Effect of Physical and Chemical Hair Removal Methods on Skin Barrier Function in vitro: Consequences for a Hydrophilic Model Permeant.

BACKGROUND: Although very common in our society, the effect of hair removal on physiological skin parameters and on the ingress of applied chemicals has not been systematically investigated. Thus, as a first step, the aim of the present study was to elucidate the effect of hair removal through epilation (electric epilation, waxing) and depilation (dry and wet shaving, depilatory cream) on skin properties in vitro using the porcine ear model. METHODS: Attenuated total reflection Fourier transform infrared spectroscopy, measurement of the transepidermal water loss (TEWL), visualization by capacitance-based contact imaging, confocal Raman spectroscopy (CRS), diffusion cell studies and tape stripping experiments were employed. RESULTS: Increased TEWL and altered skin permittivity maps were observed. Decreased stratum corneum thickness was observed after waxing. Diffusion cell studies showed increased skin permeation especially in case of dry shaving, electric epilation and waxing. CONCLUSION: Considering CRS and diffusion cell data, a moderate if significant decrease in skin barrier function was found after hair removal by dry shaving (physical skin/material interaction) and epilation methods (plucking out the entire hair, for example, by electrical epilation and waxing). Subsequent experiments will include testing of different permeants covering a broad range of physicochemical properties in vitro and confirming our findings in vivo.

Differences between evidence-based recommendations and actual clinical practice regarding tocolysis: a prospective multicenter registry study.

BACKGROUND: International guidelines recommend that tocolytic therapy be restricted to a single 48-h application. However, multiple cycles of tocolytic therapy and maintenance therapy that exceeds 48 h appear to play a role in daily clinical practice. We aimed to evaluate current trends in clinical practice with respect to treatment with tocolytic agents and to identify differences between evidence-based recommendations and daily clinical practice in Austria. METHODS: A prospective multicenter registry study was conducted from October 2013 through April 2015 in ten obstetrical departments in Austria. Women ≥18 years of age who received tocolytic therapy following a diagnosis of threatened preterm birth were included, and details were obtained regarding clinical characteristics, tocolytic therapy, and pregnancy outcome. RESULTS: A total of 309 women were included. We observed a median of 2 cycles of tocolytic therapy per patient (IQR 1-3) with a median duration of 2 days per cycle (IQR 2-5). Repeat tocolysis was administered in 41.7% of women, resulting in up to six tocolysis cycles; moreover, 40.8% of the first tocolysis cycles were maintenance tocolysis (i.e., longer than 48 h). Only 25.6% of women received one single 48-h tocolysis cycle in which they received antenatal corticosteroids for fetal lung maturation in accordance evidence-based recommendations. CONCLUSIONS: Here, we report a clear disparity between evidence-based recommendations and daily practice with respect to tocolysis. We believe that the general practice of prescribing tocolytic therapy must be revisited. Furthermore, our findings highlight the need for contemporary studies designed to investigate the effectiveness of performing maintenance and/or repetitive tocolysis treatment.

Cystine knot growth factors and their functionally versatile proregions.

The cystine knot disulfide pattern has been found to be widespread in nature, since it has been detected in proteins from plants, marine snails, spiders and mammals. Cystine knot proteins are secreted proteins. Their functions range from defense mechanisms as toxins, e.g. ion channel or enzyme inhibitors, to hormones, blood factors and growth factors. Cystine knot proteins can be divided into two superordinate groups. (i) The cystine knot peptides, also referred to - with other non-cystine knot proteins - as knottins, with linear and cyclic polypeptide chains. (ii) The cystine knot growth factor family, which is in the focus of this article. The disulfide ring structure of the cystine knot peptides is made up by the half-cystines 1-4 and 2-5, and the threading disulfide bond is formed by the half-cystines, 3-6. In the growth factor group, the disulfides of half-cystines 1 and 4 pass the ring structure formed by the half-cystines 2-5 and 3-6. In this review, special emphasis will be devoted to the growth factor cystine knot proteins and their proregions. The latter have shifted into the focus of scientific interest as their important biological roles are just to be unravelled.

Viral-Cellular DNA Junctions as Molecular Markers for Assessing Intra-Tumor Heterogeneity in Cervical Cancer and for the Detection of Circulating Tumor DNA.

