Chloroquine enhances the antimycobacterial activity of isoniazid and pyrazinamide by reversing inflammation-induced macrophage efflux.

Mycobacterium tuberculosis (MTB) is notorious for persisting within host macrophages. Efflux pumps decrease intracellular drug levels, thus fostering persistence of MTB during therapy. Isoniazid (INH) and pyrazinamide (PZA) are substrates of the efflux pump breast cancer resistance protein-1 (BCRP-1), which is inhibited by chloroquine (CQ). In this study, BCRP-1 was found to be expressed on macrophages of human origin and on foamy giant cells at the site of MTB infection. In the current in vitro study, interferon-gamma (IFNγ) increased the expression of BCRP-1 in macrophages derived from the human monocytic leukaemia cell line THP-1. Using a BCRP-1-specific fluorescent dye and radioactively labelled INH, it was demonstrated that efflux from macrophages increased upon activation with IFNγ. CQ was able to inhibit active efflux and augmented the intracellular concentrations both of INH and the dye. In agreement, CQ and specific inhibition of BCRP-1 increased the antimycobacterial activity of INH against intracellular MTB. Although PZA behaved differently, CQ had comparable advantageous effects on the intracellular pharmacokinetics and activity of PZA. The adjunctive effects of CQ on intracellular killing of MTB were measurable at concentrations achievable in humans at approved therapeutic doses. Therefore, CQ, a widely used and worldwide available drug, may potentiate the efficacy of standard MTB therapy against bacteria in the intracellular compartment.

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies.

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices.

PACAP immunoreactivity in human malignant tumor samples and cardiac diseases.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic and multifunctional neuropeptide having important roles in various physiological processes. Recent trends in PACAP research point to the clinical introduction of PACAP or its analogs/fragments possibly in the near future. Recently, we have shown the presence of PACAP in human plasma, milk, placenta, and follicular fluid samples. However, relatively few data are available on PACAP in human tissues from patients with different disorders. The aim of the present study was to determine, by radioimmunoassay, the tissue level of PACAP38-like immunoreactivity (LI) and PACAP27-LI in different primary non-small cell lung cancer, colon tumor samples, and in cardiac muscle samples from patients suffering from ischemic heart disease and valvular disorders. We also labeled the PAC1 receptors in human cardiac cells. All samples showed significantly higher PACAP38-LI compared with PACAP27-LI. We found significantly lower levels of PACAP38-LI and PACAP27-LI in tumoral and peripheral samples compared with normal healthy tissue in both lung and colon cancers. Further investigations are necessary to describe the exact function of PACAP in oncogenesis. We showed that PACAP38-LI and PACAP27-LI are significantly higher in ischemic heart diseases compared with valvular abnormalities, suggesting that PACAP might play a role in ischemic heart disorders.

Correlation between oocyte number and follicular fluid concentration of pituitary adenylate cyclase-activating polypeptide (PACAP) in women after superovulation treatment.

Follicular growth, ovulation, and luteinization are influenced by interactions of peptide and steroid hormone-signaling cascades in the ovary. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the regulation of several endocrine processes and is present in ovarian follicular fluid (FF). However, little is known about PACAP in FF with regard to maturation, ovulation, fertilization, and successful pregnancy. The aim of this pilot study was to investigate whether there is a correlation between PACAP concentration in FF and ovarian response to superovulation treatment in infertile women, performed in volunteers (n = 132; aged between 20 and 35). After treatment, the number of harvested oocytes was recorded and PACAP immunoreactivity in FF was measured by radioimmunoassay. All the corresponding PACAP concentrations were below 290 fmol/ml in cases when the number of harvested oocytes exceeded 14 per patient, while in all cases above 290 fmol/ml, the number of oocytes was below 14. Using these cutoff values, we determined three study groups: high-PACAP concentration, high-oocyte number, and low-PACAP concentration-low-oocyte number groups. Median values of PACAP concentration in these groups were 411.2, 106.5, and 101.0 fmol/ml, respectively, while the median values of harvested oocytes were 5.5, 19.0, and 5.0, respectively. Differences were significant, indicating a correlation between concentration of PACAP in FF and the number of recruited oocytes. Higher concentrations of PACAP in FF might be associated with lower number of developing oocytes, while low concentrations of PACAP might correlate with a markedly higher number of ova retrieved, thus predicting a higher chance for ovarian hyperstimulation. Our present study is among the first few human clinical studies with direct conclusions drawn for possible clinical impact of PACAP.

