Laparoscopic natural orifice specimen extraction colectomy versus conventional laparoscopic colorectal resection in patients with rectal endometriosis: a randomized, controlled trial.

BACKGROUND: The conventional laparoscopic approach for the surgical management of deep endometriosis (DE) infiltrating the rectum appears to ensure improved digestive functional outcomes. The natural orifice specimen extraction (NOSE) technique for the treatment of colorectal DE can significantly accelerate postoperative recovery; however, data on gastrointestinal function following conventional laparoscopic segmental bowel resection (CLR) compared with NOSE colectomy (NC) for DE are sparse. MATERIALS AND METHODS: Between 30 September 2019 and 31 December 2020, a randomized, open-label, two-arm, parallel-group controlled trial with women aged 18-45 years was conducted at University Hospital.Ninety-nine patients were randomized to CLR or NC, with DE infiltrating at least the muscular layer, at least 50% of the circumference of the bowel, up to 15 cm from the anal verge, exhibiting pain and bowel symptoms and/or infertility. The primary endpoint was bowel function, represented by low anterior resection syndrome (LARS). Secondary parameters included the Endometriosis Health Profile 30 (EHP30), Gastrointestinal Quality of Life Index (GIQLI), Visual Analog Scale (VAS) scores preoperatively and at set times (1 and 6 months, 1 year) following surgery. RESULTS: No significant differences were observed in the postoperative LARS scores, VAS, EHP30, and GIQLI between the NC and CLR groups. LARS scores did not reveal significant differences 12 months postoperatively compared to the preoperative values in both groups (CLR group P =0.93 versus NC group, P =0.87). GIQLI scores were significantly improved 12 months after the operation compared with baseline values in the CLR group ( P =0.002) and NC group ( P =0.001). Pain symptoms and quality of life scores significantly improved 12 months postoperatively in both groups. CONCLUSIONS: NC is a feasible surgical approach for treating patients with rectal DE. Our study did not show a statistically significant difference between CLR and NC techniques in mid-term digestive and pain outcomes.

[Laparoscopic treatment of deeply infiltrating colorectal endometriosis - ten years of single center experience]. / Mélyen infiltráló colorectalis endometriosis laparoszkópos kezelésével szerzett tízéves tapasztalataink.

INTRODUCTION: Deep infiltrating endometriosis penetrates the peritoneal surface beneath 5 mm. The bowel is affected in 3-37% of the cases. OBJECTIVE: The aim of the authors was to analyze the results of the surgical procedures performed for bowel endometriosis. METHOD: Between 2009 and 2020, 675 patients underwent bowel endometriosis surgery at the Department of Obstetrics and Gyanecology of Semmelweis University. Four surgical approaches were performed: shaving, discoid, segmental and NOSE resection. RESULTS: 182 shaving, 93 discoid, 130 NOSE and 270 segmental bowel resections were performed. Ultra-deep anastomosis was performed in 40 cases. The median operative time was 85 minutes, the shortest intervention lasted 25 minutes, the longest 585 minutes. The average operating time was 260 (± 161.3) minutes for the first, and 114 (± 47.0) minutes for the last ten operations. The average blood loss was 10 (± 20.3) mL. The average hospital stay was 6 (± 2.3) days. Serious surgical complication (Clavien-Dindo III or more severe) developed in 18 cases. In a total of 17 cases sigmoideo- or ileostomy were used. Conversion to laparotomy was necessary in 6 cases. DISCUSSION: The same team performed all the interventions, which can show the effectiveness of the surgical techniques instead of the technique of individual surgeons. The complication rate is low in the case of an experienced surgical team, and the operating time decreases significantly in proportion to the number of operations performed. CONCLUSION: Bowel endometriosis can be treated safely and effectively with both conservative (shaving or discoid) and radical (segmental or NOSE resection) approach. Orv Hetil. 2023; 164(9): 348-354.

Low anterior resection syndrome following different surgical approaches for low rectal endometriosis: A retrospective multicenter study.