The development of cervical cancer is frequently accompanied by the integration of human papillomaviruses (HPV) DNA into the host genome. Viral-cellular junction sequences, which arise in consequence, are highly tumor specific. By using these fragments as markers for tumor cell origin, we examined cervical cancer clonality in the context of intra-tumor heterogeneity. Moreover, we assessed the potential of these fragments as molecular tumor markers and analyzed their suitability for the detection of circulating tumor DNA in sera of cervical cancer patients. For intra-tumor heterogeneity analyses tumors of 8 patients with up to 5 integration sites per tumor were included. Tumor islands were micro-dissected from cryosections of several tissue blocks representing different regions of the tumor. Each micro-dissected tumor area served as template for a single junction-specific PCR. For the detection of circulating tumor-DNA (ctDNA) junction-specific PCR-assays were applied to sera of 21 patients. Samples were collected preoperatively and during the course of disease. In 7 of 8 tumors the integration site(s) were shown to be homogenously distributed throughout different tumor regions. Only one tumor displayed intra-tumor heterogeneity. In 5 of 21 analyzed preoperative serum samples we specifically detected junction fragments. Junction-based detection of ctDNA was significantly associated with reduced recurrence-free survival. Our study provides evidence that HPV-DNA integration is as an early step in cervical carcinogenesis. Clonality with respect to HPV integration opens new perspectives for the application of viral-cellular junction sites as molecular biomarkers in a clinical setting such as disease monitoring.

A core domain of the BMP2 proregion is sufficient for the biogenesis of mature homodimeric growth factor.

The large proregions of cystine knot growth factors exert several important biological functions. Previous results have shown that the proregion of BMP2 retards the activity of the mature growth factor. Hitherto, no information exists about the function of the BMP2 proregion during the biogenesis of the mature growth factor. Here, evidence is presented that the proregion is required for the secretion of BMP2 when present in covalent linkage to the mature domain. In trans complementation, i.e., with the proregion encoded by a separate plasmid, does not result in secretion of homodimeric mature growth factor. An extended variant of a previously in vitro characterized subdomain of the proregion, when being present in covalent linkage to the mature growth factor, restores secretion of mature BMP2, albeit at a reduced level.

The factor VII-activating protease (FSAP) enhances the activity of bone morphogenetic protein-2 (BMP-2).

Factor VII-activating protease (FSAP) is a circulating protease involved in the pathogenesis of atherosclerosis, calcification, and fibrotic processes. To understand how FSAP controls the balance of local growth factors, we have investigated its effect on the regulation of bone morphogenetic proteins (BMPs). BMP-2 is produced as a large pro-form and secreted as a mature heparin-binding growth factor after intracellular processing by pro-protein convertases (PCs). In this study, we discovered that FSAP enhances the biological activity of mature BMP-2 as well as its pro-form, as shown by osteogenic differentiation of C2C12 myoblasts. These findings were complemented by knockdown of FSAP in hepatocytes, which revealed BMP-2 processing by endogenous FSAP. N-terminal sequencing indicated that pro-BMP-2 was cleaved by FSAP at the canonical PC cleavage site, giving rise to mature BMP-2 (Arg(282)↓Gln(283)), as well as in the N-terminal heparin binding region of mature BMP-2, generating a truncated mature BMP-2 peptide (Arg(289)↓Lys(290)). Similarly, mature BMP-2 was also cleaved to a truncated peptide within its N-terminal region (Arg(289)↓Lys(290)). Plasmin exhibited a similar activity, but it was weaker compared with FSAP. Thrombin, Factor VIIa, Factor Xa, and activated protein C were not effective. These results were further supported by the observation that the mutation of the heparin binding region of BMP-2 inhibited the processing by FSAP but not by PC. Thus, the proteolysis and activation of pro-BMP-2 and mature BMP-2 by FSAP can regulate cell differentiation and calcification in vasculature and may explain why polymorphisms in the gene encoding for FSAP are related to vascular diseases.

Influence of the polypeptide environment next to amyloidogenic peptides on fibril formation.

Alternative folding or fibril formation of proteins is associated with many diseases. Although uncertainty remains for many diseases as to whether the fibrils themselves constitute the main pathogenicity factor, the biophysics or molecular steps leading to fibrils cannot easily be reduced to a common denominator. To date, it is known that fibrils can form (i) upon aberrant (over-)production or false processing, (ii) upon infection with prions that act as seeds and induce unfolding of a thus far native protein--as has been shockingly experienced during the bovine spongiform encephalopathy episode, (iii) when mutations are present that increase the propensity of an otherwise stable protein to aggregate, or (iv) when mutation decreases the overall stability of an individual protein. This review intends to highlight some of the biochemical and biophysical mechanisms that favor fibril formation. Special emphasis is given on the relevance of the polypeptide environment of amyloidogenic segments and the currently discussed driving forces of fibril formation.

