Altered Notch Signaling in Developing Molar Teeth of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)-Deficient Mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with neuroprotective and neurotrophic effects. This suggests its influence on the development of teeth, which are, similarly to the nervous system, ectoderm and neural crest derivatives. Our earlier studies have shown morphological differences between wild-type (WT) and PACAP-deficient mice, with upregulated sonic hedgehog (SHH) signaling in the lack of PACAP. Notch signaling is a key element of proper tooth development by regulating apoptosis and cell proliferation. In this study, our main goal was to evaluate the possible effects of PACAP on Notch signaling pathway. Immunohistochemical staining was performed of Notch receptors (Notch1, 2, 3, 4), their ligands [delta-like protein (DLL)1, 3, 4, Jagged1, 2], and intracellular target molecules [CSL (CBF1 humans/Su (H) Drosophila/LAG1 Caenorhabditis elegans transcription factor); TACE (TNF-α converting enzyme), NUMB] in molar teeth of 5-day-old WT, and homozygous and heterozygous PACAP-deficient mice. We measured immunopositivity in the enamel-producing ameloblasts and dentin-producing odontoblasts. Notch2 receptor and DLL1 expression were elevated in ameloblasts of PACAP-deficient mice compared to those in WT ones. The expression of CSL showed similar results both in the ameloblasts and odontoblasts. Jagged1 ligand expression was elevated in the odontoblasts of homozygous PACAP-deficient mice compared to WT mice. Other Notch pathway elements did not show significant differences between the genotype groups. The lack of PACAP leads to upregulation of Notch pathway elements in the odontoblast and ameloblast cells. The underlying molecular mechanisms are yet to be elucidated; however, we propose SHH-dependent and independent processes. We hypothesize that this compensatory upregulation of Notch signaling by the lack of PACAP could represent a salvage pathway in PACAP-deficient animals.

PACAP deficiency as a model of aging.

Dysregulation of neuropeptides may play an important role in aging-induced impairments. In the long list of neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) represents a highly effective cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. PACAP has neuro- and general cytoprotective effects due to anti-apoptotic, anti-inflammatory, and antioxidant actions. As PACAP is also a part of the endogenous protective machinery, it can be hypothesized that the decreased protective effects in lack of endogenous PACAP would accelerate age-related degeneration and PACAP knockout mice would display age-related degenerative signs earlier. Recent results support this hypothesis showing that PACAP deficiency mimics aspects of age-related pathophysiological changes including increased neuronal vulnerability and systemic degeneration accompanied by increased apoptosis, oxidative stress, and inflammation. Decrease in PACAP expression has been shown in different species from invertebrates to humans. PACAP-deficient mice display numerous pathological alterations mimicking early aging, such as retinal changes, corneal keratinization and blurring, and systemic amyloidosis. In the present review, we summarize these findings and propose that PACAP deficiency could be a good model of premature aging.

Protective effects of pituitary adenylate cyclase activating polypeptide against neurotoxic agents.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide highly expressed in the central and peripheral nervous system, where it exerts several neuromodulatory functions and is an important trophic and protective factor. PACAP has been shown to activate several protective pathways, mainly through its specific PAC1 receptor and protein kinase A, C and MAP kinases downstream. It has been shown to have very potent neuroprotective actions against different neurotoxic agents both in vitro and in vivo. The aim of the present review is to provide an overview on the neurotoxic injuries against which PACAP exerts protection, and to give an insight into its protective mechanism. We give a summary of the neuroprotective effects against the most commonly used neurotoxic agents, such as 6-OHDA, MPTP, glutamate and some less well-known neurotoxic compounds. Also endogenous PACAP has neuroprotective effects, known from studies in PACAP knockout mice or from blocking endogenous effects by antagonists. Altogether, the vast amount of data for the neuroprotective effects of PACAP give a firm background for its endogenous role as part of the neuroprotective machinery and its possible future therapeutic use as a neuroprotective factor.

Disturbed spermatogenic signaling in pituitary adenylate cyclase activating polypeptide-deficient mice.