INTRODUCTION: There is increasing evidence that intermediate and long-term bowel dysfunction may occur as a consequence of radical surgery for rectal deep endometriosis (DE). Typical symptoms include constipation, feeling of incomplete evacuation, clustering of stools, and urgency. This is described in the colorectal surgical literature as low anterior resection syndrome (LARS). Within this, several studies suggested that differences regarding functional outcomes could be favorable to more conservative surgical approaches, that is, excision of endometriotic tissue with preservation of the luminal structure of the rectal wall when compared with classical segmental resection techniques for DE, especially when performed for low DE. MATERIAL AND METHODS: A total of 211 patients undergoing rectal surgery for low DE (≤7 cm from the anal verge) in three different tertiary referral centers between October 2009 and December 2018 were retrospectively reviewed regarding major complications and LARS. From the 211 eligible patients, six women were excluded because of loss to follow-up. Finally, a total number of 205 patients were enrolled for the statistical analysis; 139 with nerve- and vessel-sparing segmental resection (NVSSR) and 66 operated for laparoscopic-transanal disk excision (LTADE) were included. Gastrointestinal functional outcomes of the two procedures were compared using the validated LARS questionnaire. The median follow-up time was 46 ± 11 months. As a secondary outcome, the surgical sequelae were examined. RESULTS: We found no statistically significant difference between the incidence of LARS (31.7% and 37.9%, respectively) among patients operated by LTADE when compared with NVSSR (P = .4). The occurrence of LARS was positively associated with the use of protective ileostomy or colostomy (P = .02). A higher rate of severe complications was observed in women undergoing LTADE (19.7%) when compared with patients with NVSSR (9.0%, P = .029). CONCLUSIONS: LARS is not more frequent after NVSSR when compared with a more conservative approach such as LTADE in patients undergoing rectal surgery for low DE. To confirm our findings prospective studies are required.

Investigation of pituitary adenylate cyclase activating polypeptide (PACAP) in human amniotic fluid samples.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide acting as a hormone, a neuromodulator, a neurotransmitter, a trophic factor and is involved in a variety of developmental and regenerative processes. PACAP is present in several human tissues and biological fluids. In many pathological conditions, changes in PACAP levels have been described to reflect disease progression, therefore PACAP has diagnostic value as a potential biomarker. Since PACAP has been shown to play an important role in reproductive physiology and development, it was of interest to examine whether this neuropeptide occurs in the human amniotic fluid. Amniotic fluid samples were collected between the 15-19th weeks of gestation from volunteering pregnant women undergoing amniocentesis as a prenatal diagnostic tool due to maternal age. Pathological cases were excluded after prenatal karyotype analysis. PACAP-like immunoreactivity was measured by radioimmunoassay and could be detected in all samples. The present study provides evidence for the presence of PACAP in human amniotic fluid, but determination of the exact physiological or pathological significance awaits further investigation.

Gamma-Synuclein Levels Are Elevated in Peritoneal Fluid of Patients with Endometriosis.

BACKGROUND The role of gamma-synuclein (SNCG) has been widely examined in malignant conditions due to its possible role in disease progression, but very little information is available on its theoretical function on endometriosis formation. MATERIAL AND METHODS Between January 2016 and December 2016, we collected peritoneal fluid and plasma samples from 45 consecutive female patients, of which 15 were without endometriosis, 15 had minimal to mild endometriosis, and 15 had moderate to severe endometriosis. The statistical power was 0.98. We evaluated SNCG levels in the peritoneal fluid and plasma of patients diagnosed with endometriosis, and we compared them with the levels obtained from disease-free control subjects by using enzyme-linked immunosorbent assay. RESULTS SNCG levels were statistically significantly (1.2-fold) higher in the peritoneal fluid of patients with endometriosis compared to controls (p=0.04). We did not find a significant difference between SNCG levels in the plasma of our endometriosis patients and the control group (p=0.086). However, despite previous data showing very limited expression of SNCG in healthy tissues, we found SNCG in the peritoneal fluid of all of the patients in our healthy control group. CONCLUSIONS Levels of SNCG were statistically significantly higher in the peritoneal fluid of patients with endometriosis compared to disease-free controls, which may indicate its possible role the formation and progression of the disease. Moreover, its biological function should be further investigated due to the conflicting results concerning its expression in healthy tissues.