Feasibility of newborn screening for guanidinoacetate methyltransferase (GAMT) deficiency.

Guanidinoacetate methyltransferase (GAMT) deficiency causes brain creatine deficiency characterized by developmental delays, speech delay, seizures and autism-like behavior. Identification and therapy at birth because of a positive family history has prevented intellectual disability and seizures in all cases reported. The objective of this study was to develop a method to identify patients with GAMT deficiency from newborn screening blood spots. Creatine and guanidinoacetate were extracted from 10,000 deidentified blood spots using the same protocol routinely used for newborn screening and quantified by stable isotope dilution using deuterated creatine and guanidinoacetate as internal standards. Residual dried blood spots from three infants with GAMT deficiency were used to evaluate the sensitivity of the method. A second tier test using UPLC-MS/MS was performed to analyze samples with a concentration of guanidinoacetate >2.44 µmol/L (99.5th centile of the normal population). Fifty four blood spots required second tier testing in addition to seven blood spots from three patients with GAMT deficiency retrospectively analyzed. With second tier testing, only the samples from GAMT deficiency patients had elevated concentration of guanidinoacetate. Our results show that GAMT deficiency can be identified in newborns using routine extraction methods. The cost of this additional screening is minimal, as it does not require additional instrumentation, procedure, or sample collection. The use of a second tier test can reduce the false positive rate to a minimum. Summary Brain creatine deficiency syndromes cause mental retardation that can be prevented if therapy is initiated early in life. This manuscript reports that infants with GAMT deficiency (one of the brain creatine deficiency syndromes) can be identified from elevated guanidinoacetate in newborn blood spots with virtually absent false-positive results using a second tier test.

Polyalanine-independent conformational conversion of nuclear poly(A)-binding protein 1 (PABPN1).

Oculopharyngeal muscular dystrophy is a late-onset disease caused by an elongation of a natural 10-alanine segment within the N-terminal domain of the nuclear poly(A)-binding protein 1 (PABPN1) to maximally 17 alanines. The disease is characterized by intranuclear deposits consisting primarily of PABPN1. In previous studies, we could show that the N-terminal domain of PABPN1 forms amyloid-like fibrils. Here, we analyze fibril formation of full-length PABPN1. Unexpectedly, fibril formation was independent of the presence of the alanine segment. With regard to fibril formation kinetics and resistance against denaturants, fibrils formed by full-length PABPN1 had completely different properties from those formed by the N-terminal domain. Fourier transformed infrared spectroscopy and limited proteolysis showed that fibrillar PABPN1 has a structure that differs from native PABPN1. Circumstantial evidence is presented that the C-terminal domain is involved in fibril formation.

The unresolved puzzle why alanine extensions cause disease.

The prospective increase in life expectancy will be accompanied by a rise in the number of elderly people who suffer from ill health caused by old age. Many diseases caused by aging are protein misfolding diseases. The molecular mechanisms underlying these disorders receive constant scientific interest. In addition to old age, mutations also cause congenital protein misfolding disorders. Chorea Huntington, one of the most well-known examples, is caused by triplet extensions that can lead to more than 100 glutamines in the N-terminal region of huntingtin, accompanied by huntingtin aggregation. So far, nine disease-associated triplet extensions have also been described for alanine codons. The extensions lead primarily to skeletal malformations. Eight of these proteins represent transcription factors, while the nuclear poly-adenylate binding protein 1, PABPN1, is an RNA binding protein. Additional alanines in PABPN1 lead to the disease oculopharyngeal muscular dystrophy (OPMD). The alanine extension affects the N-terminal domain of the protein, which has been shown to lack tertiary contacts. Biochemical analyses of the N-terminal domain revealed an alanine-dependent fibril formation. However, fibril formation of full-length protein did not recapitulate the findings of the N-terminal domain. Fibril formation of intact PABPN1 was independent of the alanine segment, and the fibrils displayed biochemical properties that were completely different from those of the N-terminal domain. Although intranuclear inclusions have been shown to represent the histochemical hallmark of OPMD, their role in pathogenesis is currently unclear. Several cell culture and animal models have been generated to study the molecular processes involved in OPMD. These studies revealed a number of promising future therapeutic strategies that could one day improve the quality of life for the patients.