PACAP is a neuropeptide with diverse functions in various organs, including reproductive system. It is present in the testis in high concentrations, and in addition to the stage-specific expression within the seminiferous tubules, PACAP affects spermatogenesis and the functions of Leydig and Sertoli cells. Mice lacking endogenous PACAP show reduced fertility, but the possibility of abnormalities in spermatogenic signaling has not yet been investigated. Therefore, we performed a detailed morphological analysis of spermatozoa, sperm motility and investigated signaling pathways that play a role during spermatogenesis in knockout mice. No significant alterations were found in testicular morphology or motility of sperm in homozygous and heterozygous PACAP-deficient mice in spite of the moderately increased number of severely damaged sperms. However, we found robust changes in mRNA and/or protein expression of several factors that play an important role in spermatogenesis. Protein kinase A expression was markedly reduced, while downstream phospho-ERK and p38 were elevated in knockout animals. Expression of major transcription factors, such as Sox9 and phospho-Sox9, was decreased, while that of Sox10, as a redundant factor, was increased in PACAP-deficient mice. The reduced phospho-Sox9 expression was partly due to increased expression and activity of phosphatase PP2A in knockout mice. Targets of Sox transcription factors, such as collagen type IV, were reduced in knockout mice. In summary, our results show that lack of PACAP leads to disturbed signaling in spermatogenesis, which could be a factor responsible for reduced fertility in PACAP knockout mice, and further support the role of PACAP in reproduction.

Hepatitis C viral dynamics during ribavirin priming differ according to prior treatment response and HCV type.

The mode of action of ribavirin is not completely understood. Ribavirin monotherapy has a measurable antiviral effect, which shows great variability. It might lead to an earlier steady state of plasma concentration and therefore enhance the effect of following combination treatment. The aim of this study was to evaluate the antiviral effect of ribavirin priming and its influence on sustained virologic response after combination treatment in a group of patients with different hepatitis C virus (HCV) types with or without prior treatment experience. Retrospective analysis of 75 patients (37 treatment naïve, 20 prior relapse, 16 prior nonresponse, genotype 1 present in 60 patients) from five centres who received ribavirin priming as part of an individual strategy in order to improve treatment outcome. All patients received ribavirin monotherapy with a mean dose of 14.5 mg kg-1 body weight for a mean of 28 days. After ribavirin priming, dual combination treatment with pegylated interferon alfa and ribavirin was started. The mean HCV RNA decline after ribavirin priming was 0.6 log10  IU mL-1 (P<.001). The initial viral decline depended on HCV type and previous treatment status being highest among prior relapsers (0.8 log10  IU mL-1 ; P=.002) and HCV type 2/3 (1.2 log10  IU mL-1 ; P=.05) and lowest among those with prior nonresponse (0.3 log10  IU mL-1 , P=.01). IFNL4 (formerly IL28B) genotype for rs12979860 and IFNL3 genotype rs8099917 did not influence the initial viral decline. The study demonstrates a significant variability in the viral dynamics and antiviral efficacy of ribavirin monotherapy, which is mainly influenced by prior treatment status. The fact that the lowest response pattern was observed in prior nonresponder patients to pegylated interferon alfa plus ribavirin combination therapy can be taken as a hint that not only the individual interferon, but also the ribavirin sensitivity contributes significantly to the nonresponsive state.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulates the Hypothalamo-Pituitary-Thyroid (HPT) Axis via Type 2 Deiodinase in Male Mice.