Presence of pituitary adenylate cyclase activating polypeptide and its type I receptor in the rat kidney.

Pituitary adenylate cyclase activating polypeptide (PACAP), a multifunctional neuropeptide, has 2 active forms, PACAP38 and PACAP27. It is now well-established that PACAP has several actions also in peripheral organs, including renoprotective effects. The peptide itself has not been previously identified in the rat kidney. The first aim of our study was to identify PACAP in the rat kidney using mass spectrometry and radioimmunoassay (RIA). Receptor mRNA and binding studies revealed the existence of all 3 PACAP receptors (PAC1, VPAC1, and VPAC2) in the kidney, but their exact localization in histologic sections was not evident. Because most of the cytoprotective effects of PACAP relate to its specific PAC1 receptor, our second aim was to identify the cell types wherein the PAC1 receptor is expressed in the rat kidney. Mass spectrometry revealed the presence of PACAP38 in the kidney. RIA measurements showed both PACAP38- and PACAP27-like immunoreactivities in kidney homogenates, with PACAP38 being dominant. Immunohistochemistry revealed PAC1 receptor-like immunoreactivity in kidney sections, mainly expressed in cortical tubular epithelial cells. These results showed PACAP to be endogenously present in the kidney. The tubular localization of the PAC1 receptor provides the basis for the renal effects of the peptide under physiologic and pathologic conditions.

Cone function in children with a history of preterm birth.

Color vision was examined by psychophysical tests and photopic color full-field electroretinography (ERG) in formerly preterm children, and compared with those of full-term children. In a prospective case-control study, 25 patients with a history of preterm birth 7-14 years of age were divided into three groups: group I, laser-treated retinopathy of prematurity [ROP] (n = 7); group II, spontaneously regressed ROP (n = 8); group III, no ROP (n = 10). Age-matched full-term born children comprised the control group (n = 8). Color vision was assessed by Fansworth D15 and Lanthony desaturated D15 tests. The cone function was tested using photopic full-field ERG. Besides the ISCEV standard stimuli, blue light on amber background was also used (S-cone ERG). The correlation between ERG parameters and prematurity or ROP was determined. We found no significant differences between any patient group and the control group in the results of the psychophysical tests, and implicit times of the ERG responses. The ERG b-wave amplitudes were significantly lower in group I (laser-treated ROP) compared to controls, for 2 of 4 stimulus conditions i.e. the standard (P = 0.028) and S-cone (P = 0.017) single flash ERGs. The general estimating equation model statistics found a significant effect of prematurity on the b-wave amplitudes (P = 0.025, standard, P = 0.014, S-cone ERG). A slightly reduced photopic ERG b-wave amplitude may be associated with prematurity.

Investigation of pituitary adenylate cyclase activating polypeptide in human gynecological and other biological fluids by using MALDI TOF mass spectrometry.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional and pleiotropic neuropeptide. PACAP has diverse effects in the endocrine system, among others, it plays important roles in oogenesis, implantation and development of the nervous system. However, it is not known whether PACAP is present in the fluids of the human reproductive organs. The aim of the present study was to determine, by means of mass spectrometry and radioimmunoassay, whether PACAP is present in human amniotic fluid, ovarian follicular fluid and cervico-vaginal fluid. Samples were obtained from healthy adult volunteers. Our MALDI TOF and MALDI TOF/TOF spectrometry results show that PACAP38 is present in all of the follicular fluid samples, and PACAP-like immunoreactivity was also measured by radioimmunoassay. However, we did not find the characteristic peak representing the unmodified 38 amino acid form of the peptide in normal cervico-vaginal smear and amniotic fluid samples. Furthermore, we analyzed other body fluids for comparison, such as human nasal fluid, saliva and aqueous humor. PACAP was not found in these latter samples. In summary, the present study provides evidence for the presence of PACAP in human follicular fluid, suggesting a role in oocyte function, but determination of the exact physiological significance awaits further investigation.