[The effect of surgical treatment of bowel endometriosis on fertility]. / A bélendometriosis miatt végzett mutétek hatása a fertilitásra.

Introduction: Bowel endometriosis is when endometrial-like tissue penetrates the bowel serosa, or it reaches the subserous neurovascular plexus. The effect of surgery for colorectal endometriosis on infertility and pregnancy is not fully proven. Aim: The aim of the present study was to analyse the pregnancy outcome and mode of delivery of patients who underwent 'nerve sparing' anterior resection of the colon. Method: Between 2009 and 2017, we operated 121 patients with bowel endometriosis, and built up a prospective database where we assessed their wish of pregnancy, the way of the conception, pathologies during pregnancy and mode of delivery. Statistical analysis: The relationship between endometriosis and pregnancy pathologies was tested by a χ2 probe and Fisher's exact test, additionally the odds ratio (OR) and 95% confidence interval (CI) were determined. For p<0.05, the result was considered significant. Results: Out of 121 bowel endometriosis patients, 48 (39.6%) women got pregnant, 37 (30.5%) of them with in vitro fertilisation. The control group was built from patients who underwent in vitro fertilisation because of andrological factors. We found that women with endometriosis have a significantly higher risk for praeeclampsia (p = 0.023) and placenta praevia (p = 0.045) during pregnancy. Conclusions: Our study is a unique description of pregnancy outcome and mode of delivery after surgery for bowel endometriosis, which, despite the small number of cases, has yielded similar results to the previous multicentric studies. Orv Hetil. 2019; 160(41): 1633-1638.

Natural Orifice Specimen Extraction during Laparoscopic Bowel Resection for Colorectal Endometriosis: Technique and Outcome.

STUDY OBJECTIVE: To present a detailed description of a modified natural orifice specimen extraction (NOSE) colectomy technique. We also report the postoperative outcomes of our prospective case series when compared with conventional laparoscopic bowel resection in a relatively large series of patients. DESIGN: Canadian Task Force classification II-1. SETTING: A university tertiary referral center. PATIENTS: The last 90 consecutive patients in our care with deep infiltrating endometriosis of the bowel are presented in this study. Patients were diagnosed at the 1st Department of Obstetrics and Gynecology, Semmelweis University, Budapest, Hungary. INTERVENTIONS: We performed laparoscopic bowel resection using the transrectal NOSE technique and compared the results of the new operative method (n = 30) with traditional laparoscopic bowel resection (n = 60). MEASUREMENTS AND MAIN RESULTS: The median duration of surgery was 121 minutes in the control group and 96 minutes in the NOSE group (p = .005). According to the Clavien-Dindo classification, we observed a severe, grade IIIb or higher, overall complication rate of 3.3% among all 90 patients. In the control group, anastomosis insufficiency occurred in 3.3% of patients (2/60 cases), and in 1 patient with anastomotic leakage a rectovaginal fistula was observed (1.7%). There was no significant difference in the rates of severe postoperative complications (p = .55). The length of hospital stay in the control group was a median of 7 days (range, 5-13 days), whereas in the NOSE group it was 6 days (range, 3-11 days) (p < .001). CONCLUSION: According to our findings, the use of NOSE colectomy offers a shorter recovery time and can eventually lead to a shorter surgery duration compared with traditional laparoscopic bowel resection.

Serum galectin-9 as a noninvasive biomarker for the detection of endometriosis and pelvic pain or infertility-related gynecologic disorders.