Indirect comparison of tisagenlecleucel and historical treatments for relapsed/refractory diffuse large B-cell lymphoma.

No head-to-head trials have compared the efficacy of tisagenlecleucel vs historical treatments for adults with relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). This study indirectly compared the overall survival (OS) and overall response rate (ORR) associated with tisagenlecleucel, using data from the JULIET study (Study of Efficacy and Safety of CTL019 in Adult DLBCL Patients; #NCT02445248), vs historical treatments assessed in the CORAL (Collaborative Trial in Relapsed Aggressive Lymphoma) study follow-up population. To assess treatment effects in the treated (full analysis set [FAS]) and enrolled (intention-to-treat [ITT]) study populations, the JULIET FAS vs the CORAL follow-up FAS and JULIET ITT vs CORAL follow-up ITT populations were separately compared. Propensity score weighting using standardized mortality ratio weight (SMRW) and fine stratification weight (FSW) was used to compare OS and ORR, adjusting for baseline confounders. The results indicated that tisagenlecleucel was associated with a lower hazard of death among the FAS (adjusted hazard ratio [95% confidence interval], both FSW and SMRW, 0.44 [0.32, 0.59]) and ITT populations (FSW, 0.60 [0.44, 0.77]; SMRW, 0.57 [0.44, 0.73]; all, P < .001). Median OS was 12.48 months (JULIET) vs 4.34 to 4.40 months (CORAL) for the FAS, and 8.25 (JULIET) months vs 4.04 to 4.86 (CORAL) months for the ITT populations. Tisagenlecleucel was associated with a significantly higher ORR compared with historical treatments among the FAS (adjusted response rate difference [95% confidence interval], both FSW and SMRW, 36% [22%, 0.48%]; P < .001) and among the ITT populations after SMRW adjustment (11% [0%, 22%]; P = .043). This analysis supports that improved response and OS are achieved in patients with r/r DLBCL treated with tisagenlecleucel compared with those treated with alternative historical treatments.

The pro-form of BMP-2 exhibits a delayed and reduced activity when compared to mature BMP-2.

Pro-forms of growth factors receive increasing attention since it was shown that they can affect the function(s) of the mature proteins. Here, we extend our previous investigations on the biological function of the pro-form of bone morphogenetic protein-2 (BMP-2). We demonstrate that proBMP-2, upon prolonged incubation with C2C12 cells, induces alkaline phosphatase, a marker enzyme for osteoblastic differentiation. Expression studies with three different bone marker transcripts reveal that proBMP-2 induces bone-specific transcripts, however, to a smaller extent than the mature growth factor. To resolve this finding at the protein level, the fate of proBMP-2 and BMP-2 was studied in cell culture. We demonstrate that both proteins become internalized, and proBMP-2 is processed to mature BMP-2 within the cells. The data presented here suggest that proBMP-2 elicits biological functions as mature BMP-2 at a delayed and reduced level, which might depend on intracellular cleavage and subsequent secretion as mature BMP-2.

Proneurotrophin-3 may induce Sortilin-dependent death in inner ear neurons.

The precursor of the neurotrophin (NT) nerve growth factor (NGF) (proNGF) serves physiological functions distinct from its mature counterpart as it induces neuronal apoptosis through activation of a p75 NT receptor (p75(NTR) ) and Sortilin death-signalling complex. The NTs brain-derived nerve growth factor (BDNF) and NT3 provide essential trophic support to auditory neurons. Injury to the NT-secreting cells in the inner ear is followed by irreversible degeneration of spiral ganglion neurons with consequences such as impaired hearing or deafness. Lack of mature NTs may explain the degeneration of spiral ganglion neurons, but another mechanism is possible as unprocessed proNTs released from the injured cells may contribute to the degeneration by induction of apoptosis. Recent studies demonstrate that proBDNF, like proNGF, is a potent inducer of Sortilin:p75(NTR) -mediated apoptosis. In addition, a coincident upregulation of proBDNF and p75(NTR) has been observed in degenerating spiral ganglion neurons, but the Sortilin expression in the inner ear is unresolved. Here we demonstrate that Sortilin and p75(NTR) are coexpressed in neurons of the neonatal inner ear. Furthermore, we establish that proNT3 exhibits high-affinity binding to Sortilin and has the capacity to enhance cell surface Sortilin:p75(NTR) complex formation as well as to mediate apoptosis in neurons coexpressing p75(NTR) and Sortilin. Based on the examination of wildtype and Sortilin-deficient mouse embryos, Sortilin does not significantly influence the developmental selection of spiral ganglion neurons. However, our results suggest that proNT3 and proBDNF may play important roles in the response to noise-induced injuries or ototoxic damage via the Sortilin:p75(NTR) death-signalling complex.

