Vcan null mouse called heart defect (hdf). Total absence of the Vcan gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, Vcan(tm1Zim), of heart defects that results from deletion of exon 7 in the Vcan gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the Vcan gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of Vcan exon 7. The Vcan(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the  During embryonic development, the extracellular matrix plays a central role in restructuring the single heart tube into a mature multi-chambered organ. To date, only two structural components of the extracellular matrix, versican and its binding partner hyaluronan, have been shown to be indispensably required for successful completion of this morphogenetic transition. Complete loss of either of these matrix molecules results in early embryonic lethality with a failure of endocardial cushion formation and a highly dilated myocardium of the primitive heart tube Vcan; formerly Cspg2) consists of 15 exons and spans a region of approximately 100 kb Versican is a chondroitin sulfate proteoglycan that was first identified in fibroblastic extracts heart defect mouse (hdf), the absence of versican protein results in early embryonic lethality hdf allele display severe cardiac defects including absence of pre-valvular endocardial cushions, loss of anterior (second) heart field structures and a thin, dilated myocardium. Similarly, loss of a subdomain of the G1 domain  results in embryonic lethality of homozygous embryos on a congenic and mixed background. However, on the mixed background some embryos survive longer and have ventricular septal defects Vcan’s alternative splice forms at later stages of heart development using a versican alternative splice form deficient mouse model. The Vcan(tm1Zim) mouse, expresses only the V1 and V3 splice forms of versican in the heart and survive embryogenesis Vcan(tm1Zim) deletion have less severe cardiac defects than the hdf homozygous mice. Differential protein expression profiling using isobaric tag reagents for absolute and relative quantitation (iTRAQ) As shown by the (tm1Zim)Vcan hearts were collected from timed pregnant dams and staged according to Theiler This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol (AR#1572) was approved by the Institutional Animal Care & Use Committee (IACUC) at the Medical University of South Carolina. Wild-type C57BL6/J and Vcan(tm1Zim) mice were created and characterized as described elsewhere 5′-GAGAGGACAGAAACACCAAG-3′, 5′-ACTGTGGGTCAAATGAACTC-3′ designed to yield a 454 bp product from the mutated allele and a 312 bp product from the wild-type allele. The absence of the V0 and V2 splice forms was independently confirmed in our laboratory by immunohistochemistry and western blot analysis.The To evaluate the whole mouse embryo hearts by optical sectioning, we adapted a confocal fluorescence imaging technique originally used with avian hearts Embryos (E10.5 pc) were dissected from the mouse decidua and extra embryonic tissues and pooled into Earle’s balanced salt solution (Gibco). Hearts were dissected from the embryos and the AV regions were removed, and cut to expose the lumen. The AV explants were placed immediately onto the surface of drained collagen gels with 1 explant per well, and incubated at 37°C for 3–4 hr to allow attachment of the explants onto the collagen gel surface as previously described Hearts dissected from postnatal mice or embryos were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned as previously described Vcan(tm1Zim) mutant and wild-type E13.5 p.c. embryos obtained from multiple litters were removed by micro-dissection, bisected and rinsed with PBS, frozen in liquid nitrogen and then finely crushed in a liquid nitrogen cooled stainless steel mortar and pestle . Protein profiling was performed on the pooled hearts of 5 animals from each group in order to obtain sufficient protein for analysis. The samples were homogenized  in ice cold 20 mM HEPES, pH 8/4°C, containing 1 M urea in a 1 mL ground glass tissue grinder then subjected to 770×g for 10 min at 4°C to remove nuclei and undisassociated tissue. The resulting supernatants were subjected to centrifugation at 150,000×g for 90 min at 4°C (Beckman Optima TLX ultracentrifuge with TKS-55 rotor) to obtain a combined urea soluble and insoluble fraction. The protein concentration in the urea soluble fraction was determined by the BCS method  using bovine serum albumin as standard. The samples were reduced and alkylated prior to digestion with LysC and trypsin at a ratio of 1∶10 Pregnant females at E13.5 post coitus (pc) were anesthetized and euthanized following approved protocols of the Division of Laboratory Animal Resources at MUSC and AAALAC guidelines. Hearts of homozygous Vcan(tm1Zim) mutant E13.5 pc hearts (n = 5 per group) were labeled with the “isobaric tag reagents for absolute and relative quantitation” (iTRAQ™) . Duplicate samples (90 µg of protein each) from wild-type and Vcan(tm1Zim) mutant hearts were each labeled with one of the 4 unique isobaric tags  sample 1; 117- Vcan(tm1Zim) sample 2) and all run in parallel through the iTRAQ analysis. This strategy created an internal control for the analysis since the ratio for any one detected protein when compared to the value for the same protein in the duplicate sample should equal 1 (114-wt/116-wt and 115-Vcan(tm1Zim)/117-Vcan(tm1Zim)). The combined samples were subjected to cationic exchange/reversed-phase HPLC fractionation and mass spectrometry analysis for protein identification and quantification. Protein quantification for all of the iTRAQ peptides found was calculated using a GPS software and the statistical model for iTRAQ proteomic analysis Heart preparations from the Wt (wild type), and Vcan(tm1Zim) mutant and wild-type E13.5 p.c. embryos were obtained by microdissection and proteins extracted as described for iTRAQ analysis. The protein concentration in the urea soluble fraction was determined by the BCS method  using bovine serum albumin as standard. Protein samples (20 µg) from individual hearts were electrophoresed on 4–15% SDS–PAGE gels essentially as we have previously described Hearts of homozygous Vcan(tm1Zim) hearts showed an altered external morphology and the presence of heart defects. External examination of the postnatal Vcan(tm1Zim) hearts showed a dilated right ventricular wall that was most pronounced in the subpulmonary infundibulum adjacent to the right atrium (Vcan(tm1Zim) hearts a more rounded external appearance than in wild-type hearts  hearts achieved normal internal septation. However, AV cushions in all the Vcan(tm1Zim) hearts were smaller than those found in the wild-type hearts (see below) and ventricular septal defects were observed  hearts at E13.5 pc showed significant differences in the size of the central AV endocardial cushions of the heart (Vcan(tm1Zim) hearts as determined by AMIRA 3D reconstruction measurements (Vcan(tm1Zim) and wild type littermates (Vcan(tm1Zim) (Vcan(tm1Zim) compared to the wild-type animals (Vcan(tm1Zim) and wild-type animals (Vcan(tm1Zim) leaflets also appeared smaller than those found in the wild-type littermate controls (Vcan(tm1Zim) hearts . Cushion explants from the AV regions of Vcan(tm1Zim) hearts cultured on 3D hydrated collagen gels (EMT assay) Histological three-dimensional (3D) reconstructions of the he heart . Both thurements . Similartermates . The dortermates . In the (tm1Zim) . Whereas(tm1Zim) . In addi animals  The lume animals . In newbcontrols . The denVcan(tm1Zim) mouse. Results confirmed that, as expected, the GAG-α/exon 7 antibody did not react with the Vcan(tm1Zim) hearts since the polypeptide region containing the epitope recognized by this antibody is missing in the Vcan(tm1Zim) mouse  mouse . Localizm) mouse .Vcan(tm1Zim) hearts (Vcan(tm1Zim) hearts.Immunolocalization of the GAG-β/exon 8 epitope, found in both V0 and V1 versican, was localized throughout all valvular leaflets in both the wild-type and the ) hearts . HoweverVcan(tm1Zim) extracellular matrix the protein periostin was evaluated in embryonic and adult hearts because it is expressed in a close pattern with versican in developing cushions and adult structures Vcan(tm1Zim) and wild-type hearts. Staining for periostin appeared more abundant and fibrillar in the Vcan(tm1Zim) (Vcan(tm1Zim) hearts, periostin was detected in a similar pattern, but with more intensity in the walls of the aorta (Vcan(tm1Zim) hearts   B,D than(tm1Zim)  A,C endo(tm1Zim) . Stainin(tm1Zim) . In the he aorta . Also, tVcan(tm1Zim) hearts (E13.5 pc) showed a lack of cells positive for α-sarcomeric actin within the mesenchyme of the right parietal leaflet and reduced amounts in the central mesenchymalized cushions (Vcan(tm1Zim) become muscularized during the late stages of embryonic development through a process of differentiation or myocardial cell invasion Vcan(tm1Zim) E13.5 hearts, cells positive for of α-sarcomeric actin staining are completely absent within the cushion mesenchyme of the right parietal leaflet (Vcan(tm1Zim) hearts have a diminished level of α-sarcomeric actin staining in the central regions of mesenchyme formed by the spina vestibule and dorsal mesenchymal protrusion  was used. Protein extractions were made of separately pooled E13.5 pc Vcan(tm1Zim) (n = 5) hearts and wild-type (n = 5) hearts. Replicate aliquots of the WT and Vcan(tm1Zim) heart homogenates were labeled with unique isobaric tags for separate analysis. The separate protein fractions were then analyzed by iTRAQ to profile the identities and relative changes in abundance. The replicate aliquots from the same groups allowed for controlled comparisons within each group. Therefore, as an internal control the ratio of protein abundance for each identified protein within the same (Vcan(tm1Zim) or wt) replicate aliquot should yield a value of 1.0. Only those that deviated from a ratio of 1.0 were considered to represent a unique protein fingerprint for Vcan(tm1Zim) hearts compared to wild-type. Identities, relative abundance and statistical analysis of multiple peptides mapping to individual proteins were performed on the data using methods and software previously described To identify some of the resulting differences in cellular protein abundance that results from the loss of the extracellular Vcan(tm1Zim) and WT fractions. A total of 2882 unique peptides were identified, corresponding to 940 proteins. Well over 50% of the proteins identified were represented by two or more unique peptides. Only those proteins with at least two unique peptides are reported. Forty-seven proteins showed significant increases in the mean expression ratio of abundance by more than 1.20-fold in the Vcan(tm1Zim) mouse heart and 74 were decreased by more than 0.85X (Vcan(tm1Zim) hearts was reduced (0.45x) in abundance compared to the wild-type. Other proteins of interest were surveyed in western blots to verify the relative change in abundance detected by iTRAQ. For example, Annexin A6 (4 unique peptides) and Stathmin 1 (9 uniques peptides) together showed a relative decrease (0.85 and 0.73 respectively) in the Vcan(tm1Zim) heart. Also, all 3 peptides mapped to Serpinh1 (Hsp 47) showed an increased abundance (1.55) in the Vcan(tm1Zim). The relative decreases or increase was confirmed by western blots of protein in more than one animal  hearts into standard gene ontology (GO) categories  mice revealed septal defects, changes in the size of some mesenchymalized cushions, dilated right ventricle with dilation of the sub-pulmonary infundibulum and altered integration of the aortic and pulmonary trunks into the AV cushion complex. These combined altered phenotypes result in a failure of the outflow tract to integrate and align correctly with the ventricles during development.Since the initial identification and characterization of versican (tm1Zim) hearts. Closure of the interventricular foramen occurs at approximately E13–14 p.c. in the wild-type mice on the C57B6 background. In the E13.5 p.c. hearts, analyzed by AMIRA 3d reconstruction, forming cushions of the dorsal mesenchymal protrusion, septal and aortic leaflets were significantly smaller and did not merge as in wild-type. This reduction in cushion tissue could contribute to the higher incidence of VSD at E13.5, however many of these defects eventually