OBJECTIVE: To investigate the usefulness of soluble galectin-9 (Gal-9) in the noninvasive laboratory diagnosis of endometriosis and various gynecologic disorders. DESIGN: Prospective case-control study. SETTING: University medical centers. PATIENT(S): A total of 135 women of reproductive age were involved in the study, 77 endometriosis patients, 28 gynecologic controls, and 30 healthy women. INTERVENTION(S): Diagnostic laparoscopy and collection of tissue biopsies, peritoneal cells, and native peripheral blood from different case groups of gynecology patients and healthy women. MAIN OUTCOME MEASURE(S): The expression of mRNA and serum concentration of Gal-9. RESULT(S): Semiquantitative reverse transcription-polymerase chain reaction analysis and serum soluble Gal-9 ELISA were performed on three different cohorts of patients: those with endometriosis, those with benign gynecologic disorders, and healthy controls. Differences in the Gal-9 concentrations between the investigated groups and the stability of Gal-9 in the serum and diagnostic characteristics of Gal-9 ELISA were determined by statistical evaluation and receiver operating characteristic (ROC) curve analysis. Significantly elevated Gal-9 levels were found in both minimal-mild (I-II) and moderate-severe (III-IV) stages of endometriosis in comparison with healthy controls. At a cutoff of 132 pg/mL, ROC analysis revealed an excellent diagnostic value of Gal-9 ELISA in endometriosis (area under the curve = 0.973) with a sensitivity of 94% and specificity of 93.75%, indicating better diagnostic potential than that of other endometriosis biomarkers. Furthermore, various pelvic pain or infertility-associated benign gynecologic conditions were also associated with increased serum Gal-9 levels. CONCLUSION(S): Our results suggest that Gal-9 could be a promising noninvasive biomarker of endometriosis and a predictor of various infertility or pelvic pain-related gynecologic disorders.

[Prospective study to determine the diagnostic sensitivity of sigmoidoscopy in bowel endometriosis]. / Prospektív vizsgálat a sigmoideoscopia diagnosztikai érzékenységének meghatározására vastagbelet infiltráló endometriosisban.

INTRODUCTION AND AIM: In the treatment of colorectal endometriosis a multidisciplinary laparoscopic resection is suggested, for this reason the correct selection of bowel infiltration is essential before surgery. PATIENTS AND METHOD: Between 2009 and 2015, 383 sigmoidoscopies were performed in patients with endometriosis. Where mucosal invasion was absent secondary signs (wall rigidity, impression, kinking, pain during the examination, suffusion) were analysed. In endoscopically confirmed cases multidisciplinary surgery was performed, the remaining patients were operated by a gynecologic team only. RESULTS: Endometriosis was endoscopically confirmed in 224 patients (58.49%), 108 of them underwent multidisciplinary operation, the negative 135 cases received gynaecological surgery. Bowel endometriosis was confirmed in 103 out of 108 cases intraoperatively, while in 8 cases of the sigmoidoscopically negative patients bowel infiltration was diagnosed intraoperatively by the gynaecological team. Complete sigmoidoscopy was performed in 43.47% of the cases. Intraluminal endometriosis was found in 4.91%, secondary signs as rigidity in 38.39%, impression in 45.54%, kinking in 57.14%, pain (in cases of examination without narcosis) in 26.06% and suffusion in 3.82% of the cases was found during sigmoidoscopy. Sigmoidoscopic examination has a 92.8% specificity and 96.2% sensitivity in cases of bowel endometriosis. CONCLUSION: Sigmoidoscopy performed by an experienced gastroenterologist is a highly sensitive examination for the diagnosis of bowel endometriosis. Orv. Hetil., 2017, 158(7), 264-269.

[Importance of nerve-sparing surgical technique in the treatment of deep infiltrating endometriosis]. / Az idegkímélo mutéti technika jelentosége a mélyen infiltráló endometriosis sebészetében.

