (HPA) axis

Urocortin II (Ucn II) and Urocortin III (Ucn III) are selective agonists of the CRF receptor type 2 (CRFR2). The aim of the present experiments was to investigate the effects of Ucn II and Ucn III on the central CRF and peripheral glucocorticoids in rats. Increasing doses (0.5-1-2-5 μg/2 μl) of Ucn II or Ucn III were administered intracerebroventricularly, then CRF concentration was determined by immunoassays in two different brain regions, the amygdala and the hypothalamus, and in two different time paradigms, 5 and 30 min after the administration of peptides. In parallel with the second determination, plasma corticosterone concentration was measured by chemofluorescent assay. The amygdalar CRF amount was increased significantly by 0.5 and 5 μg of UCN II and 2 and 5 μg of UCN III in the 5 min experiments and by 5 μg of UCN II and 0.5 and 5 μg of UCN III in the 30 min experiments. The hypothalamic CRF content was not affected considerably in the 5 min paradigm, but it was influenced significantly in the 30 min paradigm, with 0.5 and 1 μg of UCN II and 0.5-2 μg of UCN III decreasing, and 2 and 5 μg of UCN II and 5 μg of UCN III increasing the hormone concentration, respectively. The plasma corticosterone concentration was decreased by 1 and 2 μg of UCN II and UCN III and increased by 0.5 and 5 μg of UCN III. The present results demonstrate that central administration of Ucn II and Ucn III modulate time-dependently and dose-dependently the amygdalar and the hypothalamic CRF concentration, and, directly or indirectly, the plasma corticosterone concentration. The present experiments suggest that the role of CRFR2 in the regulation of the HPA axis can be inhibitory or stimulatory, depending on the actual concentration of their agonists.

Characterization of late-pregnant rat uterine contraction via the contractility ratio in vitro Significance of α1-adrenoceptors

The aim of this study was to characterize the ability of late-pregnant (days 15-22) rat uterine tissue rings to contract in response to electric field stimulation in vitro. For this purpose, maximum rhythmic contractions were elicited by optimum choice of the period time and the pulse width, the two main parameters of electric field stimulation. In parallel, the plasma 17beta-estradiol and progesterone levels were determined. It was found that the contractility ratio, i.e. the quotient of the optimum pulse width and the period time, is a good parameter with which to express the contractility. The larger the contractility ratio, the better the ability to contract. Evaluation of the area under the curve did not furnish information relating to the contractility in this method. A very close correlation was observed between the contractility ratio and the quotient of the 17beta-estradiol and progesterone levels on different days, demonstrating that the in vitro ability characterized by the contractility ratio is in keeping with the physiological regularity. There was also a very close correlation between the contractility ratio and the quotient of the alpha1- and beta-adrenergic receptors, suggesting the main role of the numbers of alpha1-receptor in pregnant uterine contractility. It is believed that this is the first in vitro model to give a numerical measure concerning the ontogeny of uterine contractility in late pregnancy.

Rosuvastatin enhances anti-inflammatory and inhibits pro-inflammatory functions in cultured microglial cells

Microglial activation results in profound morphological, functional and gene expression changes that affect the pro- and anti-inflammatory mechanisms of these cells. Although statins have beneficial effects on inflammation, they have not been thoroughly investigated for their ability to affect microglial functions. Therefore the effects of rosuvastatin, one of the most commonly prescribed drugs in cardiovascular therapy, either alone or in combination with bacterial lipopolysaccharide (LPS), were profiled in pure microglial cultures derived from the forebrains of 18-day-old rat embryos. To reveal the effects of rosuvastatin on a number of pro- and anti-inflammatory mechanisms, we performed morphometric, functional and gene expression studies relating to cell adhesion and proliferation, phagocytosis, pro- and anti-inflammatory cytokine (IL-1β, tumor necrosis factor α (TNF-α) and IL-10, respectively) production, and the expression of various inflammation-related genes, including those related to the above morphological parameters and cellular functions. We found that microglia could be an important therapeutic target of rosuvastatin. In unchallenged (control) microglia, rosuvastatin inhibited proliferation and cell adhesion, but promoted microspike formation and elevated the expression of certain anti-inflammatory genes (Cxcl1, Ccl5, Mbl2), while phagocytosis or pro- and anti-inflammatory cytokine production were unaffected. Moreover, rosuvastatin markedly inhibited microglial activation in LPS-challenged cells by affecting both their morphology and functions as it inhibited LPS-elicited phagocytosis and inhibited pro-inflammatory cytokine (IL-1β, TNF-α) production, concomitantly increasing the level of IL-10, an anti-inflammatory cytokine. Finally, rosuvastatin beneficially and differentially affected the expression of a number of inflammation-related genes in LPS-challenged cells by inhibiting numerous pro-inflammatory and stimulating several anti-inflammatory genes. Since the microglia could elicit pro-inflammatory responses leading to neurodegeneration, it is important to attenuate such mechanisms and promote anti-inflammatory properties, and develop prophylactic therapies. By beneficially regulating both pro- and anti-inflammatory microglial functions, rosuvastatin may be considered as a prophylactic agent in the prevention of inflammation-related neurological disorders.

The effect of urocortin I on the hypothalamic ACTH secretagogues and its impact on the hypothalamic-pituitary-adrenal axis

Urocortin I (UCN I) is a structural analogue of corticotropin-releasing factor (CRF), which, together with arginine-vasopressin (AVP), are the principle adrenocorticotropic hormone (ACTH) secretagogues in mammals. The aim of the present study was to investigate the effects of UCN I on the hypothalamic CRF and AVP concentration and its impact on the hypothalamic-pituitary-adrenal (HPA) axis. First, male Wistar rats were injected intracerebroventricularly (ICV) with 0.5, 1, 2 and 5 μg of UCN I. After 30 min hypothalamic CRF and AVP concentrations were determined by immunoassays. In parallel, the trunk blood was collected and plasma ACTH and corticosterone concentration was determined by ELISA and chemofluorescent assay, respectively. Second, rats were pretreated ICV with selective antagonists of receptors being implicated in the regulation of the HPA axis (0.1 μg antalarmin for CRFR1, 1 μg astressin 2B for CRFR2 or 0.1 μg deamino-Pen1,Tyr2,Arg8-vasopressin for AVPR3) and treated ICV with the most effective dose of UCN I (5 μg). After 30 min plasma corticosterone concentration was determined by chemofluorescent assay. UCN I induced dose-dependent augmentation of the hypothalamic CRF and AVP concentration, associated with dose-dependent elevation of the plasma ACTH and corticosterone concentration. The most significant effect of UCN I on the plasma corticosterone concentration was inhibited by antalarmin, but was not influenced by astressin 2B or deamino-Pen1,Tyr2,Arg8-vasopressin. The present study demonstrates that UCN I modulates the concentration of the hypothalamic ACTH secretagogues in parallel with the concentration of the plasma ACTH and corticosterone. Our results suggest that UCN I may activate the HPA axis by stimulation of the hypothalamic CRF production, and this process is mediated by CRFR1, and not by CRFR2. UCN I may stimulate the AVP production, as well, but, based on the results with AVPR3 antagonist, this effect is not involved in the regulation of the HPA axis.