The hypothalamic activation of thyroid hormones by type 2 deiodinase (D2), catalyzing the conversion of thyroxine to T3, is critical for the proper function of the hypothalamo-pituitary-thyroid (HPT) axis. Regulation of D2 expression in tanycytes alters the activity of the HPT axis. However, signals that regulate D2 expression in tanycytes are poorly understood. The pituitary adenylate cyclase-activating polypeptide (PACAP) increases intracellular cAMP level, a second messenger known to stimulate the DIO2 gene; however, its importance in tanycytes is not completely characterized. Therefore, we tested whether this ubiquitously expressed neuropeptide regulates the HPT axis through stimulation of D2 in tanycytes. PACAP increased the activity of human DIO2 promoter in luciferase reporter assay that was abolished by mutation of cAMP-response element. Furthermore, PAC1R receptor immunoreactivity was identified in hypothalamic tanycytes, suggesting that these D2-expressing cells could be regulated by PACAP. Intracerebroventricular PACAP administration resulted in increased D2 activity in the mediobasal hypothalamus, suppressed Trh expression in the hypothalamic paraventricular nucleus, and decreased Tshb expression in the pituitary demonstrating that PACAP affects the D2-mediated control of the HPT axis. To understand the role of endogenous PACAP in the regulation of HPT axis, the effect of decreased PACAP expression was studied in heterozygous Adcyap1 (PACAP) knockout mice. These animals were hypothyroid that may be the consequence of altered hypothalamic T3 degradation during set-point formation of the HPT axis. In conclusion, PACAP is an endogenous regulator of the HPT axis by affecting T3-mediated negative feedback via cAMP-induced D2 expression of tanycytes.

Ischemia/reperfusion-induced Kidney Injury in Heterozygous PACAP-deficient Mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with very diverse distribution and functions. Among others, PACAP is a potent cytoprotective peptide due to its antiapoptotic, anti-inflammatory, and antioxidant actions. This also has been shown in different kidney pathologies, including ischemia/reperfusion-induced kidney injury. Similar protective effects of the endogenous PACAP are confirmed by the increased vulnerability of PACAP-deficient mice to different harmful stimuli. Kidneys of homozygous PACAP-deficient mice have more severe damages in renal ischemia/reperfusion and kidney cell cultures isolated from these mice show increased sensitivity to renal oxidative stress. In our present study we raised the question of whether the partial lack of the PACAP gene is also deleterious, i.e. whether heterozygous PACAP-deficient mice also display more severe damage after renal ischemia/reperfusion. Mice underwent 45 or 60 minutes of ischemia followed by 2 weeks reperfusion. Histological evaluation of the kidneys was performed and individual histopathological parameters were graded. Furthermore, we investigated apoptotic markers, cytokine expression, and the activity of superoxide dismutase (SOD) enzyme 24 hours after 60 minutes of renal ischemia/reperfusion. We found no difference between the intact kidneys of wild-type and heterozygous mice, but marked differences could be observed following ischemia/reperfusion. Heterozygous PACAP-deficient mice had more severe histological alterations, with significantly higher histopathological scores for most of the tested parameters. Higher level of the proapoptotic pp38 MAPK and of some proinflammatory cytokines, as well as lower activity of the antioxidant SOD could be found in these mice. In conclusion, the partial lack of the PACAP gene results in worse outcomes in cases of renal ischemia/reperfusion, confirming that PACAP functions as an endogenous protective factor in the kidney.

Structural and morphometric comparison of the molar teeth in pre-eruptive developmental stage of PACAP-deficient and wild-type mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.

Examination of calcium-binding protein expression in the inner ear of wild-type, heterozygous and homozygous pituitary adenylate cyclase-activating polypeptide (PACAP)-knockout mice in kanamycin-induced ototoxicity.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with diverse biological effects. It also occurs and exerts protective effects in sensory organs; however, little is known about its effects in the auditory system. Recently, we have shown that PACAP protects cochlear cells against oxidative-stress-induced apoptosis and homozygous PACAP-deficient animals show stronger expression of Ca(2+)-binding proteins in the hair cells of the inner ear, but there are no data about the consequences of the lack of endogenous PACAP in different ototoxic insults such as aminoglycoside-induced toxicity. In this study, we examined the effect of kanamycin treatment on Ca(2+)-binding protein expression in hair cells of wild-type, heterozygous and homozygous PACAP-deficient mice. We treated 5-day-old mice with kanamycin, and 2 days later, we examined the Ca(2+)-binding protein expression of the hair cells with immunohistochemistry. We found stronger expression of Ca(2+)-binding proteins in the hair cells of control heterozygous and homozygous PACAP-deficient mice compared with wild-type animals. Kanamycin induced a significant increase in Ca(2+)-binding protein expression in wild-type and heterozygous PACAP-deficient mice, but the baseline higher expression in homozygous PACAP-deficient mice did not show further changes after the treatment. Elevated endolymphatic Ca(2+) is deleterious for the cochlear function, against which the high concentration of Ca(2+)-buffers in hair cells may protect. Meanwhile, the increased immunoreactivity of Ca(2+)-binding proteins in the absence of PACAP provide further evidence for the important protective role of PACAP in ototoxicity, but further investigations are necessary to examine the exact role of endogenous PACAP in ototoxic insults.