close as was observed shortly after weaning of the newborn mice . The presence of ventricular septal defects have been described in other mouse models of Vcan partial depletion that include the Crtl-1 null, hdf heterozygous and the Vcan subdomain A null mouse (Δ3/Δ3Vcan) (tm1Zim)Vcan exon mouse heart defects with the Crtl1-null and the hdf heterozygote models show this overlap in structural defects (e.g. VSD) (tm1Zim)Vcan reported here. We also know that hdf heterozygous (tm1Zim)Vcan heart have reduced levels (approximately 50%) of versican. Similarly, the expression of the truncated Vcan gene in Δ3/Δ3Vcan is approximately 55% of the wild type, The hdf heterozygote is able to express all splice forms of Vcan from its unaffected allele, the  Δ3/Δ3Vcan expresses an altered form containing the G3 domain and the (tm1Zim)Vcan can only expresses 2 of the 4 alternative splice form variants. Taken together the observations suggest that the VSDs observed in all 3 models are the result of an overall reduction in versican expression and not specifically due to the splice variants expressed.We observed the presence of VSDs in the VcanThe process by which the outflow tract becomes aligned with the ventricles involves the integration of the proximal outflow tract cushion into the central mesenchymalized cushion of the E13.5 p.c. heart. This positions the aortic and pulmonary arterial trunks in proper alignment between the left and right ventricular canals . The mecVcan(tm1Zim) heart show evidence of less myocardialization. The Vcan(tm1Zim) hearts at E13.5 pc showed reduced numbers of cells positive for α-sarcomeric actin within both these cushions (Vcan splice forms V0/V2. This could be due to 1) a non-permissive matrix environment in the cushion that does not promote or prevents myocardial differentiation and/or invasion of needed precursor cells; 2) inability of the cushion matrix to sustain the myocardial phenotype within the cushion. The loss of Vcan exon 7 containing alternative splice forms V0/V2 may directly result in a “non-permissive” matrix, or indirectly alter other ECM molecules to interfere with invasion and/or myocardial phenotypic stability. We have shown that there are overall fewer cushion mesenchymal cells in the Vcan(tm1Zim) heart. It should be noted that the time points chosen for this study might not be entirely sufficient to fully understand the mechanisms of the valvular defects. Others have shown that a sufficient threshold level of cushion mesenchyme is essential for myocardialization to occur using in vitro assays that model the process Both the right mural and the central AV mesenchymalized cushions in cushions  & 12. Th(tm1Zim) mutant cushions. Vcan induced changes in periostin distribution may effect the developing valves by altering the structural integrity of the ECM through altered interaction with other molecules such as tenascin-C, fibronectin, collagen and other proteoglycans Vcan(tm1Zim) hearts correlated with protein abundance changes identified in the proteomic results. Among these, a protein that showed changes in abundance by the iTRAQ analysis was LIM domain binding 3 isoform b also known as cypher/ZASP. Cypher/ZASP is a member of the family of proteins containing a PDZ domain at their amino terminus and LIM domains at their COOH terminus. Cypher null mice display a severe form of congenital myopathy and die postnatally from functional failure in multiple striated muscles Vcan(tm1Zim) and may be related to the observed lower mesenchyme cell density in the endocardial cushions. The versican V1 (exon 8 containing) splice form still present in the Vcan(tm1Zim) down regulates the expression of vimentin in NIH 3T3 cells Because versican is an extracellular matrix molecule, the cellular responses reflected by the differentially expressed proteins are likely due to the cells interaction with an altered ECM. Periostin is an important molecule of the heart cushion extracellular matrix In conclusion, the defects in cardiac phenotype and alterations in protein abundance reported in this study show that the versican splice forms V1/V2 containing exon 7 are important for regulation of cardiac cushion size and the myocardial remodeling required for the proper integration of the outflow tract into the base of the heart.File S1Supplemental iTRAQ report.(PDF)Click here for additional data file.File S2Supplemental GO file.(PDF)Click here for additional data file.


Our results show that RASSF2, RASSF5C and RASSF10 are aberrantly hypermethylated in MCC to a varying degree and this might contribute to Merkel cell carcinogenesis.Merkel cell carcinoma (MCC) is one of the most aggressive cancers of the skin. RASSFs are a family of tumor suppressors that are frequently inactivated by promoter hypermethylation in various cancers. We studied CpG island promoter hypermethylation in MCC of RASSF2, RASSF5A, RASSF5C and RASSF10 by combined bisulfite restriction analysis (COBRA) in MCC samples and control tissue. We found RASSF2 to be methylated in three out of 43 (7%), RASSF5A in 17 out of 39 , RASSF5C in two out of 26 (8%) and RASSF10 in 19 out of 84 (23%) of the cancer samples. No correlation between the methylation status of the analyzed RASSFs or between RASSF methylation and MCC characteristics (primary  Merkel Cell Carcinoma (MCC) is a rare but aggressive cutaneous malignancy  of the eC-terminally (RASSF1–6) or N-terminally (RASSF7–10). The functions of the C-terminal or classical members range from apoptosis induction, cell cycle inhibition to microtubule stabilization [Tumor suppressor genes (TSG) are commonly inactivated by promoter hypermethylation in cancer. Methylation occurs on the DNA level at 5' position of cytosines, when found as dinucleotides with guanine. CpGs are overrepresented in the promoter region of TSG forming so called CpG islands. DNA methylation in CpG islands of TSG leads to epigenetic silencing of the according transcript . Wellization . The focus of our current work was on RASSF2, RASSF5A, RASSF5C and RASSF10, due to the fact that epigenetic inactivation of these tumor suppressors of the RASSF family was already reported in different cancer types. We and others showed promoter hypermethylation of RASSF2 ,19,20, ROur aim in the present study was the comparative analysis of RASSF promoter hypermethylation in a set of primary Merkel cell carcinoma and controls. Therefore the promoter regions of RASSF2, RASSF5A, RASSF5C and RASSF10 were analyzed by combined bisulfite restriction analysis (COBRA). We show that the degree of promoter hypermethylation in MCC varies between the RASSFs, but is present for different RASSFs at the same time.Taq1 sites at position 111. For RASSF5A COBRA PCR product is 334 bp after semi-nested PCR with Taq1 sites at position 94, 234 and 260. COBRA PCR product of RASSF5C is 322 bp after nested PCR with Taq1 sites at position 221 and 284. The RASSF10 COBRA PCR product is 241 bp (with Taq1 sites at 50 and 141) or with alternative primer pair 167 bp (with Taq1 sites at 67). A Summary of COBRA PCR products, CpG islands, primer positions and Taq1 restriction sites is shown in The promoter regions of RASSF2, RASSF5A, RASSF5C and RASSF10 were analyzed by CpG plot  to show We used retrospectively sampled Merkel cell carcinomas from the tumor registries of the University of Heidelberg, the University of Halle , DermPath Friedrichshafen, and the Institute of Pathology of Halle . 87 samples of 85 tumors  from 79 patients  were studied . Skin coTissue specimens were deparaffinized by xylene and ethanol treatment. DNA was isolated with a QIAamp DNA extraction kit  after a proteinase K restriction and concentrations of DNA were determined by UV-photospectrometery.2O and followed by 10 min incubation with 5 µL 3 M NaOH at 37 °C. DNA was then precipitated with 100% ethanol and 7.5 M ammonium acetate and redissolved in 1 × TE buffer. 200 ng were subsequently used for 25 µL PCR reaction with COBRA primers  1 h at 65 °C and resolved on 2% TBE gel together with mock digest.Genomic DNA from MCC or control tissue (2 µg) was bisulfite treated  and incubated overnight at 50 °C. Then DNA was purified using MSB Spin PCRapace , eluted in 50 µL HThe RASSF family members RASSF2, RASSF5A, RASSF5C and RASSF10 all contain CpG islands in their promoter region, as analyzed by CpG plot and UCSC Genome browser and as shown in CpG island promoter methylation was analyzed for RASSF2, RASSF5A, RASSF5C and RASSF10 in Merkel cell tumor  and skinin vitro methylated DNA (ivm) was used as positive control. Representative samples are shown for each RASSF promoter analyzed. In case of RASSF2 promoter methylation in MCCs no sample shows digestion products as compared to ivm DNA . The according control tissue also showed a high degree of RASSF5A promoter methylation (43%). RASSF5C is methylated to a degree of 8% in 26 of tumor samples and unmethylated in control tissue. The RASSF10 CpG island promoter methylation was studied in 84 Merkel cell tumor samples of which 19 were methylated. Control tissue (14 samples) was unmethylated at the RASSF10 promoter region. p > 0.05). For details of tumors analyzed regarding MCPyV status and promoter methylation results see The aim of this work was to perform a comparative analysis of CpG island promoter hypermethylation of four Ras-association domain family members in Merkel cell carcinoma. MCC represents one of the most aggressive kinds of skin cancer , of whicThe RASSF members analyzed in this study have in common a frequent promoter hypermethylation of their CpG island in cancer. The RASSF2 transcript was detected in normal tissue , but wasThe RASSF members chosen for analysis were shown to harbor different functional properties. The mouse model suggests a role of RASSF2 in bone development , but alsC-terminal and RASSF10 as a member of the N-terminal family contribute to carcinogenesis through independent mechanisms. In our study we showed that the RASSF2 promoter region was hypermethylated in 7% of cancer samples, but unmethylated in control tissue. With a total of 43 analyzed tumor samples RASSF2 seems to be hypermethylated in only a small subset of samples. RASSF5A was found to be frequently hypermethylated in Merkel cell carcinoma (44%), but also in control tissue (43%). Therefore promoter hypermethylation of RASSF5A seems not to be restricted to MCC. However, previous studies showed that RASSF5A can be epigenetically inactivated by promoter hypermethylation in other cancer types . The RASTo date only limited functional data exist for RASSF10. However it was shown that the RASSF10 promoter was hypermethylated in cancers. e.g., malignant melanoma of the skin . The curThe present study is the first comparative analysis of RASSF promoter methylation in Merkel cell carcinoma. In summary we were able to show different RASSFs like RASSF2, RASSF5C and RASSF10 are tumor specifically methylated at their promoter region in MCC, additional to the earlier reported presence of RASSF1A hypermethylation in MCC . Promote