INTRODUCTION: Traditional surgeries performed in cases of deep infiltrating endometriosis lead to impaired quality of life. AIM: To summarize the postoperative outcome and to compare the rate of postoperative complications after different therapeutic approaches applied in deep infiltrating endometriosis. METHOD: The authors analized the articles published between March 31, 2004 and March 31, 2015, in the database http://www.pubmed.org using the following keywords: endometriosis, deep infiltrating, nerve sparing, surgery. RESULTS: Non-nerve sparing surgery resulted in temporary urinary dysfunction in 19.1-38.5% of patients, while it occurred in 0.61-33.3% of patients after nerve-sparing surgery. Non-nerve sparing surgical technique resulted in an average of 121 days of need for self-catheretisation. When nerve-sparing surgeries were performed the duration of self-catheterisation varied between 7 to 39.8 days. After nerve sparing surgeries, permanent bladder dysfunction was not detected in any case. CONCLUSIONS: Because of the successful treatment of the patients symptoms and the lower postoperative complication rate, nerve-sparing surgical technique leads to a significant improvement of the quality of life.

[Epigenetic background of the most common non-oncologic gynecological diseases]. / A leggyakoribb nem onkológiai eredetu nogyógyászati kórképek epigenetikai háttere.

Epigenetic effects influence the function of genes regulating the main physiological mechanisms. Some of these environmental factors may reduce or inhibit the function of these genes. The environmental effects on gene function may result in a change of the DNA structure leading to non-heritable phenotype changes. Epigenetic factors play an important etiological role in the development of numerous diseases in obstetrics and gynecology. Uterine fibroids probably have a complex etiological background including epigenetic mechanisms. The multifactorial aetiology of endometriosis suggests key roles for immunological and hormonal factors in the development of the diseases. These mechanisms are influenced by epigenetic factors, which may serve as therapeutic targets in the future. The possible in utero origin of polycystic ovary syndrome determines the main directions of research concerning epigenetic factors in the etiological background, with the hope of eventual prevention and/or treatment in the preconceptional period as well as during pregnancy care.

[Experience with multidisciplinary laparoscopic surgery in patients with deep infiltrating colorectal endometriosis]. / Mélyen infiltráló colorectalis endometriosis miatt végzett multidiszciplináris laparoszkópos mutétek során szerzett tapasztalataink.

INTRODUCTION: Deep infiltrating endometriosis is a particular form of endometriosis that penetrates the peritoneal surface or it reaches the subserosal neurovascular plexus. AIM: The aim of the authors was to analyze the results of segmental colorectal resections performed for deep infiltrating endometriosis. METHOD: Between 2009 and 2012, 50 patients underwent segmental rectum or/and sigmoid resection for endometriosis. RESULTS: 21 patients had ultralow rectal resection and 29 patients had low colorectal anastomosis or anterior resection. Concomitant intervention in other organs was required in all cases, including gynecologic procedures (n = 50), additional gynecologic (n = 47), vesical (n = 9) and ureteral (n = 18) resections. The mean number of endometriosis lesions was 2.4±1.8 per patient. In all patients fertility was preserved. Severe surgical complications (Clavien-Dindo stage III or more severe) occurred in 3 patients (6%). CONCLUSIONS: The results confirm that segmental bowel resection is an efficient and safe method for the treatment of deep infiltrating colorectal endometriosis. Orv. Hetil., 2014, 155(5), 182-186.

Placental gene expression patterns of endoglin (CD105) in intrauterine growth restriction.

OBJECTIVE: In this study, we describe placental gene expression patterns of endoglin in pregnancies with intrauterine growth restriction (IUGR) compared to normal pregnancies. METHODS: Placental samples were obtained from 101 pregnancies with IUGR using 140 normal pregnancy cases as control. Gene expression patterns and protein levels of the endoglin were compared between the two groups. For the gene expression analysis real-time PCR was applied, while for the estimation of placental protein level we performed Western analysis. RESULTS: The placental endoglin gene was significantly overexpressed in the IUGR group versus the control group (Ln2(α): 1.69). The placental endoglin protein level proved to be significantly higher in case of IUGR (endoglin/ß-actin ratio: 13.8 ± 2.3) versus the control cases (5.3 ± 1.1). The placental gene expression as well as the protein levels of endoglin showed no significant difference between female and male newborns. Concerning the placental gene expression and protein level, no significant difference was justified between the more (0-5 percentile) and less (5-10 percentile) severe cases of IUGR. CONCLUSION: Increased placental gene expression of endoglin may result in vascular dysfunction leading to chronic fetal hypoxia, which may induce VEGF-A to stimulate angiogenesis. This can be explained as feed back response to restore fetal placental circulation.

