Effects of pituitary adenylate cyclase activating polypeptide on small intestinal INT 407 cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the gastrointestinal tract. It has been shown to exert actions on intestinal functions, mainly affecting glandular secretion and motility. PACAP has several different effects on cell survival depending on the cell type and the applied stimulus. Its influences on small intestinal epithelial cells are not yet elucidated, therefore the aim of the present study was to investigate the effects of PACAP on intestinal epithelial cells having high turnover (INT 407) against different harmful stimuli, such as oxidative stress, in vitro hypoxia and gamma radiation. We tested the effect of PACAP on proliferation and cell survival using MTT assay. Moreover, various cancer-related factors were evaluated by oncology array. PACAP did not influence the proliferation rate of INT 407 cells. Its cell survival-enhancing effect could be detected against oxidative stress, but not against in vitro hypoxia or gamma irradiation. Clonogenic survival assay was performed to analyze the effect of PACAP on clonogenic potential of cells exposed to gamma radiation. Surprisingly, PACAP enhanced the clone-forming ability decrease induced by irradiation. Western blot analysis of ERK1/2 phosphorylation was performed in order to obtain further information on the molecular background. Our data showed phospho-ERK1/2 suppression of PACAP in irradiated cells. Furthermore, the role of endogenous PACAP against oxidative stress was also investigated performing ADCYAP1 small interfering RNA transfection. We found significant difference in the cell vulnerability between cells undergoing silencing and cells without transfection suggesting the protective role of the endogenously present PACAP against oxidative stress in INT 407 cells. In summary, PACAP seems to be able to exert contradictory effects in INT 407 cells depending on the applied stressor, suggesting its regulatory role in the cellular household.

Investigation of the possible functions of PACAP in human trophoblast cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide having a widespread distribution both in the nervous system and peripheral organs including the female reproductive system. Both the peptide and its receptors have been shown in the placenta but its role in placental growth, especially its human aspects, remains unknown. The aim of the present study was to investigate the effects of PACAP on invasion, proliferation, cell survival, and angiogenesis of trophoblast cells. Furthermore, cytokine production was investigated in human decidual and peripheral blood mononuclear cells. For in vitro studies, human invasive proliferative extravillous cytotrophoblast (HIPEC) cells and HTR-8/SVneo human trophoblast cells were used. Both cell types were used for testing the effects of PACAP on invasion and cell survival in order to investigate whether the effects of PACAP in trophoblasts depend on the examined cell type. Invasion was studied by standardized invasion assay. PACAP increased proliferation in HIPEC cells, but not in HTR-8 cells. Cell viability was examined using MTT test, WST-1 assay, and annexin V/propidium iodide flow cytometry assay. Survival of HTR-8/SVneo cells was studied under oxidative stress conditions induced by hydrogen peroxide. PACAP as pretreatment, but not as co-treatment, significantly increased the number of surviving HTR-8 cells. Viability of HIPEC cells was investigated using methotrexate (MTX) toxicity, but PACAP1-38 could not counteract its toxic effect. Angiogenic molecules were determined both in the supernatant and the cell lysate by angiogenesis array. In the supernatant, we found that PACAP decreased the secretion of various angiogenic markers, such as angiopoietin, angiogenin, activin, endoglin, ADAMTS-1, and VEGF. For the cytokine assay, human decidual and peripheral blood lymphocytes were separated and treated with PACAP1-38. Th1 and Th2 cytokines were analyzed with CBA assay and the results showed that there were no significant differences in control and PACAP-treated cells. In summary, PACAP seems to play various roles in human trophoblast cells, depending on the cell type and microenvironmental influences.

Autologous whole blood injections to patients with chronic urticaria and a positive autologous serum skin test: a placebo-controlled trial.

BACKGROUND: Patients with chronic urticaria (CU) frequently exhibit positive skin test reactions to autologous serum (ASST). Therapies aimed at inducing tolerance to circulating histamine-releasing factors in ASST+ CU patients, e.g. by treatment with autologous whole blood (AWB), have not yet been tested. OBJECTIVE: To test whether ASST+ CU patients can benefit from repeated low-dose intramuscular injections of AWB. METHODS: We characterized CU severity and duration, anti-Fc(epsilon)RI and anti-IgE expression, use of antihistamines, and quality of life in 56 CU patients (ASST+: 35, ASST-: 21) and assessed the therapeutic effects of 8 weekly AWB injections in a randomized, placebo-controlled, single-blind, parallel-group trial. RESULTS: Numbers, size, intensity, and/or duration of CU symptoms, quality of life, as well as expression of anti-Fc(epsilon)RI or anti-IgE were similar in ASST+ and ASST- CU patients. However, CU in ASST+ patients was of longer duration and required markedly more antihistaminic medication. Interestingly, ASST+ patients, but not ASST- patients, showed significantly (1) reduced CU activity, (2) decreased use of antihistamines, and (3) improved quality of life after AWB treatment. Placebo treatment was ineffective in both groups, but differences of AWB and placebo treatment responses did not achieve statistical significance in either group, most likely due to the limited number of patients treated. CONCLUSION: Our findings suggest that ASST+ CU is clinically different from other CU subforms and that ASST+ CU patients can benefit from AWB therapy.