[Prognostic factors and visual outcome for open globe injuries with intraocular foreign bodies]. / Prognostische Faktoren und klinische Ergebnisse in der Behandlung von offenen Augenverletzungen mit intraokularen Fremdkörpern.

AIMS: The aim of this study was to evaluate the clinical features as well as the visual results and determine the prognostic factors after the removal of retained intraocular foreign bodies (IOFBs). PATIENTS AND METHODS: At our centre we conducted a non-randomised, non-comparative retrospective analysis of records of 31 patients with intraocular foreign bodies treated using pars plana vitrectomy and other conventional surgical techniques during a 3-year period between January 2006 and December 2008. Using the OTS classification for ocular injuries (United States Eye Injury Registry [USEIR]), we evaluated the visual outcome and various pre-, intra- and postoperative clinical factors. RESULTS: Almost all patients (96.8 %) were male with a mean age of 32.29 years ± 13.38 SD (SD = standard deviation). The most common cause of ocular injury was hammering (58 %), in 27 cases the IOFB was magnetic. Follow-up ranged from 1 to 34 months with a mean of 4.5 months. In 30 eyes, the IOFBs could be removed during the first surgery. The average time between injury and surgery was 15.78 ± 23.45 SD hours for 27 patients (87.1 %). In 3 cases the intraocular foreign body (IOFB) was removed 7, 9, 14 days after the injury. In one case the IOFB was removed in a second surgery 40 days after the injury. In 23 cases we performed pars plana vitrectomy, for 8 patients we performed anterior segment surgery, in 21 cases the lens was also removed. At the time of surgery initial vitreous haemorrhage was present in 17 patients (54.8 %). OTS score for ocular injury was calculated as 2.45 ± 0.78 SD. Initial visual acuity was below 20 / 200 in 61.3 % of the cases. Subsequent to surgical treatment over a mean follow-up of 2.5 months, 25 patients (80.64 %) achieved a visual acuity of 20 / 200 or better (0.69 ± 0.35 SD). Best corrected visual acuity was significantly better for ocular injuries with a higher OTS score (p = 0.000095). Other significant factors for good visual outcome were good initial visual acuity (p = 0.002), the absence of vitreal haemorrhagies (p = 0.024) and small size of the IOFB (p = 0.043). Postoperative complications included retinal detachment (9.67 %), endophthalmitis (3.22 %) and PVR (3.22 %). CONCLUSION: Acceptable visual results could be achieved after the removal of posterior segment IOFBs by vitrectomy. A reliable classification of ocular injuries may provide significant information about the prognosis of injuries with intraocular foreign bodies.

[Prospective study of quality of life after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy using oxaliplatin for peritoneal carcinomatosis]. / Étude prospective de la qualité de vie après une CHIP par oxaliplatine pour carcinose péritonéale.

BACKGROUND: Cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is now the reference technique for limited peritoneal carcinomatosis (PC). Operative mortality is actually at 3 or 4%, and decrease as morbidity. Together, they did not limit acceptation of HIPEC. However, one of the major limitation of this aggressive treatment is that patient can be afraid to impair overall quality of life (QoL). The aim of this article was to assess QoL in patients at least 12 months after HIPEC using Oxaliplatin. METHOD: Patients completed a questionnaire before and after surgery at 1, 3, 6 and 12 months. QoL was assessed with the EORTC QLQ-C30 questionnaire. RESULTATS: Between September 2006 and October 2008, 32 of 35 patients who had undergone HIPEC were interviewed. PC originated in primary lesions of the colon/rectum (N = 17), ovary (N = 3), stomach (N = 3), appendix (N = 2), mesothelium (N = 2), pseudomyxoma peritonei (N = 3) and primary carcinoma of peritoneum (N = 2). The percentage of patients completing the questionnaire at each time point was: baseline = 87% (N = 28); 1 and 3 months = 46% (N = 15); 6 months = 62% (N = 20); and 12 months = 59% (N = 19). Morbidity and mortality were respectively 35 and 5%. Median hospital stay was 19 days. QoL score had decreased considerably in 60% of patients in the early postoperative assessment period after HIPEC (1 month), as compared with baseline score. Forty five per cent had reported significant pain and limitations on physical functioning. QoL score had returned to baseline at 3 months in 53,3% of patients: 20% reported lack of energy and fatigue. Fifty-five and 73% of patients had recovered their overall QoL at 6 and 12 months, respectively. Also, psychosocial problems, diarrhea and constipation, and peripheral neuropathy of oxaliplatin were reported in 20% of survivors over the course of the first year after HIPEC. CONCLUSION: Short-term QoL with physical and functional well-being are impaired in the first few months after surgery plus HIPEC using oxaliplatin. Long-term QoL returns to baseline at 3 months; however 20% of patients still report psychosocial problems, gastrointestinal symptoms and oxaliplatin-induced neuropathy. It is useful and important for patients to see this HIPEC QoL data at the time of consultation before treatment.