Clinical benefit from CO2 inhalation in patients with craniofacial pain caused by a putative activation of TRPV1 receptor positive trigeminal neurons has also been reported. These effects are probably mediated via an activation of TRPV1 receptor - positive neurons in the nasal mucosa with subsequent central inhibitory effects (such as conditioned pain modulation). In this study, we aimed to examine the effects of intranasal CO2 on a human model of craniofacial pain elicited by nasal application of capsaicin.Nasal insufflation of CO2 or air was insufflated alternatingly into the nasal cavity at a flow rate of 1 l/min for 60 sec each. In the subsequent experiment, all participants were randomized into 2 groups of 24 each and received either continuous nasal insufflation of CO2 or placebo for 18:40 min after nociceptive stimulation with intranasal capsaicin. In both experiments, pain was rated on a numerical rating scale every 60 sec.In a first experiment, 48 healthy volunteers without previous craniofacial pain received intranasal capsaicin to provoke trigeminal pain elicited by activation of TRVP1 positive nociceptive neurons. Then, CO2 on experimental trigeminal pain were only marginal. In the first experiment, CO2 reduced pain ratings only minimally by 5.3% compared to air if given alternatingly with significant results for the main factor GROUP  and the interaction term TIME*GROUP  in the repeated-measures ANOVA. However, these effects were abrogated after continuous insufflation of CO2 or placebo with no significant changes for the main factors or the interaction term.Contrary to previous animal studies, the effects of CO2 could be seen in this human model of TRPV-1 mediated activation of nociceptive trigeminal neurons, utility is limited as observed changes in pain ratings are clinically non-significant.Although mild modulatory effects of low-flow intranasal CO Preliminary data also show efficacy of nasal CO2 insufflation in migraine patients [Phasic nasal insufflation of COn humans  to recorn humans  and chemn humans . However minutes . Likewistentials . These rtentials  upon propatients ,12.2 is a potent vasodilator, the first studies attributed possible therapeutic effects to the vasodilating properties on cerebral vessels. More recently, CO2 has been shown to be a powerful modulator of activated nociceptive trigeminal neurons [2 or air nasally [2 flow rates of 0.8 l/min but not of 0.4 l/min CO2 or air. Based on additional pharmacological experiments the authors concluded that CO2 exerts its antinociceptive, respectively antihyperalgesic effects by activation of mucosal primary trigeminal afferents through a decreased mucosal pH within the nasal cavity.The putative mode of action is uncertain. As CO neurons . Tzabazi nasally . Nocifenin vivo models of migraine [These findings are well in line with the hypothesis that TRPV1 receptor positive trigeminal C and A delta fibres (which are activated by application of capsaicin) may play a relevant role in the pathogenesis of primary headaches such as migraine  althoughmigraine .2 could have some positive effect on acute headache. We therefore aimed to examine the modulatory efficacy of intranasal CO2 on experimental TRPV1-mediated trigeminal pain elicited by intranasal application of capsaicin in healthy volunteers to answer the following questions:In summary, there are some clinical but also pre-clinical data that nasal instillation of CO2 on a numerical rating scale at a flow rate of 1 l/min?1. How painful is prolonged intranasal application of CO2 lead to relevant systemic changes of pH and pCO2 in capillary blood samples?2. Does intranasally applied CO2 modulate pain ratings after intranasal application of capsaicin?3. Does intranasal insufflation of CO2 on TRPV1-mediated trigeminal pain in healthy volunteers. All participants provided written informed consent prior to inclusion into the study. Our study was approved by the local Ethics Committee (protocol number PV3814) and conformed to the Declaration of Helsinki.We conducted a controlled randomized parallel-group study to investigate the effects of intranasal COHealthy volunteers were recruited among medical students at the Medical Faculty of Hamburg University  for 18:40 minutes and rated their pain on a numeric rating scale (NRS) from 0 (no pain) to 10 (worst imaginable pain) every 80 seconds. To assess possible systemic changes in pH and CO2 levels, pH and pCO2 were additionally determined in 10 patients by a capillary blood gas analyses. This was taken from the earlobe before nasal installation of CO2 started and immediately after the last pain rating while the participants were still exposed to CO2.In a pilot study Figure  designed2 (1 l/min) and air (1 l/min) alternatingly. If volunteers had already participated in the pilot study, both experimental sessions were separated by at least 6 weeks. Again, subjects rated the magnitude of pain verbally on a numerical rating scale (NRS) ranging from 0 (non-painful) to 10 (worst imaginable pain).In the main study Figure , 48 heal2 and air were insufflated for 60 sec each in an alternating fashion via a nasal cannula. During an interval of 20 sec between exposure to either CO2 or air, no gas was applied and the participant was asked to rate the resulting pain on a NRS. After 4 cycles, insufflation was interrupted for 1 min to allow participants to remove excessive nasal discharge. However, subjects were asked not to blow their noses to avoid early removal of the intranasal capsaicin. Thereafter, 4 identical cycles of CO2, respectively air were given and subjects were asked to rate the pain on the NRS.Initially, participants received one puff of capsaicin spray  into the left nostril to trigger TRPV1-mediated trigeminal nociception. After three initial pain ratings on a NRS every 60 seconds  or placebo in 2 subgroups of 24 subjects each.In the subsequent second experiment separated from the first experiment by at least 4 weeks all participants from the first experiment were randomly allocated to receive either CO2 for a total of 18:40 min via a nasal cannula or placebo  and rated the pain on the NRS as in the pilot study.Again, participants received 1 puff of capsaicin spray into the left nostril. Pain ratings on a NRS were noted every 60 sec over the entire duration of this experiment  was administered from compressed gas cylinders with a volume of 10 l. By using a combined pressure reducing valve (200 mbar/4.5 mbar) and flowmeter  a constant flow of 1 l/min was maintained. Medical grade compressed air was obtained from the hospital gas reticulation system and delivered at a flow of 1 l/min by a flow meter . Nasal cannulae  were used to apply both gases locally into both nasal cavities.Medical grade CO2 should only be insufflated nasally and not inhaled due to safety concerns, all subjects were trained before the first experiment. CO2 was applied bilaterally into the anterior nasal cavity, where highest mucosal responsiveness of evoked potentials to short pulses of CO2 could be found [As CObe found , namely One puff of a nasal spray containing 200 μg of capsaicin (prepared from 1.42 ml capsaicin liquid extract in 10 ml of refined sesame oil) was applied to the left nostril with the head in a stooped position to avoid contamination of the pharynx. Participants were allowed to remove excessive nasal discharge by soaking cotton swaps, but were asked not to blow their noses to avoid early removal of intranasal capsaicin.2 males: 35-48, females: 32-45.Blood gas analyses were collected in from the earlobe in capillary tubes after pretreatment with an ointment containing 5% benzyl nicotinate  for 10 mins. Immediately afterwards samples were tested with a blood gas analyzer . The manufacturer’s reference values for capillary blood gas analysis were as follows: pH 7.35- 7.45; pCO2 or air). Comparison of categorical data (gender) was done by means of the chi square test. In all tests p values <0.05 were considered significant.Descriptive statistics are given as mean values and standard errors of the mean. Differences on mean values were either examined by paired t-tests or by means of a repeated measures analysis of variance (ANOVA). For analysis of pain ratings over time in the pilot study, the factor TIME (pain ratings 1-15) was used, for other comparisons of pain ratings a two-way repeated measures ANOVA with the factors TIME (corresponding pain ratings) and GROUP .2 was given exclusively to 20 healthy subjects , mean pain ratings over time were 0.6 (± 0.06) out of 10 on the NRS. Pain ratings reached their maximum after 1 min of inhalation with 0.8 (±0.21) out of 10 on the NRS . Seven (35%) of the participants indicated that they had not perceived any pain at all.When CO2 nasally or started talking during the training phase, they mostly complained of a highly unpleasant irritation spreading from the nasal cavity into the nasal sinus and the pharynx which led to interruption of the training.If subjects accidentally inhaled CO2 levels (difference between pCO2 after – pCO2 before insufflation) did not correlate with corresponding pain ratings in the pilot study (Pearson’s bivariate correlation: p > 0.05).Net changes of pCO2 (3.6 ± 0.3) and air (3.8 ± 0.3) respectively differed significantly (t(47) = -2.107; p = 0.041). Likewise, a repeated-measures ANOVA for the factors TIME (pain ratings for insufflation 1 to 4) and GROUP (CO2 or air) yielded no significant main effect for TIME , but significant results for both the main factor GROUP  and the interaction term TIME*GROUP . Mean values are given in Figure For epidemiological details see Table 2 .6 ± 0.3 2 or air in experiment 2 (for further details see Table Age and gender distribution did not differ significantly between the randomly assigned subgroups receiving either CO2 or air) yielded a significant main effect for TIME , but not for the factor GROUP  = 0.089; p = 0.767) or the interaction term TIME*GROUP .A repeated-measures ANOVA with the factors TIME (pain ratings 1 to 17) and GROUP  = 0.15; p = 0.902).There was no baseline difference defined as comparison of the first pain rating between the CO2. Most notably, no patient complained about dyspnea or other clinical side effects. After 20 min of CO2 insufflation, a significant shift of capillary pH and capillary pCO2 readings could be found. pH decreased and pCO2 levels increased significantly , alternating application of CO2 reduced pain ratings only minimally compared to a sham paradigm with air, but this effect was abrogated during continuous insufflation of CO2.Contrary to previous animal studies, the effects of intranasally insufflated COCapsaicin (8-Methyl-N-vanillyl-trans-6-nonenamide) increases release of substance P and simultaneously blocks its reuptake exerting its effects by activation of the TRPV1 receptor . This lein vitro experiments with slices of rat trigeminal nucleus caudalis [It has been shown in caudalis  that actcaudalis ,28. A recaudalis ,30. Accocaudalis . In contcaudalis .Regardless of whether TRPV1 receptor positive V1 neurons are indeed involved in migraine pathophysiology or not, they have been associated with other craniofacial pain syndromes such as dental pain where an upregulation of TRPV1 expression in rat trigeminal ganglia was observed in a model of lipopolysaccharide-induced pulpitis . We note2 insufflation was rated as mildly painful by 65% of patients. We found systemic changes in capillary pH and pCO2 levels but no relevant alterations beyond the normative range and conclude that nasal application at 1 l/min is safe. Insufficient delivery of CO2 into the nasal cavity seems unlikely as changes of pCO2 levels after nasal insufflation did not correlate with pain ratings in our pilot study.CO2 induced unpleasantness respectively pain with vaporized ammonia to establish a robust sham condition were in vain (data not shown). Ammonia induced a more stabbing and - at higher concentrations - unbearable pain in the nasal cavity with variable pain thresholds. The unpleasantness of intranasal CO2 in our sample implies that true blinding is not feasible at flow rates of 1 l/min and above as most patients will notice an unpleasant or painful perception. As clinical effects were negligible in our sample, potential sources of bias such as a relevant placebo effect are unlikely.Our attempts to mimic COintranasal flow rates of 0.6 l/min, blinding seemed to be less problematic in prior studies. Casale et al. [insufflation of CO2 at 0.6 l/min for only 1 minute – similar to the setup used in the therapeutic studies in migraine patients by Spierings [Inhalation of CO2 was less well tolerated raising doubts about effective blinding in prior studies reporting efficacy of inhaled CO2.At lower e et al.  reportedpierings ,12. Inha2 in our study was small and reached statistical significance only when CO2 and placebo were given in an alternating fashion within the same subject (experiment 1) – as opposed to application of either CO2 or air only in experiment 2. It may be easier for the participants to sense a subtle analgesic or anti-hyperalgesic efficacy of CO2 if it is given in a contrasting fashion with air as placebo.The antihyperalgesic efficacy of CO2 with flow rates of 0.8 l/min attenuated nocifensive behaviour after sensitization with capsaicin unlike lower flow rates of CO2 (0.4 l/min) or air. In the nasal mucosa CO2 decomposes into protons and carbonate catalyzed by mucosal carbonic anhydrase and activates TRPV1- and ASIC- positive neurons by proton accumulation. Subsequent, central inhibitory effects are proposed such as a widespread inhibition of afferent trigeminal input though inhibitory interneurons, trans-segmental inhibitory