The role of the alcohol dehydrogenase-1 (ADH1) gene in the pathomechanism of uterine leiomyoma.

OBJECTIVE: To describe alterations of gene expression patterns of the alcohol dehydrogenase-1 (ADH1) gene in human leiomyoma tissue. We correlated changes in ADH1 gene activity with several clinical and demographic variables. STUDY DESIGN: We compared gene expression patterns of ADH1 in leiomyoma tissue samples obtained from 101 hysterectomy cases to 110 cases of hysterectomy performed for non-oncological indications. Gene expression was determined by standard PCR technique. Clinical and epidemiological data were extracted from the computerized database of the 1st Department of Obstetrics and Gynecology of Semmelweis University and from patient questionnaires. RESULTS: Median age in the leiomyoma group was significantly lower than in the control group (47.5 ± 12.1 vs. 54.7 ± 10.2 years). The incidence of uterine leiomyoma was highest (48%) in the 41-50 year age group. In the obstetric history, cumulative gestational age in the leiomyoma group was significantly lower (105.1 ± 8.2 weeks) than in the control group (127.2 ± 9.1 weeks) and cumulative lactation length was also significantly shorter (2.4 ± 1.2 months vs. 5.1 ± 2.2 months). Surgical treatment of the fibroid was myomectomy in 39.6% of the cases and hysterectomy in 60.4%. The ADH1 gene was significantly underexpressed in the leiomyoma group compared to the control group. There was no significant association between ADH1 gene expression and family history. Within the leiomyoma group, there was no significant difference in ADH1 gene expression between subgroups of cases with different number of fibroid tumors found in the hysterectomy sample, but individual tumor number did correlate with the degree of underexpression of the ADH1 gene. There was no significant association between ADH1 gene expression and cumulative history of lactation. CONCLUSIONS: Underexpression of the ADH1 gene, which influences the transformation of the extracellular matrix, plays a probable role in the etiology of uterine fibroid. Although significant differences in ADH1 gene activity were not seen, a negative correlation between tumor number and degree of ADH1 underexpression was found. Neither family history nor cumulative lactation length was a significant predictor of uterine leiomyoma.

Peripheral and central alterations of pituitary adenylate cyclase activating polypeptide-like immunoreactivity in the rat in response to activation of the trigeminovascular system.

Pituitary adenylate cyclase activating polypeptide (PACAP) is present in the cranial arteries and trigeminal sensory neurons. We therefore examined the alterations in PACAP-like immunoreactivity (PACAP-LI) in a time-dependent manner in two rat models of trigeminovascular system (TS) activation. In one group chemical stimulation (CS) was performed with i.p. nitroglycerol (NTG), and in the other one the trigeminal ganglia (TRG) were subjected to electrical stimulation (ES). The two biologically active forms, PACAP-38 and PACAP-27, were determined by means of radioimmunoassay (RIA) and mass spectrometry (MS) in the plasma, the cerebrospinal fluid (CSF), the trigeminal nucleus caudalis (TNC), the spinal cord (SC) and the TRG. The tissue concentrations of PACAP-27 were 10 times lower than those of PACAP-38 in the TNC and SC, but about half in the TRG. PACAP-38, but not PACAP-27, was present in the plasma. Neither form could be identified in the CSF. PACAP-38-LI in the plasma, SC and TRG remained unchanged after CS, but it was increased significantly in the TNC 90 and 180 min after NTG injection. In response to ES of the TRG, the level of PACAP-38 in the plasma and the TNC was significantly elevated 90 and 180 min later, but not in the SC or the TRG. The alterations in the levels of PACAP-27 in the tissue homogenates in response to both forms of stimulation were identical to those of PACAP-38. The selective increases in both forms of PACAP in the TNC suggest its important role in the central sensitization involved in migraine-like headache.