Changes in pituitary adenylate cyclase-activating polypeptide following renal ischemia-reperfusion in rats.

PACAP (pituitary adenylate cyclase-activating polypeptide) occurs in two biologically active forms, with 38 and 37 amino acid residues (PACAP38 and PACAP27). In mammalian tissues, PACAP38 is the dominant form. Diverse effects have been described in the cardiovascular, respiratory, gastrointestinal, and urogenital systems. PACAP is known for its strong cytoprotective effects, which are present endogenously as well, as proven by knockout studies and results showing that PACAP is up-regulated following diverse injuries. Little is known about such effects in the kidney. We have previously shown that PACAP is protective in renal ischemia-reperfusion injury. Therefore, the aim of the present study was to investigate the changes of endogenous PACAP following 60-minute renal ischemia using radioimmunoassay. Changes were observed within 24 hours following renal vessel clamping. In the cortex, an acute decrease was followed by an increase on the intact side, and levels returned to original ones on the operated side. In the medulla, changes were only observed on the clamped side: a marked up-regulation was detected in PACAP38-like immunoreactivity within the first 24 hours. The present study showed that PACAP38- and PACAP27-like immunoreactivities sensitively react to renal ischemia-reperfusion, the physiological importance of which awaits further investigation.

Alterations of pituitary adenylate cyclase-activating polypeptide-like immunoreactivity in the human plasma during pregnancy and after birth.

BACKGROUND: Since its discovery, several distinct effects of pituitary adenylate cyclase activating polypeptide (PACAP) have been established - predominantly in animal studies - in the nervous system, various peripheral organs as well as in the endocrine regulation. It is unknown whether PACAP has any effect on human pregnancy regarding either utero-maternal or perinatal aspects of the gestation. AIM: We investigated alterations of PACAP38-like immunoreactivity (PACAP38-LI) in the human plasma throughout normal pregnancy, during and after delivery, and its level in the umbilical vessels, as well as in the peripheral blood of term healthy newborns. MATERIALS AND METHODS: A 2 ml blood sample was used for each test, PACAP38-LI was determined by radioimmunoassay. RESULTS: In the 2nd and 3rd trimester significant elevation was observed in the PACAP38-LI compared to the earlier gestation and non-pregnant conditions. During delivery its level significantly decreased and returned to the original values 3 days after birth. In the neonates PACAP38-LI level of the peripheral blood was similar to that of healthy adults, but umbilical arteries and veins contained significantly lower concentrations of PACAP38-LI. Besides, the levels were lower in the umbilical vein compared to the artery. CONCLUSIONS: PACAP38-LI levels show sensitive change during normal pregnancy and delivery. Our findings suggest that the fetal organs actively synthesize PACAP. Further investigations are required to elucidate the physiological importance of the alterations observed.