control circuits or conditioned pain modulation.As shown by Tzabazis and colleagues in a rat model , nasal i2 or capsaicin resulted in an acidification of culture medium and a consecutive nociceptive activation with CGRP release [2 attenuated CGRP release by pretreatment with capsaicin if cultured under isohydric conditions which prevents extracellular but allows intracellular acidification.Vause and colleagues showed that incubation of cultured trigeminal ganglions with CO release . Similar2 insufflation on non-sensitized skin as compared to air insufflation [2, so that efficacy could have been better in patients with chronic craniofacial pain. Alternatively, CO2 inhalation with a potentially different locus of action could represent a more powerful alternative although tolerability seems to limit feasibility [Tzabazis and co-workers observed less intense and only short-lived antinociceptive or antihyperalgesic effects of COfflation . These fsibility ,10.2 exerts antihyperalgesic effects in animal models but resulted in only minor clinical effects in our human model of trigeminal pain elicited by activation of nociceptive TRPV1 receptors in healthy volunteers. These moderate effects question the clinical utility of intranasal CO2 in TRPV1-mediated pain at flow rates of 1 l/min.In summary, intranasal insufflation of CO2 in either air or oxygen for 5 min [2 mixed with oxygen for 10 min daily which was repeated up to 2 times if the headache was not relieved [2 suggesting a potentially different mode of action. In addition, translating these clinical results to our human model is difficult as the role of the TRPV1 receptor in migraine pathophysiology has been challenged [2 provoked a headache attack or increased headache intensity in 18 of 40 subjects with mainly posttraumatic headache and migraine. CO2-induced headache attacks or aggravation were less intense than headaches triggered by histamine and adrenaline [2 for 6 min [Marcussen and Wolff successfully treated aura symptoms in migraine patients termed as “vasoconstrictor symptoms” by inhalation of 10% COor 5 min . Likewisrelieved . After 3allenged . Furtherrenaline . Likewisor 6 min .2 in primary and secondary headaches is difficult despite promising data from animal experiments. Trials on the efficacy of inhaled CO2 in various headache syndromes are relatively old and yielded ambiguous results. Evidence for the efficacy of intranasal CO2 in migraine has been published in preliminary form.At present, a firm conclusion on the clinical efficacy of CO2 at flow rates of 1 l/min could be seen in a human model of TRPV1 mediated activation of nociceptive trigeminal neurons which is in line with previous studies. While application was safe, clinical utility at low flow rates was limited in our model as the therapy is uncomfortable and changes in pain ratings are therapeutically irrelevant.Only mild modulatory effects of intranasal insufflation of COhttp://www.linde-healthcare-realfund.com).TPJ and RR: no relevant conflicts of interest, AM: has received unrestricted scientific grant support from Linde Gas (RealFund; TPJ conceived of the study, participated in the conception and design, performed the statistical analysis, interpreted and discussed the data and drafted the manuscript. RR participated in the design of the study and data acquisition and helped in the interpretation of it. AM participated in the conception and design of the study, interpreted and discussed the data and drafted the manuscript. All authors read and approved the final manuscript.


Short accessory pathway (AP) effective refractory period (ERP) is one of the risk factors in Wolff-Parkinson-White syndrome (WPW). The purpose of study was to evaluate the reproducibility of APERP measurement during a same electrophysiological study (EPS).EPS consisted of 2 APERP measurements performed prospectively in 77 patients for a WPW in control state (CS) at a cycle length of 400 ms (n=76) and after isoproterenol (n=56).In CS, 18 patients (24 %) had the same APERP at both measurements; 41 (54.6 %) had differences from 10 to 40 ms, 17 (22.4 %) had differences > 40 ms. Among 45 patients with initial APERP > 240 ms, 7 had an APERP ≤ 240 ms at 2nd study. Among 31 patients with initial APERP ≤ 240 ms, 5 had an APERP > 240 ms at 2nd study. Pearson's product-moment correlation was 0.75. After isoproterenol, 5 patients (9 %) had the same APERPs; 37 (66 %) had differences from 10 to 40 ms and 14 had differences > 40 ms. Among 38 patients with initial APERP > 200 ms, 12 had an AP ERP ≤ 200 ms at 2nd study. Among 18 patients with initial APERP ≤ 200 ms, 10 had still APERP ≤ 200 ms at 2nd study. Pearson's product-moment correlation was 0.54.There are important variations of APERPs during EPS mainly after isoproterenol infusion. Therefore the values of APERPs should be interpreted cautiously. The prevalence of a typical Wolff-Parkinson-White (WPW) pattern is estimated to be 0.1% . A recenOther factors are discussed such as the presence of multiple accessory pathways and the male gender. Therefore, the measurement of the AP ERP during an electrophysiological study is important. Actually AP ablation is largely indicated and a short APERP might be sufficient to indicate AP ablation. The aim of this study is to evaluate the reproducibility of the measurement of AP ERP during the same electrophysiological study.Seventy seven patients, 56 males (73 %) and 21 females (27 %) aged from 8 to 57 years (mean age 31 ± 12 years), with a ventricular preexcitation syndrome were consecutively included in this study; 46 patients were asymptomatic (60 %), 20 patients had palpitations (27 %), 7 patients had history of unexplained syncope (7 %), 2 had spontaneous documented atrial fibrillation (3 %). Only 2 patients (3 %) had a history of life-threatening arrhythmic events .Accessory pathway was septal located in 51 patients . Three patients had an anteroseptal location (4 %); 19 patients had a left lateral accessory pathway location (25 %) and 4 a right lateral location (5 %).This was a prospective study performed between 2004 and 2010 after informed consent. An electrophysiological study was performed in patients not sedated and after cessation of treatment. Patients were studied by transesophageal route and/or intracardiac route . The claThe disappearance of the pattern of preexcitation indicated when the APERP was reached. The longest A1A2 that fails to conduct at the atria was considered as the effective APERP. This protocol was reproduced again after several minutes to study the reproducibility of the measure of anterograde effective refractory period.The method was used to induce supraventricular tachycardia. The protocol was performed during 80 consecutive electrophysiological studies. Three patients were excluded from this protocol because sustained atrial fibrillation was induced at first programmed atrial stimulation and flecainide injection was required to stop it. In one patient the AP did not conduct in control state and the measurement of APERP was made only after isoproterenol.In the absence of induction of a tachycardia conducted through the accessory pathway at a rate higher than 250 bpm, isoproterenol (0.02 to 10 μg.min-1) was then infused to increase the sinus rate to at least 130 bpm and the pacing protocol was repeated. APERP was determined twice at a basic cycle length of 400 ms. Isoproterenol was infused in only 56 patients: 9 patients did not require isoproterenol infusion because they had an electrophysiological malignant form in control state with the induction of an atrial fibrillation and short cycle lengths conducted by AP. Two other patients remained in atrial fibrillation without signs of malignancy after second programmed stimulation in control state. Poorly-tolerated orthodromic tachycardia was induced in 5 symptomatic patients during the basal study and catheter ablation was indicated without evaluation after isoproterenol. In 6 children, isoproterenol infusion was poorly-tolerated and programmed atrial stimulation was not repeated.Conduction over the accessory atrioventricular connection was evaluated by the measurement of the maximal heart rate with a 1 to 1 conduction over the accessory connection and the shortest atrial tachycardia cycle length at which there was 1 to 1 conduction over the accessory connection. A short APERP was defined in the present study as less than or equal to 240 ms in control state (CS) and less than or equal to 200 ms after isoproterenol.Results were presented as mean and standard deviation and compared with the paired Student t test. A value of p < 0.05 was considered to be significant. Correlations were performed between 2 determinations of AP ERP and agreement was expressed according to the Pearson's correlation coefficient. It was obtained by dividing the covariance of the two variables by the product of their standard deviations.The mean fastest cardiac rate conducted by the accessory pathway was 214±55 bpm in control state and the ranges were 100 and 300 bpm. The mean sinus cycle length did no differ significantly during both measurements (805±12 vs 819±11msec). The dispersion of the two measurements of APERP is represented on a graphic in Figure 2. The mean value of APERP at the first study was 264±52 ms. Mean value of the second measure was 265±50 ms (NS). However, there were important individual changes of APERP values between both studies.At the first determination, APERP was more than 240 ms (mean 299± 37) in 45 patients (59 %); mean value was the same in the second study (299± 37 ms); APERP became less than or equal to 240 ms at second measurement in 7 patients (15.5 %) and remained more than 240 ms in 38 patients (84.5 %). At the first APERP determination, 31 patients (41 %) had an APERP less than or equal to 240 ms: 24 patients had an APERP less than or equal to 240 ms at both studies (77 %); 7 patients had an APERP more than 240 ms at second study (22.5 %). The mean absolute difference between the two trials was 25.7 ms (±25.2 ms). The variations are reported in Twenty-eight measurements (37%) were shorter in the second trial than in the first with a mean difference of 34±24 ms. Thirty measurements (40 %) were shorter in the first trial than in the second, with a mean difference of 34±23 ms. The Pearson's product - moment correlation was 0.75. The mean fastest cardiac rate conducted by the accessory pathway was 265±18 bpm after isoproterenol infusion. The mean cycle length between 2 measurements does not differ significantly (475±12 mesc vs 479±14 ms). The dispersion of the two measurements with isoproterenol infusion was represented on a graphic in The mean value of the first measurement was 223±40 ms. Mean value of the second measurement was 224±48 ms (NS). Individual data differed. At the first APERP determination, 18 APERPs (32%) were less than or equal to 200 ms; 10 measures (56%) were less than or equal to 200 ms and reproducible at 2 measurements; 8 APERPs were more than 200 ms at the second determination (46%). The mean absolute difference between two trials was 34±25.4 ms. Among 38 patients with initial APERP more than 200 ms, 12 measures were less than or equal to 200 ms at the second determination (33%). The individual variations are reported in For 30 patients (53.4 %) the APERP was shorter at the second measurement than at the first, with a mean difference of 30±18 ms. For 21 patients (37.5 %) the first measure of APERP was the shortest with a mean difference of 47.6 ± 28.4ms. The Pearson's product - moment correlation was 0.54.Fourteen patients underwent transesophageal study and later intracardiac study to perform the accessory pathway ablation. The initial mean values of APERP measured at esophageal study were significantly shorter than the values measured at intracardiac study, but other measurements did not differ significantly. The mean variations were similar either in control state (31±29 ms and 30±27 ms) or after isoproterenol (26±16 and 24±15 ms) .The mean age of our population was 31±12 years. In the youngest population (30 years old or less), representing 36 patients, the Pearson correlation coefficient was 0.675 in control state and 0.58 after isoproterenol infusion. In the oldest population (more than 30 years old), representing 36 patients, the Pearson correlation coefficient was 0.78 in control state