High-dose silibinin rescue treatment for HCV-infected patients showing suboptimal virologic response to standard combination therapy.

Incomplete suppression of hepatitis C virus (HCV) replication with persistence of minimal viremia (partial virologic response) leading to treatment failure can be observed in a significant proportion of HCV type 1-infected patients during antiviral therapy. Recently, high-dose intravenous silibinin has demonstrated strong antiviral activity against HCV. We were therefore interested in whether patients with partial virologic response can be rescued by the on-treatment addition of a short-term course of high-dose intravenous silibinin infusions. Twenty patients who failed to achieve a complete virologic response to different interferon-based regimens qualified for the rescue strategy and received 1400 mg/day silibinin infusions on two consecutive days. Complete viral suppression (below the limit of detection <6 IU/mL, TMA assay) could be induced in 13 of 20 patients within the first week after the short-term silibinin infusion, and all but one of them also remained HCV RNA negative during the subsequent follow-up period on continued peginterferon plus ribavirin treatment. In the remaining seven patients, no complete suppression could be achieved although four showed a significant HCV RNA reduction in response to silibinin. Silibinin infusions were generally well tolerated, and activation of abdominal peristalsis with nausea, diarrhoea and vomiting were the most prominent side effects. Of the twelve patients who exhibited a durable response to peginterferon and ribavirin treatment, three achieved an SVR, two achieved a week 12 SVR and four suffered a viral relapse. Three patients could not complete the assigned antiviral treatment with peginterferon alpha and ribavirin for nonvirological reasons. Short-term administration of high-dose intravenous silibinin might be an interesting approach to rescue patients with ongoing minimal residual viremia while on interferon-based therapy. These preliminary findings may stimulate further studies to evaluate more refined therapeutic strategies.

Comparative examination of inner ear in wild type and pituitary adenylate cyclase activating polypeptide (PACAP)-deficient mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide with well-known neuroprotective and neurotrophic effects. The involvement of PACAP in sensory processing has also been documented, but little is known about its effects in the auditory system. PACAP and its specific receptor (PAC1) are present in the cochlea and in brain structures involved in auditory pathways. Recently, we have shown that PACAP protects cochlear cells against oxidative stress-induced apoptosis. The endolymphatic Ca(2+) concentration controlled by Ca(2+) buffers of the hair cells is essential for the normal hearing processes. In this study we examined the localization of PAC1 receptor and Ca(2+) buffering proteins (parvalbumin, calretinin, calbindin) in the inner ear of 5-day-old PACAP-deficient mice compared with wild-type mice in order to get a closer insight into the effect of endogenous PACAP in the cochlear function. We did not find differences in the distribution pattern of PAC1 receptors between the two groups, but wild-type animals showed significantly higher PAC1 receptor expression. In contrast, inner and outer hair cells of PACAP-deficient mice showed more pronounced parvalbumin, calbindin, and calretinin immunopositivity compared with wild-type mice. Elevated endolymphatic Ca(2+) is deleterious for cochlear function, while the high concentration of Ca(2+) buffers in hair cells may offer protection. The increased immunoreactivity of Ca(2+) binding proteins in the absence of PACAP provide further evidence the important role of PACAP in the hearing processes.

[The role of MIBI scintigraphy in the early detection of breast cancer]. / A MIBI szcintigráfia szerepe az emlórákok korai diagnosztikájában.