Mice deficient in pituitary adenylate cyclase activating polypeptide (PACAP) show increased susceptibility to in vivo renal ischemia/reperfusion injury.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with well-known cytoprotective effects. We have reported earlier that PACAP decreases mortality and the degree of tubular atrophy in a rat model of renal ischemia/reperfusion injury. Recently, we have shown that kidney cultures isolated from PACAP deficient mice show increased susceptibility to renal oxidative stress. Based on these previous studies, we raised the question whether PACAP deficient mice display increased sensitivity to in vivo kidney ischemia/reperfusion. PACAP⁻/⁻ mice underwent 45 or 60 min of renal ischemia followed by 2 weeks reperfusion. Kidneys were processed for histological analysis. Sections stained with PAS-haematoxylin were graded for the following parameters: degree of tubular dilation, Bowmann's capsule dilation, lymphocyte and macrophage infiltration, thyroidization and the disappearance of the PAS-positive glycocalyx from under the brush border. In other sets of experiments, tissue cytokine expression and the level of the endogenous antioxidant superoxide dismutase (SOD) were also determined after 60 min ischemia/reperfusion. Our results show that while intact kidneys were not different between wild-type and PACAP deficient mice, marked differences were observed in the histological structures in groups that underwent ischemia/reperfusion. PACAP deficient mice had a worse histological outcome, with significantly higher histological scores for all tested parameters. Cytokine expression was also markedly different between wild-type and PACAP deficient mice. In addition, the level of SOD was significantly lower in PACAP⁻/⁻ animals after ischemia/reperfusion. In conclusion, the lack of endogenous PACAP leads to higher susceptibility to in vivo renal ischemia/reperfusion, suggesting that PACAP has an endogenous renoprotective effect.

Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals.

Milk contains a variety of proteins and peptides that possess biological activity. Growth factors, such as growth hormone, insulin-like, epidermal and nerve growth factors are important milk components which may regulate growth and differentiation in various neonatal tissues and also those of the mammary gland itself. We have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), an important neuropeptide with neurotrophic actions, is present in the human milk in much higher concentration than in the plasma of lactating women. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance. Therefore, the aim of the present study was to determine the presence and concentration of PACAP in the plasma and milk of three ruminant animal species. Furthermore, the presence of PACAP and its specific PAC1 receptor were investigated in the mammary glands. Radioimmunoassay measurements revealed that PACAP was present in the plasma and the milk of the sheep, goat and the cow in a similar concentration to that measured previously in humans. PACAP38-like immunoreactivity (PACAP38-LI) was 5-20-fold higher in the milk than in the plasma samples of the respective animals, a similar serum/milk ratio was found in all the three species. The levels did not show significant changes within the examined 3-month-period of lactation after delivery. Similar PACAP38-LI was measured in the homogenates of the sheep mammary gland samples taken 7 and 30 days after delivery. PAC1 receptor expression was detected in these udder biopsies by fluorescent immunohistochemistry suggesting that this peptide might have an effect on the mammary glands themselves. These data show that PACAP is present in the milk of various ruminant domestic animal species at high concentrations, the physiological implications of which awaits further investigation.

Effects of PACAP on oxidative stress-induced cell death in rat kidney and human hepatocyte cells.