and 0.46 after isoproterenol infusion. The subgroups according to the age of the patient were too small to verify possible relationships between the age and the variability of APERP.There were no significant differences of APERP according to the location of AP: AP was left-sided in 19 patients and the variations were 27±25 ms in control state and 25±13.5 ms after isoproterenol. AP was septal in 54 patients and the variations were 24±25.5 ms in control state and 35±27 ms after isoproterenol. AP was right lateral side in only 4 patients and variations (42.5±22 ms in control state and 35±21 ms after isoproterenol) can not be interpreted.Among the 2 patients who have presented life-threatening arrhythmic events and 2 other patients with syncope and malignant form of preexcitation syndrome at electrophysiological study; all had a maximal heart rate with a 1 to 1 conduction over the accessory pathway was more than 240 bpm in control state during induced sustained atrial fibrillation. AF stopped spontaneously and the protocol was performed in these patients. Two patients had an APERP of 250 and 260 ms at the first measurement and a short value (210 and 230 ms) at the second measurement. Other patients had a short value (190 ms) at both measurements.We reported important variations of APERPs during electrophysiological study mainly after isoproterenol infusion. This may mean that ERP measurement with isoproterenol cannot be relied upon for assessing risk. The variations could be partially explained by the various conditions of the autonomic nervous system. The vagal and sympathetic activities constantly interact, and modulate the electrophysiological behaviour of the heart under different conditions such as postural changes, effort, and psychological activation ,13. A caFenici and al  studied As showed by Castellanos et al , if the The classical values of 240 ms in controls state and 200 ms after isoproterenol as cut-off for the evaluation of the arrhythmic risk should be discussed and the values of APERP's interpreted carefully in association with other data of electrophysiological study. Male gender, young age, sport, septal accessory pathway (AP), multiple accessory pathways, short AP refractory period, atrial fibrillation (AF) were reported as risk factors of sudden death in WPW syndrome ,18,19. PSome of our studies were performed by transesophageal route. However 14 of these patients had later an intracardiac study and the variations of APERPs were similar. Nanthakumar et al  have comThree patients were excluded after induction of atrial fibrillation at the first measurement, requiring flecainide to stop it. We can not prove the role of autonomic nervous system because no changes in sinus cycle length were found in the present study. We have not studied the reproducibility of the ventricular rate during induced atrial fibrillation, because AF was not always inducible or required flecainide to stop it.Only 3 patients had a spontaneous malignant form. More, the ages of our population are heterogeneous and a difference between younger and older patients can not be excluded. The data are only applicable to non sedated patients. Accessory pathways were mainly septal located probably because the majority of our patients were asymptomatic. Similar data were noted by our group  in a larIn conclusion this prospective study reports important variations of APERPs during electrophysiological study mainly after isoproterenol infusion. The study highlights the limitation of measurement of ERP as a predictor of the future risk. The APERP in patients with WPW should be interpreted with carefulness in association with other data of electrophysiological study. We suggest repeating the measurement in patients complaining of palpitations or syncope to avoid missing short values of APERP.


In this paper, we evaluate the ability of G4 (flexible) and G5 (rigid) dendrimers to complex model siRNA molecules at low +/− ratio of 2/1 upon incubation for 20 minutes and 24 hours. We examine the ability of the formed G4 and G5 dendriplexes to shield the loaded siRNA molecules and protect them from degradation by RNase V1 enzymes using atomic force microscopy (AFM). Results show that G4 and G5 dendrimers form similar hexagonal complexes upon incubation with siRNA molecules for 20 minutes with average full width of 43±19.3 nm and 62±8.3 at half the maximum height, respectively. AFM images show that these G4 and G5 dendriplexes were attacked by RNase V1 enzyme leading to degradation of the exposed RNA molecules that increased with the increase in incubation time. In comparison, incubating G4 and G5 dendrimers with siRNA for 24 hours led to the formation of large particles with average full width of 263±60 nm and 48.3±2.5 nm at half the maximum height, respectively. Both G4 and G5 dendriplexes had a dense central core that proved to shield the loaded RNA molecules from enzymatic attack for up to 60 minutes. These results show the feasibility of formulating G4 and G5 dendriplexes at a low N/P (+/−) ratio that can resist degradation by RNase enzymes, which reduces the risk of inducing non-specific toxicity when used in vivo.Cationic polymers such as poly(amidoamine), PAMAM, dendrimers have been used to electrostatically complex siRNA molecules forming dendriplexes for enhancing the cytoplasmic delivery of the encapsulated cargo. However, excess PAMAM dendrimers is typically used to protect the loaded siRNA against enzymatic attack, which results in systemic toxicity that hinders the  Many cationic peptides, lipids, and polymers have been used to condense siRNA via electrostatic interaction forming ionic complexes with variable size and surface charge, which proved effective in delivering the RNA cargo into the cytoplasm of mammalian cells in vitroin vivo delivery of siRNA required the use of excess cationic carrier to shield and protect the RNA cargo against nucleases leading to non-specific distribution of the formed complexes to the reticular endothelial system  Preclinical investigations showed the potential of small interfering RNA (siRNA) molecules in selectively silencing the expression of the genes implicated in the development of cancer, cardiovascular, neurodegenerative, and infectious diseases indicating their therapeutic potential Poly(amidoamine), PAMAM, dendrimers are a family of water-soluble polymers that is characterized by a unique, highly-ordered, three dimensional, tree-like branching architecture with a large number of primary, secondary, and tertiary amine groups embedded in their structure, which become ionized at physiologic pH conferring a high positive charge density Earlier studies showed that DNA condensation has two kinetic phases starting with an initial rapid binding (within 15 seconds) of DNA to multivalent cations followed by slower structural rearrangement that reaches an apparent equilibrium typically within 1–2 hours and exhibit insignificant changes at longer incubation times It is important to note that pDNA molecules exist in solution as long flexible chains with an average length of ∼1.2 µm, which allow them to wrap around cationic carriers forming compact particles that resist degradation by DNase enzymes and achieve high transfection In this article, we describe the complexation of anti-GAPDH siRNA molecules with G4 (flexible) and G5 (rigid) dendrimers based on the size and morphology of the formed dendriplexes at different incubation times (20 minutes and 24 hours). We also investigate the stability of the formed dendriplexes upon incubation with RNase V1 enzymes compared to naked siRNA molecules as a function of exposure time. We used atomic force microscopy (AFM) to visualize the morphology of the formed complexes and monitor the attack of RNase V1 enzymes in solution as a function of time. We relied on established AFM protocols used to study the dynamics of DNA binding to PAMAM dendrimers G4  and G5  with ethylene diamine cores were purchased from Dendritic Nanotechnologies, Inc.  as 10% w/v solutions in methanol. G4 and G5 solutions were dialyzed using Slide-A-Lyzer dialysis cassettes with a 7 kDa MWCO  against DI water for 24 hours to remove polymer debris. The aqueous solutions of G4 and G5 were lyophilized and stored at 4°C till used. Anti-GAPDH siRNA and RNase V1 enzyme were purchased from Ambion Inc. .G4 and G5 dendrimers were dissolved in RNase-free water and mixed with 0.7 µg of anti-GAPDH siRNA molecules dissolved in 1 µl of RNase-free water at a nitrogen/phosphate  ratio of 2/1. Each mixture was vortexed and allowed to stand at room temperature for 20 minutes or 24 hours before loading onto a 1% w/v agarose gel containing ethidium bromide (EtBr). The gel was immersed in a Tris-acetate-EDTA (TAE) buffer and run at 60 V for 1 hour and visualized under UV exposure .2 where MgCl2 ions would allow weak adsorption of anionic siRNA molecules to the negatively charged mica surface through electrostatic interaction following established protocols All AFM images were acquired using Nanoscope III MultiMode AFM with a sharp nitride lever  in the tapping mode at a 256×256 pixel resolution. Selected fields were scanned at scan rates ranging from 3–4 Hz where each image was acquired within 90 seconds and tapping frequencies ranged from 8–10.5 kHz in solution. Images were flattened to account for Z offsets and sample tilts. Tapping set points were selected close to the free oscillation amplitude to reduce forces exerted on the interfacial species. All imaging experiments started with scanning the mica substrate in DI water to confirm the absence of any adsorbed contaminants. Naked anti-GAPDH siRNA was imaged using 30 µl of siRNA solution (3.92 µg/ml) in 1 mM PBS containing 2 mM MgClBoth G4 and G5 dendrimers successfully complexed the loaded anti-GAPDH siRNA molecules (0.7 µg) at an N/P (+/−) ratio of 2/1 via the electrostatic interaction between the cationic amine groups (N) and the anionic phosphate groups (P). The gel image shows that siRNA molecules were retained in the wells after mixing with G4 and G5 dendrimers for 20 minutes and 24 hours indicating rapid condensation of the loaded siRNA molecules by the cationic carriers Figure 1+2 ions and multiple anionic siRNA molecules t = 0 minutes) (Atomic Force Microscopy (AFM) is a powerful surface analytical technique that is routinely used to image and characterizes objects in the sub nanometer scale Incubation of G4 dendrimers with anti-GAPDH at an N/P ratio of 2/1 for 20 minutes yielded dendriplexes that were visualized using AFM Figure 3In comparison, G4 dendriplexes prepared by mixing of G4 dendrimers with anti-GAPDH siRNA at an N/P ratio of 2/1 for 24 hours appeared as large globular particles with a dense central core surrounded by “loose” network Figure 4Similarly, incubation of G5 dendrimers with anti-GAPDH at an N/P ratio of 2/1 for 20 minutes yielded compact hexagonal dendriplexes that formed a densely packed monolayers on the surface of freshly cleaved mica with an average full width of 62±8.3 nm at half the maximum height and an average height of 1.1±0.05 nm (+2 cations to allow weak electrostatic adsorption of free siRNA and G4/G5 particles to mica's surface following previously published protocols Particles prepared by ionic complexation of PAMAM dendrimers with siRNA molecules were examined using AFM under air-dry conditions &&&&AFM images show that despite the difference in size, number of cationic amine groups, and flexibility of G4 and G5 dendrimers, they formed similar hexagonal particles when incubated for 20 minutes with anti-GAPDH siRNA &5A. IncAFM images show that increasing the incubation time of G4 and G5 dendrimers with siRNA molecules to 24 hours results in formation of ionic complexes that can protect the loaded RNA cargo against enzymatic degradation without using excess cationic dendrimers. Further, the size of the formed complexes can be tuned by controlling the flexibility of the cationic carrier with flexible G4 forming larger particles than the more rigid G5 dendrimers while maintaining the desired enzymatic stability. These findings provide insight on potential formulation strategies to develop enzymatically-resistant complexes with tunable size for enhanced intracellular delivery of therapeutic siRNA molecules without inducing undesirable side effects due to the use of excess cationic carrier.