The authors investigate the role of MIBI scintigraphy in the early diagnosis of breast cancer. The importance of early diagnosis is emphasized by the fact that breast cancer has the highest morbidity and mortality preceding cervical carcinoma amongst women. Retrospective examinations were made in case of 42 patients operated before because of breast cancer in order to examine accuracy of both mammography and scintigraphy in comparison with the results of histological diagnosis. In these cases sensitivity of scintigraphy turned out to be 69%, while its specificity 42%. In cases of mammographical investigations the sensitivity proved to be 74% and specificity was 61%. Besides mammography, scintigraphy has a very important additional role in the diagnosis of breast cancer. Because of its results and costs scintigraphy is not able to take over the mammography's dominant position (as a popular diagnostic method) but in selected patients' groups it can help to realise tumors as well as to avoid unnecessary operations or needle biopsis. Based on our results this method can give significant additional information in T1b and especially in T1c states of tumors. Therefore this method can be offered as an additional investigation in cases of physically realised or mammographically screened, but not-palpable, larger than 1 cm size lumps when there is little or moderate risk of malignancy.

[Changes in the surgical treatment of breast cancer]. / Szemléletváltozás az emlórák sebészi kezelésében.

Changes in radicality of breast cancer surgery between 1991 and 2000 are issued in this paper. While breast conserving surgery was performed in 26 cases (25%) out of 103 in 1991, in the year 2000 165 patients were operated of breast cancer and the breast was preserved in 143 cases (87%). These results are partly approved by the breast cancer screening performed by physical examination since 1981 and also by mammography since 1998. The five-year overall survival of patients who had been operated between 1991 and 1994 is 90%, the local recurrency rate is 6%. Considering these data breast preserving surgery is a reliable method.

[Follow up examination of the endometrium during tamoxifen therapy]. / Az endometrium ellenörzése tamoxifen kezelés alatt.

Tamoxifen is a non-steroidal estrogen antagonist used mainly in the adjuvant therapy of breast cancer. Tamoxifen acts primarily as an antiestrogen, but also carries estrogenic effect, mainly to the endometrium. Although diverse pathological uterine findings has been described in connection with the long term use of this drug, there is no widely accepted protocol for the monitoring of the endometrium in these patients. All patients referred for gynecologic examination at the Department of Obstetrics and Gynecology University Medical School Debrecen, Hungary with previous treatment for breast cancer were involved in a screening programme. The gradual screening programme consists of: transvaginal sonography, color doppler imaging, hysteroscopy, and endometrial curettage or sampling. If any of the first steps proved to be negative, further evaluation could be avoided. Altogether 31 patients were referred for examination and transvaginal sonography alone excluded endometrial abnormality in 9 cases, while the rest of the patients went through further evaluation. The ratio of false positive results using transvaginal sonography alone was high and could be lowered with additional color doppler imaging. Endometrial pathology could be excluded in about 50% of cases without tissue sampling. Positive histology was found in 25% of patients receiving Tamoxifen. The most frequent pathological finding was endometrial polyp. In conclusion endometrial changes found by transvaginal sonography, should be further evaluated. The gradual screening procedures enable us to the proper use of more invasive procedures.

Integrin expression on normal and neoplastic human breast epithelium.

Integrin adhesion receptor expression of different benign and malignant breast tumours was examined by means of immunohistochemical techniques. A panel of seven different anti-alpha and two different anti-beta subunit antibodies was used. Normal breast epithelium displayed a well characterized and constant pattern of integrin expression consisting of strong alpha 1,2,3,6 and alpha v, and a relatively weaker beta 1 and beta 3 staining. No staining for alpha 4 or alpha 5 could be detected on the epithelial cells. Benign fibroadenomas did not show changes in their receptor expression compared to normal tissues. In the cases of different types of breast cancer, there was a significant downregulation of all subunits. The staining pattern was distinct if there could a basement membrane like structure be detected around the invading tumour nodules. When laminin and collagen type IV surrounded the tumour cells, those cells in contact with the extracellular matrix components still displayed strong positivity for the integrin subunits. Other cells inside the tumour cell nests or not surrounded by basement membrane did not express integrins. The positively staining cells might be more differentiated owing to the effect the basement membrane. Myoepithelial labeling of the integrin expressing cells gave negative results. The observed integrin expression heterogeneity renders the histologic picture difficult to interpret with regard to clinical behavior of the tumour.