Changes in the expression of pituitary adenylate cyclase-activating polypeptide in the human placenta during pregnancy and its effects on the survival of JAR choriocarcinoma cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with survival-promoting actions, has been observed in endocrine organs and is thought to play a role in reproductive functions, including pregnancy. PACAP occurs in two forms, 27 and 38 amino acid residues, with PACAP38 being the predominant form in human tissues. In the present study, we determined the concentrations of PACAP38 and PACAP27 in first-trimester and full-term human placentas using radioimmunoassay. We found high levels of PACAP38 and lower levels of PACAP27 in different parts of the full-term human placenta. PACAP38 content increased in the placenta during pregnancy, both on the maternal side and on the fetal side. The effects of PACAP on the survival of JAR human choriocarcinoma cells were investigated using flow cytometry and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assay in cells exposed to the widely used chemotherapeutic agent methotrexate (MTX). It was found that PACAP neither influenced the survival of JAR cytotrophoblast cells nor affected cellular response to the death-inducing effect of the chemotherapeutic agent MTX. The present observations further support the significance of PACAP in the human placenta. The observation that PACAP did not influence the effects of MTX may have future clinical importance, showing that PACAP does not decrease the effects of certain chemotherapeutic agents.

[Imaging of gastrointestinal stromal tumors]. / Imagerie des tumeurs stromales digestives.

Gastrointestinal stromal tumors (GIST) arising from interstitial cells of Cajal, represent the first type of solid tumor, which is very sensitive to a specific molecularly targeted tyrosine kinase receptor blocker (i. e., imatinib). On CT, which is considered as the reference technique, GISTs typically present as large, well-delineated, heterogeneous and sometimes exophytic masses. In contrast with the absence of lymph node involvement, hepatic metastasis as well as mesenteric involvement can be observed. MR-enteroclysis is indicated to investigate the local extent of the disease in specific cases whereas MR imaging is used to detect hepatic metastasis. Because of a specific treatment, contrast-enhanced imaging is needed for the follow-up of treated tumors. Evaluation of tumor response to treatment is best assessed with CT which still remains the reference imaging technique whereas FDG-PET imaging is used in specific cases.

Changes and effect of PACAP-38 on intestinal ischemia-reperfusion and autotransplantation.

Tissue injury caused by cold preservation and reperfusion during small bowel transplantation remains an unsolved problem. Increasing evidence suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) has protective effects in several ischemia-reperfusion (I/R) models. This study investigated the effect of PACAP-38 on oxidative stress in autotransplanted intestine. We established sham-operated, I/R, and autotransplanted groups in Wistar rats (n = 55). We applied ischemia for 1 (GI), 2 (GII), or 3 hours (GIII). In autotransplanted groups, we performed total orthotopic intestinal autotransplantation. Grafts were preserved in University of Wisconsin (UW) solution for 1 (GIV), 2 (GV), 3 (GVI), or 6 (GVII) hours and in PACAP-38-containing UW for 1 (GVIII), 2 (GIX), 3 (GX), or 6 (GXI) hours. Reperfusion lasted 3 hours in each group. Endogenous PACAP-38 values were measured by radioimmunoassay. Oxidative stress parameters malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) were measured in tissue homogenates. Concentration of endogenous PACAP-38 significantly decreased in GI to GIII compared with the sham-operated animals following I/R periods (P < .05). Cold preservation in UW and reperfusion of the intestine increased the level of tissue MDA in GIV to GVII, which correlated with the duration of cold storage. The content of GSH decreased in GIV to GVII to levels that were significantly different between GIV and GVIII and between GVII and GXI. SOD activity decreased dramatically in GIV to GVII with significantly higher activity in GIX to GXI. Our findings confirmed that I/R decreased endogenous PACAP-38 concentration. Administration of PACAP-38 to UW solution mitigated the oxidative injury during intestinal autotransplantation.

[Ultrasonographic findings in endophthalmitis following cataract surgery : a review of 81 cases]. / Ultrasonographische Befunde bei Endophthalmitis nach Kataraktoperationen : Rückblick auf 81 Fälle.

PURPOSE: Review and analysis of ultrasonographic data of patients with endophthalmitis following cataract surgery. PATIENTS AND METHODS: We conducted a retrospective analysis of data and ultrasound findings of 81 patients with endophthalmitis following cataract surgery between 2000 and 2005. We evaluated the type of surgery, time of onset of endophthalmitis, and different ultrasonographic findings. RESULTS: During the study period, acute endophthalmitis following cataract surgery developed in 41 eyes, subacute endophthalmitis in 25 eyes, and late-developing endophthalmitis in 20 eyes. In 51% of the cases, the endophthalmitis occurred after the phacoemulsification method. Ultrasonographic findings such as membrane formation were found in 23 eyes (28%), and dense vitreous opacities were detected in nine eyes. Posterior wall thickness was measured in 73 eyes. Two initial echography findings were associated with acute and subacute endophthalmitis: dense vitreous opacities and detachment of the posterior vitreous limiting membrane. CONCLUSION: Ultrasonographic findings such as increased thickness of the posterior wall and dense organization of the vitreous can be useful in the clinical evaluation, prediction, and treatment of postoperative endophthalmitis.