LAMTOR2 and LAMTOR3 as genetic risk factors for breast cancer.The late endosomal LAMTOR complex serves as a convergence point for both the RAF/MEK/ERK and the PI3K/AKT/mTOR pathways. Interestingly, both of these signalling cascades play a significant role in the aetiology of breast cancer. Our aim was to address the possible role of genetic polymorphisms in LAMTOR2 (p14) and LAMTOR3 (MP1) in 50 prospectively collected pairs of cancerous tissue and blood samples from breast cancer patients and compared their genetic variability. We found one single nucleotide polymorphism (SNP) in LAMTOR2 (rs7541) and two SNPs in LAMTOR3 (rs2298735 and rs148972953) in both tumour and blood samples, but no somatic mutations in cancerous tissues. In addition, we genotyped all three SNPs in 296 samples from the Risk Prediction of Breast Cancer Metastasis Study and found evidence of a genetic association between rs148972953 and oestrogen (ER) and progesterone receptor negative status (PR) (ER: OR = 3.60 (1.15–11.28); PR: OR = 4.27 (1.43–12.72)). However, when we additionally genotyped rs148972953 in the MARIE study including 2,715 breast cancer cases and 5,216 controls, we observed neither a difference in genotype frequencies between patients and controls nor was the SNP associated with ER or PR. Finally, all three SNPs were equally frequent in breast cancer samples and female participants (n = 640) of the population-based SAPHIR Study.We sequenced the exons and exon–intron boundaries of LAMTOR2 and LAMTOR3 do not seem to play a relevant role in breast cancer. Our work does not exclude a role of other not yet identified SNPs or that the here annotated polymorphism may in fact play a relevant role in other diseases. Our results underscore the importance of replication in association studies.The identified polymorphisms in  LAMTOR3 (MP1) was identified in a yeast two-hybrid screen as a specific binding partner of MEK1 Anchorage of the p14/MP1/MAP kinase pathway to late endosomes is mediated by a small lipid raft adaptor called LAMTOR1 (p18), which directly binds endosomal lipids Progression, proliferation, and hormone independent growth of breast cancer cells is dependent on MAP kinase (ERK) activity The PI3K/AKT signalling cascade is another major player in cancer progression. Mutations in the PI3K subunit p110 are found in roughly 25% of breast cancers It has been recently shown that treatment of breast cancer cells with MEK or mTOR inhibitors and either Doxorubicin or Tamoxifen results in a synergistic response that highlights the advantages of combining classical chemotherapy with targeted adjuvant treatments Interestingly, in 2007, Conrad et al. submitted a patent on LAMTOR3 (Mp1) as a diagnostic and therapeutic target for breast cancer treatment and prevention . In addition, a recent publication from the same group reports that LAMTOR3 (Mp1) is required for the survival of estrogen receptor positive breast cancer cell lines LAMTOR3 and LAMTOR2 in the aetiology of breast cancer.Taking into consideration the above report and recent findings identifying the LAMTOR complex as a convergence point for both the ERK and mTORC1 pathways, we aimed to investigate the potential role of mutations in This study was approved by the ethics committee of the Innsbruck Medical University (study code UN3377).For mutation screening, tissue samples of 50 consecutive breast cancer patients were prospectively collected at the Innsbruck Medical University starting in July 2009. Patients aged 18 or older, who had signed an informed consent, were consecutively included in the study. The following clinical parameters were collected: age; menopausal status; tumour histology; tumour size; tumour grade; lymph node status; oestrogen receptor status; progesterone receptor status; HER2  status; and presence of metastasis.Genomic DNA was extracted from frozen tumour tissue or from peripheral blood collected on EDTA on a BioRobot EZ1 advanced Workstation with the EZ1 DNA tissue or blood kit  and quantified with a NanoDrop spectrophotometer . Amplification and sequencing primes were designed with Visual OMP .LAMTOR2 gene  were amplified in 2 PCR reactions and sequenced with 8 primers  were amplified in 4 PCR reactions and sequenced with 14 primers , 5 µl of PCR reaction buffer, 250 µM each dNTP, and 0.25 µM each primer. The reaction cocktails were heated to 95°C (2 min) and then put through 35 amplification cycles: 95°C for 20 s, 55°C for 20 s, and 72°C for 60 sec and a final extension phase at 72°C for 10 min. Fragment MP1-1 was amplified in a total reaction volume of 25 µl, containing 60 ng of DNA, 0.5 µl of KAPA HiFi HotStart DNA Polymerase , 5 µl of 5X KAPA HiFi GC Buffer, 0.5 µl KAPA dNTP Mix (10 mM each dNTP), and 0.3 µM each primer. The reaction cocktails were heated to 95°C (5 min) and then put through 30 amplification cycles: 98°C for 20 s, 56.7°C for 15 s, and 72°C for 45 sec and a final extension phase at 72°C for 5 min.PCR products were purified using QIAvac vacuum manifolds (QIAGEN) and eluted in 50µl distilled water according to the manufacturer’s protocol. For cycle sequencing, 3 µl of purified PCR product were combined with the sequencing master mix  and cycled  for 30 cycles of 30 s at 96°C, 20 s at 55°C, and 1 min at 60°C. Purification of cycle-sequencing products with Sephadex  was performed according to the procedure described in Brandstätter et al. xl capillary sequencer with POP-7 and a 36 cm capillary array.Electrophoretic separation was carried out on an ABI3130LAMTOR2 (rs7541) and two SNPs in LAMTOR3 (rs2298735 and rs148972953) were genotyped using a TaqMan® SNP Genotyping Assay on a 7900HT Fast Real-Time PCR System  in 296 samples of the Risk Prediction of Breast Cancer Metastasis Study. This study is a multicenter study including prospectively collected breast cancer samples from the cities Innsbruck (Austria), Salzburg (Austria) and Meran . Patients aged 18 or older, who had signed an informed consent, were consecutively included in the study. Detailed information on tumour characteristics  and treatment  were collected using clinical and pathological records. The Risk Prediction of Breast Cancer Metastasis Study was approved by the ethics committee of the Innsbruck Medical University (study code AM330a). The genotyping success rates were 97.3% for rs7541, 99.3% for rs2298735, and 97.3% for rs148972953.For studying the effects of the identified variants in a breast cancer sample, one single nucleotide polymorphism (SNP) in For a more detailed analysis, the SNP rs148972953 was genotyped using a TaqMan® SNP Genotyping Assay on a 7900HT Fast Real-Time PCR System  in 7,931 samples of the MARIE Study. The MARIE (Mammary carcinoma Risk factor Investigation) study population comprises breast cancer patients who participated in a population-based case-control study conducted in two German study regions (Hamburg and Rhine-Neckar-Karlsruhe) LAMTOR2 (rs7541) and two SNPs in LAMTOR3 (rs2298735 and rs148972953) were genotyped using a TaqMan® SNP Genotyping Assay on a 7900HT Fast Real-Time PCR System  in female participants of the SAPHIR Study. The Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR) is an observational study conducted in the years 1999–2002 involving 1,770 healthy unrelated subjects: 663 females from 39 to 67 years of age and 1,107 males from 39 to 66 years of age For studying the frequency of the identified variants in a healthy working population, one SNP in The genotype distribution was used to calculate minor allele frequencies and deviations from Hardy-Weinberg equilibrium were evaluated using a Chi-square test. Due to the low minor allele frequency of rs148972953, heterozygous and homozygous minor allele carriers were assessed combined in comparison to major allele carriers. Differences in allele frequencies between carriers and non-carriers of rs148972953 by oestrogen receptor status, progesterone receptor status, metastasis at baseline and metastasis after treatment were calculated by using a Chi-squared test. Odds ratios with a 95% confidence interval were calculated for estimating the relationship between rs148972953 and oestrogen receptor status (ER), progesterone receptor status (PR) and metastasis Following functional considerations based on the SNP position, the potential effects of the three SNPs were investigated using selected bioinformatic applications LAMTOR2 and LAMTOR3 revealed three SNPs to occur in a sequencing sample of 50 breast cancer patients, but no novel mutations. One SNP was found in LAMTOR2 (rs7541) and two SNPs within LAMTOR3 (rs2298735 and rs148972953)  and cancerous tissue.Sequencing of the exons and exon-intron boundaries of both 8972953) . Our hypLAMTOR2 could possibly affect splicing regulation   of 4%, we investigated whether a healthy working population sample would show different genotype distributions than the breast cancer sample. Therefore, we genotyped all three SNPs in female participants of the SAPHIR population (n = 640). The allele and genotype frequencies in SAPHIR women were similar to those in our sequencing sample , and as the SNP rs148972953 in  p>0.05; .LAMTOR3 was found to be strongly associated with negative progesterone receptor status (OR = 4.27 (1.43–12.72); For the analysis of genetic association, we combined the samples for sequencing and those in the Risk Prediction of Breast Cancer Metastasis Study since they were collected at the same University Clinic in the same manner. In the combined breast cancer sample, rs148972953 in Encouraged by the findings from the combined breast cancer sample and the bioinformatic analyses we performed a replication in the MARIE Study. The minor allele of rs148972953 was not found to show differential association by oestrogen receptor or progesterone receptor status (ER: OR = 1.18 (0.69–2.02); PR: OR = 0.97 (0.60–1.58); Based on the results of the bioinformatics analysis, we hypothesized that rs148972953 could be associated with tumour metastasis. However, our assumption was not supported in the MARIE Study. Compared to patients carrying the wildtype of rs148972953 polymorphism, carriers of the minor allele of rs148972953 were neither more likely to have metastasis at baseline (OR = 1.76 (0.62–4.96)) nor were they at higher risk of distant disease-free survival (HR = 0.96 (0.75–1.23)) . The genLAMTOR2 (p14) is significantly up-regulated in breast cancer cells. Interestingly, two independent publications also reported an up-regulation of p14 in invasive ductal breast carcinomas LAMTOR3 (Mp1) was also found to be up-regulated in invasive ductal breast carcinomas LAMTOR3 (Mp1) was also reported to exhibit reduced levels in the stroma of invasive breast carcinoma LAMTOR3 (Mp1) was required for the survival of oestrogen receptor positive breast cancer cell lines LAMTOR2 and LAMTOR3 that could contribute to the aetiology of breast cancer by altering any of the above mentioned processes.Bioinformatic analysis of high-throughput cancer microarrays available on the public domain Oncomine LAMTOR2 and LAMTOR3 could