Oxidative stress plays an important role in various renal and hepatic pathologies, and reduction of oxidative stress-induced processes is an important protective strategy in tissues of diverse origins against harmful stimuli. Pituitary adenylate cyclase activating polypeptide (PACAP) is a well-known cytotrophic and cytoprotective peptide. PACAP promotes cell survival in numerous cells and tissues exposed to various stimuli. Protective effects of PACAP have been shown in the kidney, but it is not known whether PACAP is protective against oxidative stress in renal cells. Little is known about the effects of PACAP in the liver. The aim of the present study was to investigate whether PACAP is protective against oxidative stress in primary rat kidney cell culture and whether PACAP has any effect on cell survival in human WRL-68 hepatocytes and HEP-G2 hepatocellular carcinoma cells subjected to oxidative stress. Cells were exposed to various concentrations of H(2)O(2) with or without PACAP co-treatment and cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test (MTT). We found that oxidative stress induced a significant decrease in cell viability in both cell lines. PACAP could dose-dependently increase the percentage of living cells in kidney cells, but it failed to do so in hepatocytes. Given the survival-promoting effects of PACAP against oxidative stress in rat kidney, we conducted a further experiment to determine whether PACAP influences the markers of oxidative stress in vivo. We have proven earlier that PACAP was effective in kidney ischemia/reperfusion injury in vivo. In the present study, we determined the levels of the oxidative stress marker malondialdehyde and the activity of the scavenger molecules glutathione (GSH) and superoxide dismutase (SOD) following kidney ischemia/reperfusion in rats. We found that PACAP significantly increased the level of GSH and counteracted the marked reduction of SOD activity after ischemia/reperfusion in vivo. In summary, the present study showed that while PACAP was able to significantly increase the cell survival in primary kidney cell cultures exposed to oxidative stress, possibly involving interaction with the endogenous scavenger system, it failed to influence the viability of normal or cancerous hepatocytes.

Effects of PACAP on the oxidative stress-induced cell death in chicken pinealocytes is influenced by the phase of the circadian clock.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with highly potent neuro- and general cytoprotective actions. PACAP is also an important modulator of circadian rhythmic functions, including time-dependent effects in the pineal gland. It is not known whether PACAP influences the survival of pinealocytes. The present study had two aims. First, we tested whether the cytoprotective effects of PACAP are present also in the pineal cells. As the pineal gland is the main circadian master clock in birds, we also tested whether this effect depends on the time of day. Using flow cytometry, we detected a significant decrease of cell viability after hydrogen peroxide-induced oxidative stress in chicken pinealocytes. PACAP alone did not influence cell survival. Co-incubation with PACAP in the dark phase (9 PM) was able to attenuate the toxic effect of H2O2. The survival-promoting effect could be counteracted by simultaneously applied PACAP antagonist, PACAP6-38. However, co-treatment with PACAP during the light phase (9 AM) did not result in significant differences in the percentage of living cells. In summary, our results show that PACAP has a protective effect against the oxidative stress-induced cell death in chicken pinealocytes, but this effect is dependent on the phase of the circadian biological clock.

Mice deficient in pituitary adenylate cyclase activating polypeptide display increased sensitivity to renal oxidative stress in vitro.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide, showing widespread occurrence in the nervous system and also in peripheral organs. The neuroprotective effects of PACAP are well-established in different neuronal systems against noxious stimuli in vitro and in vivo. Recently, its general cytoprotective actions have been recognized, including renoprotective effects. However, the effect of endogenous PACAP in the kidneys is not known. The main aim of the present study was to investigate whether the lack of this endogenous neuropeptide influences survival of kidney cells against oxidative stress. First, we determined the presence of endogenous PACAP from mouse kidney homogenates by mass spectrometry and PACAP-like immunoreactivity by radioimmunoassay. Second, primary cultures were isolated from wild type and PACAP deficient mice and cell viability was assessed following oxidative stress induced by 0.5, 1.5 and 3mM H(2)O(2). Our mass spectrometry and radioimmunoassay results show that PACAP is endogenously present in the kidney. The main part of our study revealed that the sensitivity of cells from PACAP deficient mice was increased to oxidative stress: both after 2 or 4h of exposure, cell viability was significantly reduced compared to that from control wild type mice. This increased sensitivity of kidneys from PACAP deficient mice could be counteracted by exogenously given PACAP38. These results show, for the first time, that endogenous PACAP protects against oxidative stress in the kidney, and that PACAP may act as a stress sensor in renal cells. These findings further support the general cytoprotective nature of this neuropeptide.