Agonistic behavior of PACAP6-38 on sensory nerve terminals and cytotrophoblast cells.

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) are mediated through G-protein-coupled receptors, the specific PAC1 receptor and VPAC1 and VPAC2 receptors which bind vasoactive intestinal peptide with similar affinity. Based on binding affinity studies, PACAP6-38 was discovered as a potent antagonist of PAC1 and it has been used by hundreds of studies as a PACAP antagonist. Recently, we have found that in certain cells/tissues, PACAP6-38 does not antagonize PACAP-induced effects, but surprisingly, it exerts similar actions to PACAP1-38, behaving as an agonist. In the present study, we report on the agonistic behavior of PACAP6-38 on neuropeptide release from sensory nerves of the isolated rat trachea and on the MAPK signaling pathways in cytotrophoblast cells. In isolated rat tracheae, PACAP6-38, similarly to PACAP1-38, induced significant inhibitory effects on the release of three simultaneously measured sensory neuropeptides, substance P, calcitonin gene-related peptide, and somatostatin evoked by both chemical excitation and electrical field stimulation of capsaicin-sensitive afferents. Effects of PACAP6-38 were the same as those of PACAP1-38 on MAPK signaling in human cytotrophoblast cells. Western blot analysis showed that both peptide forms stimulated ERK1/2 and JNK phosphorylation, while they both inhibited p38 MAPK phosphorylation. The most pronounced effects were observed when both peptides were present. In summary, our results show that PACAP6-38, which is a PACAP receptor antagonist in most cells/tissues, can behave as an agonist in other systems. The increasing interest in the effects of PACAP requires further studies on the pharmacological properties of the peptide and its analogues.

The presence and distribution of pituitary adenylate cyclase activating polypeptide and its receptor in the snail Helix pomatia.

The aim of this study was to show the presence, distribution and function of the pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors in the CNS and peripheral nervous system of the mollusk, Helix pomatia. PACAP-like and pituitary adenylate cyclase activating polypeptide receptor (PAC1-R)-like immunoreactivity was abundant both in the CNS and the peripheral nervous system of the snail. In addition several non-neuronal cells also revealed PACAP-like immunoreactivity. In inactive animals labeled cell bodies were mainly found and in the neuropile of active animals dense immunostained fiber system was additionally detected suggesting that expression of PACAP-like peptide was affected by the behavioral state of the animal. RIA measurements revealed the existence of both forms of PACAP in the CNS where the 27 amino acid form was found to be dominant. The concentration of PACAP27 was significantly higher in samples from active animals supporting the data obtained by immunohistochemistry. In Western blot experiments PACAP27 and PACAP38 antibodies specifically labeled protein band at 4.5 kDa both in rat and snail brain homogenates, and additionally an approximately 14 kDa band in snail. The 4.5 kDa protein corresponds to PACAP38 and the 14 kDa protein corresponds to the preproPACAP or to a PACAP-like peptide having larger molecular weight than mammalian PACAP38. In matrix-assisted laser desorption ionization time of flight (MALDI TOF) measurements fragments of PACAP38 were identified in brain samples suggesting the presence of a large molecular weight peptide in the snail. Applying antibodies developed against the PACAP receptor PAC1-R, immunopositive stained neurons and a dense network of fibers were identified in each of the ganglia. In electrophysiological experiments, extracellular application of PACAP27 and PACAP38 transiently depolarized or increased postsynaptic activity of neurons expressing PAC1-R. In several neurons PACAP elicited a long lasting hyperpolarization which was eliminated after 1.5 h continuous washing. Taken together, these results indicate that PACAP may have significant role in a wide range of basic physiological functions in snail.