be associated with breast cancer progression and metastasis. We additionally expected that several rare instead of common variants would be detected and those rare variants were not expected to be already listed in databases. Therefore, we applied a discovery stage by sequencing the exons of LAMTOR2 and LAMTOR3 in 50 cancerous tissues and the corresponding peripheral leukocyte DNA and observed no differences in the identified mutations between the two tissues. The identified mutations were investigated in two different case samples (Risk Prediction of Breast Cancer Metastasis Study and MARIE cases) as well as two different control samples (SAPHIR women and MARIE controls).We initially hypothesized that somatic, cancer-tissue-specific mutations in LAMTOR3 and both oestrogen and progesterone receptor status and metastasis. The present work does not exclude that the observed polymorphisms may play a role in other disease contexts or that other not yet identified polymorphisms in LAMTOR2 and LAMTOR3 may in fact contribute to breast cancer aetiology. We would also like to emphasize that genetic variation is not the only factor contributing to disease progression. Many regulatory aspects, in particular those controlling protein stability, posttranslational modifications and association with binding partners, play a fundamental role in determining how much and how active a protein actually is. As discussed before, previous publications identified the LAMTOR complex as a convergence point of key signalling pathways: MAPK and mTOR. Due to the well established role of both signalling cascades in breast cancer progression, and the recent implication of LAMTOR3 in oestrogen receptor positive breast cancer, we leave open the possibility of therapeutically targeting the complex as previously proposed by others Despite the promising results in the initial study, the replication study failed to support an association between the SNP rs148972953 in LAMTOR2 nor LAMTOR3 came up as hits in genome-wide association studies (GWAS) with any kind of disease or trait as indexed by the catalogue of published GWAS LAMTOR3 is located, showed signals of associations with upper aerodigestive tract cancers per se is interesting for cancer. Therefore, future studies including a dense map of SNPs from the LAMTOR complex including the regulatory regions might reveal implications of genetic associations of LAMTOR2 and LAMTOR3 with breast cancer.Although the LAMTOR complex was independently demonstrated to be involved in breast cancer The present study constitutes a good example for the necessity and importance of replication in genetic association studies. Small cohorts intrinsically increase the number of false positives that are accepted. Replication in larger populations is therefore fundamental to separate the wheat from the chaff.Figure S1LAMTOR2.Amplification and sequencing strategy of (DOC)Click here for additional data file.Figure S2LAMTOR3.Amplification and sequencing strategy of (DOC)Click here for additional data file.Table S1LAMTOR2.Primers used for amplification and sequencing of (DOC)Click here for additional data file.Table S2LAMTOR3.Primers used for amplification and sequencing of (DOC)Click here for additional data file.


BMPR1A or SMAD4 are found in 50% of affected individuals. Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease diagnosed upon the presence of epistaxis, visceral arteriovenous malformations (AVM) or mucutaneous telangiectasias. HHT is diagnosed when there are ≥ 3 manifestations and is suspected when there are at least 2 manifestations. Most HHT cases are caused by a germline mutation in ALK1 or ENG, members of the TGFβ signaling pathway. Approximately 22% of patients with Juvenile Polyposis Syndrome (JPS) due to a SMAD4 mutation have been reported to also have HHT [SMAD4 mutation including those who underwent systematic screening for AVM’s.Juvenile Polyposis Syndrome (JPS) is defined by the presence of ≥ 5 colorectal juvenile polyps or any number of juvenile polyps in an individual with a family history of JPS. Genetic alterations including either point mutations or large rearrangements in SMAD4 mutation were studied by screening affected patients for cutaneous telangiectases and with cardiac bubble ECHO, CAT scan chest, or MRI of brain for other AVMs.JPS patients were identified from a comprehensive polyposis database using Cologene© software. Families carrying a germline SMAD4 mutation. These families include 21 affected relatives, 11 men and 10 women, with a current mean age of 36.3 years (range 4 - 70). Fourteen affected relatives, from 6 families, underwent HHT screening . Eleven of 14 (79%) had ≥ 3 HHT manifestations and two of 14 (14 %) had at least 2. In addition, 3 of 7 unscreened affected relatives have presented with at least 2 manifestations of HHT. Of the 24 families that have not had genetic testing or HHT screening one affected family member presented with ≥ 3 HHT manifestations, and two had at least 2 manifestations.Fourteen of 38 JPS families underwent genetic testing. Nine families were identified to have a SMAD4 mutations had clinically diagnosed or suspected HHT. Genetic testing should be performed in all JPS patients. In addition, systematic HHT screening is recommended for JPS patients with SMAD4 mutations.Greater than 90% of our patients with JPS due to


MSH2 account for 1-2% of all CRCs and up to 20% of these are large germline deletions [MSH2 deletion of exons 1-6 has been characterized as a North American Founder Mutation (AFM) [Lynch syndrome (LS) is a genetic disorder that accounts for approximately 3% of all colorectal cancers (CRC) . ClinicaMLH1 and MSH2 genes [MSH2 gene. This results in the premature truncation of the MSH2 protein and confirms the diagnosis of LS.A 68-year-old Caucasian male presented to cancer genetics following a second primary diagnosis of infiltrating poorly differentiated adenocarcinoma of the colon. His history included; moderately differentiated invasive adenocarcinoma of the sigmoid colon at age 40; left ureteral carcinoma diagnosed at 54; and a bladder carcinoma diagnosed at age 59. Additionally, colonoscopy revealed multiple adenomatous polyps within a ten year period. Family history is significant for a son diagnosed with colon cancer at age 34, father with gastric cancer, two paternal aunts and paternal grandfather with colon cancer, and German ancestry. Peripheral blood was sent for analysis of the MSH2 mutations are at a higher risk of developing extracolonic cancers than individuals with MLH1 mutations [MSH2 mutations and male carriers have up to a 28% lifetime risk of developing uroepithelial cancers [This case reveals a LS patient with a history of four metachronous cancers. Phenotypic variations exist amongst the different MMR genes causative for LS -7. Indivutations ,6. There cancers . The sev cancers . A bette


PROP1 mutations have multiple pituitary hormone deficiencies (MPHD) that typically advance from growth insufficiency diagnosed in infancy to include more severe growth hormone (GH) deficiency and progressive reduction in other anterior pituitary hormones, eventually including adrenocorticotropic hormone (ACTH) deficiency and hypocortisolism. Congenital deficiencies of GH, prolactin, and thyroid stimulating hormone have been reported in the nullProp1 (-/-Prop1) and the Ames dwarf (df/dfProp1) mouse models, but corticotroph and pituitary adrenal axis function have not been thoroughly investigated. Here we report that the C57BL6 background sensitizes mutants to a wasting phenotype that causes approximately one third to die precipitously between weaning and adulthood, while remaining homozygotes live with no signs of illness. The wasting phenotype is associated with severe hypoglycemia. Circulating ACTH and corticosterone levels are elevated in juvenile and aged Prop1 mutants, indicating activation of the pituitary-adrenal axis. Despite this, young adult Prop1 deficient mice are capable of responding to restraint stress with further elevation of ACTH and corticosterone. Low blood glucose, an expected side effect of GH deficiency, is likely responsible for the elevated corticosterone level. These studies suggest that the mouse model differs from the human patients who display progressive hormone loss and hypocortisolism.Humans with  PROP1, POU1F1 (PIT1), HESX1, LHX3, LHX4, OTX2, SOX2, SOX3, and GLI2 , and thyroid stimulating hormone (TSH) in addition to overall pituitary hypoplasia Prophet of PIT1 (PROP1) are the most common known causes of MPHD in humans. The hormone deficiencies are similar to those caused by POU1F1 mutations, except that the deficiencies include reduced gonadotropin production requiring sex hormone substitution and there is a strong tendency toward progressive hormone loss leading to lower circulating adrenocorticotropic hormone (ACTH) later in life, requiring glucocorticoid replacement therapy PROP1 and POU1F1 patients is the tendency of patients with PROP1 mutations to undergo apparent degeneration of the pituitary gland during childhood PROP1 mutations are not well understood.Congenital pituitary hormone deficiency in humans occurs with a frequency of approximately 1 in 4000 live births and is caused primarily by mutations in genes important for pituitary development Prop1 action in pituitary development and function. The Ames dwarf (df/dfProp1) and the nullProp1 (-/-Prop1) mouse mutants recapitulate the human MPHD phenotype in that adult mutants are profoundly deficient in TSH, GH, PRL, have low circulating gonadotropins, and pituitary hypoplasia Prop1 mutant mice show that precursor cells fail to colonize the anterior lobe resulting in reduced cell proliferation and enhanced apoptosis after birth leading to hypoplasia that becomes evident in the weeks after birth Prop1 mouse mutants differ from humans with PROP1 mutations in that the hormone deficits are consistently congenital rather than progressive, thyroid hormone and growth hormone replacement are sufficient for fertility, and there is no clear evidence for transient pituitary hyperplasia.Several mouse models have been used to dissect the mechanism of Prop1 deficient mice, although both alleles, df/dfProp1 and -/-Prop1, have the same features when normalized for genetic background PROP1 can have different clinical presentations Prop1 mouse mutants die of respiratory distress. The lack of pituitary TSH results in fetal hypothyroidism, reduced expression of the thyroid hormone inducible transcription factor TTF1 in the lung, and inadequate production of surfactants, known target genes of TTF1. The lungs fail to inflate, causing respiratory distress and lethality Prop1 deficient mice to lethality after weaning. The reason for this juvenile lethality has not been explored.The genetic background exerts a considerable influence on the phenotype of the Prop1 deficient mice, and corticosterone levels are not reduced in newborn mutants PROP1 patients who have been closely followed appear to have evolving hypocortisolism Prop1 mutant mice is not known, it is necessary to investigate pituitary adrenal function in young and old Prop1 deficient mice on a sensitized (B6) genetic background.Corticotroph development does not appear to be affected in the Prop1 deficient mice. In contrast, our results show increased serum ACTH and corticosterone levels in young and old Prop1 mutants. The pituitary-adrenal axis is functional in young adult nullProp1 mice as demonstrated by elevated activity in response to restraint stress. Prop1 mutants have significantly reduced blood glucose levels, as expected for GH deficient animals, which could trigger the activation of the pituitary-adrenal axis. Untreated hypoglycemia can cause mortality in both humans and mice Prop1 mouse alleles we tested on various genetic backgrounds differ from human patients by maintaining elevated pituitary adrenal axis activity through 1 year of age, with no evidence for evolving hypocortisolism.We report no evidence for progressive ACTH loss in juvenile and young adult All mice were housed in a 12-h light, 12-h dark cycle with unlimited access to tap water and Purina 5008 or 5020 chows. All procedures using mice were approved by the University of Michigan Committee on Use and Care of Animals, and all experiments were conducted in accordance with the principles and procedures outlined in the NIH Guidelines of the Care and Use of Experimental Animals.Sactm1Prop1 heterozygous null mice, referred to here as Prop1+/−, were generated from R1 (129/Sv x 129/Sv-CP) ES cells by replacing the coding region of exon 1, intron 1, and a portion of exon 2 with cassettes encoding β-galactosidase and neomycin resistance . The chimeras were mated to C57BL/6J mice (B6)  to generate F1 heterozygous animals and were first analyzed on a mixed F2 C57BL/6J-129S1/SvImJ background (B6/129). The F2 +/Prop1− heterozygous mice were backcrossed to B6 mice for four generations to establish the +/Prop1− N4 B6 breeding colony, which is theoretically 93.75% pure B6. Mice used in the study of pituitary-adrenal function were from the N4 B6 genetic background unless expressly stated otherwise. -/-Prop1 mice were determined by PCR as previously described The +/dfProp1 stock is not inbred. It was obtained from Dr. A. Bartke at Southern Illinois University in 1988 and maintained at University of Michigan. This stock was backcrossed to B6 to N4 The DF/B-Young adult mice  were housed individually 12 hours prior the experiment, with special precautions to avoid stress associated with noise and cage handling. The blood samples were collected in the morning (between 9:00am and 10:30am) by retro orbital bleeding in heparinized collection tubes . The retro orbital bleeding was done in less than one minute after initial mouse handling to prevent stress-induced corticosterone release. Animals were subjected to restraint stress for 30 minutes, after which another blood sampling was performed by the same method For ACTH measurements in non-stressed conditions, animals of various ages were anesthetized with metaphane, rapidly decapitated within less than 1 min from the time of initial handling, and blood samples collected.125I RIA kit  according to the manufacturer's protocol http://www.abbottdiabetescare.com). If glucose levels were below the level of detection, an arbitrary number of 19 mg/dL was assigned for the purpose of statistical analysis.ACTH and corticosterone were measured by radioimmunoassay (RIA) in plasma using a Adrenals were collected immediately after euthanizing and rinsed in ice-cold PBS prior to 1 h fixation in 4% paraformaldehyde on ice . Samples were washed in PBS, dehydrated in a graded series of ethanol, and embedded in paraffin. Seven-micrometer sections were prepared and either stained with hematoxylin and eosin. The 20α-hydroxysteroid dehydrogenase antibody was generously provided by Yacob Weinstein and used at 1:2000-3000 dilution Data were processed and plotted using StatView software , with the exception of the qRT-PCR data that was processed using Microsoft Excel Software. ANOVA  and Fisher's exact test were used to evaluate the data. All data are shown as +/− 1 SEM (standard error of the mean). P-values of less than 0.05 were considered to be statistically significant.Prop1 on several genetic backgrounds. The Ames dwarf mutant, df/df,Prop1 arose spontaneously on a poorly defined genetic background (DF/B), and it carries a missense mutation in the homeodomain, Ser83Pro +/Prop1−, on a mixed background comprised of C57BL/6J (B6) and 129S1/SvImJ (129) Prop1-/- animals exhibited lethargy, wasting, and death between 3 and 7 weeks of age. Death usually occurred within 3-5 days of initial signs of distress. More males were affected than females (p = 0.03). A similar wasting and lethality phenotype was also observed in 27% (6/22) of compound heterozygotes, Prop1df/-, on a mixed background.We analyzed the viability of two different mutant alleles of Prop1df/+ and 129/B6-Prop1+/−, four times to B6 to be able to compare the phenotypes of the two alleles on a consistent genetic background. We observed identical viability of the homozygous mutants for each allele at two weeks of age: 17.5% Prop1df/df and 19.5% Prop1-/- for each on N4 B6, p = 0.69 Prop1 mutant alleles.We back-crossed both strains, DF/B-Prop1-/- and normal mice on the N4 B6 background by RIA to address the ability of Prop1 mutant corticotrophs to secrete ACTH .To determine whether the observed post-weaning lethality on the N4 B6 background could arise from evolving hypocortisolism, we examined ACTH and corticosterone production. We analyzed the serum of 3.5 to 5 week old Prop1 mutants exhibit evolving hypocortisolism at older ages we aged Prop1 mutant animals with three different genotypes and genetic backgrounds  to 7–12 months old and measured both ACTH and corticosterone. All genotype combinations of Prop1 mutants had significantly elevated ACTH and corticosterone . The fold increase in corticosterone from basal to post-stress measurements is less for Prop1-/- animals compared to the wild type (2-3 fold compared to 11-16 fold). While this could be described as a blunted response, the absolute value of circulating corticosterone following restraint was higher in mutants than normal littermates. Post stress, the corticosterone values for male and female mutants were 504 +/− 29 ng/ml and normal littermates were 373 +/− 18 ng/ml. The post stress values in the 500 ng/ml range may be the maximal response. Thus, there is no evidence for impaired pituitary-adrenal axis function.Stress increases pituitary ACTH release and subsequent corticosterone secretion by the adrenal gland hallenge Fig. 2. -/-Prop mice were compared to +/−Prop1 and wild type to determine the consequence of elevated ACTH on adrenal growth. The absolute size of the adrenal gland is smaller in the Prop1-/- dwarf males compared to wild type. However, the ratio of adrenal weight to body weight is actually increased in the Prop1-/- males compared to wild type . Similar levels of 21-hydroxylase enzyme, which is important for corticosterone biosynthesis Prop1-/- adrenals compared to +/−Prop1 or wild type. These results are consistent with functioning adrenal glands in -/-Prop1 mice.We used Western blotting to evaluate the levels of steroidogenic enzymes in Prop1 deficient mice. Prop1 deficient mice produce very few somatotrophs and lack detectable circulating GH Igf1) Igf1 expression in the Prop1-/- mouse livers compared to wild type (data not shown). Growth hormone is important for metabolism and glucose homeostasis though its role in modulating Igf1 production Prop1 mutant genotypes at several ages (Prop1-/- mice (N4 B6 background) is similar to that of heterozygous littermates and wild types, 140 +/− 14 mg/dL vs. 177 +/− 16 mg/dL, p = 0.048 , 193 +/− 55 in Prop1df/- (N = 12), and 211 +/− 36 in healthy Prop1-/- (N = 12), and 393 +/− 86 in sick Prop1-/- mice, (N = 7). The very high corticosterone levels support the idea that the wasting phenotype is not due to failure of the pituitary adrenal axis. The elevated levels are consistent with a response to metabolic stress, but it is difficult to determine whether the cachexia is the cause or the effect of severe hypoglycemia.We hypothesized that reduced glucose levels secondary to growth hormone deficiency could cause the elevated basal levels of ACTH and corticosterone in the blood of Prop1 deficiency mice. At 8–10 weeks the N4 B6 Prop1-/- mice had lower glucose levels (ave. 117 +/− 4 mg/dL) than controls (178 +/− 10 mg/dL) . It is possible that severe hypoglycemia contributes to the increased susceptibility of Prop1 mutants to lethality on the B6 background, although other differences in metabolism may be responsible. For example, the livers of healthy Prop1 deficient mice resemble livers of normal fasted mice, and sickly mutant livers are more affected (data not shown) Prop1 mutants that survive to adulthood have significantly longer life spans than their normal littermates, like other strains with reduced insulin like growth factor activity Both Prop1 deficient mice are consistent with clinical data from human patients with GH deficiency. Approximately 5% of humans with GH deficiency also had hypoglycemia, and 10% of the hypoglycemic patients died The lower glucose levels we observed in Prop1 deficient mice. In direct contrast to the human MPHD cases with progressive ACTH loss and hypocortisolism, Prop1 deficient mice exhibit elevated ACTH and corticosterone and reduced glucose levels at 6 mo and 1 yr of age. Young adult Prop1 deficient mice respond to restraint stress with further elevation of ACTH, corticosterone and glucose levels, and show no reduction in adrenal content of steroidogenic enzymes, indicating that the pituitary-adrenal axis can react functionally to this challenge. In addition, the adrenals of the Prop1-/- mice are enlarged relative to normal mice when normalized to body weight, as expected for chronic ACTH secretion in rodents and other mammals, including primates Prop1 mutants have even higher corticosterone levels than healthy mutants.We found no evidence for disruption of the pituitary-adrenal axis in PROP1 deficient patients remains elusive. It is tempting to speculate that it arises from depletion of progenitors, but species differences in function are also possible. Genetic background affects the viability of young Prop1 deficient mice, largely due to different responses of target organs to pituitary hormone deficiency. Multiple Prop1 mutant alleles and genetic backgrounds support elevated ACTH and corticosterone levels and lower glucose levels that persist with age. Although mice with MPHD have been invaluable for understanding the molecular basis for human disorders of hormone-deficiency and dwarfism, pituitary growth, and pituitary cell specification, they may be less pertinent for understanding the nature of progressive hormone deficiency that characterizes humans with PROP1 mutations.The basis for the evolving nature of the hormone deficiencies, including hypocortisolism, in human