Identification of a core domain within the proregion of bone morphogenetic proteins that interacts with the dimeric, mature domain.

Proregions of bone morphogenetic proteins (BMPs) fulfill important biological functions as intramolecular chaperones and for extracellular targeting of the mature signal ligand. Knowledge of the structures of the proregions would contribute to a more comprehensive picture of the biological activities of the pro-forms of BMP growth factors. In this study, a protease resistant core domain of the proregions of three BMPs was identified. For a more detailed analysis, the core domain of the BMP-2 proregion was recombinantly produced. Unfolding/refolding experiments and spectroscopic analyses proved that the core domain can be obtained as a folded entity. Binding of the core domain to the mature growth factor was demonstrated by SPR. Via peptide microarray analysis, residues within the core fragment could be identified that engage in binding to the dimeric, mature domain. Our study reveals that diverse members of the BMP family share a common, independently folding core domain within the large proregions peculiar to TGF-ß superfamily members that may serve as a scaffold for folding and assembly of the dimeric proprotein complexes.

Multiple increased osteoclast functions in individuals with neurofibromatosis type 1.

Skeletal abnormalities including scoliosis, tibial dysplasia, sphenoid wing dysplasia, and decreased bone mineral density (BMD) are associated with neurofibromatosis type 1 (NF1). We report the cellular phenotype of NF1 human-derived osteoclasts and compare the in vitro findings with the clinical phenotype. Functional characteristics (e.g., osteoclast formation, migration, adhesion, resorptive capacity) and cellular mechanistic alterations (e.g., F-actin polymerization, MAPK phosphorylation, RhoGTPase activity) from osteoclasts cultured from peripheral blood of individuals with NF1 (N = 75) were assessed. Osteoclast formation was compared to phenotypic, radiologic, and biochemical data. NF1 osteoprogenitor cells demonstrated increased osteoclast forming capacity. Human NF1-derived osteoclasts demonstrated increased migration, adhesion, and in vitro bone resorption. These activities coincided with increased actin belt formation and hyperactivity in MAPK and RhoGTPase pathways. Although osteoclast formation was increased, no direct correlation of osteoclast formation with BMD, markers of bone resorption, or the clinical skeletal phenotype was observed suggesting that osteoclast formation in vitro cannot directly predict NF1 skeletal phenotypes. While NF1 haploinsufficiency produces a generalized osteoclast gain-in-function and may contribute to increased bone resorption, reduced BMD, and focal skeletal defects associated with NF1, additional and perhaps local modifiers are likely required for the development of skeletal abnormalities in NF1.

Prevalence of iron deficiency in pregnant women: A prospective cross-sectional Austrian study.

The aim of the study was to determine, for the first time, in a prospective cross-sectional multicenter study, the prevalence of iron deficiency (ID) in an Austrian pregnant population. A cohort of 425 pregnant women was classified into four groups of different weeks of gestation. Group 1 was monitored longitudinally, while groups 2-4, iron status, were sampled only once. Evaluation of the prevalence of ID was performed by comparing the diagnostic criteria of the WHO to the cutoff proposed by Achebe MM and Gafter-Gvili A (Achebe) and the Austrian Nutrition Report (ANR). In comparison with the ANR, the prevalence of ID was lower in group 1 and higher in groups 2-4 (17.2% vs. 12.17%, 25.84%, 35.29%, and 41.76%, respectively) (p-values < .01 except group 1). According to WHO, the prevalence in group 1 was 12.17% at inclusion, 2 months later 31.7%, and further 2 months later 65.71%, respectively. According to Achebe, the number of cases doubled; for group 1, the number of cases rose from 13 to 42 (115 patients total); for groups 2-4, we observed an increase from 112 to 230 (340 patients total). This study reported a prevalence of around 12% at the beginning of pregnancy, which increased during pregnancy up to 65%. ID can have a massive impact on quality of life, justifying screening, as iron deficiency would be easy to diagnose and treat.

Evaluation of Integrated HPV DNA as Individualized Biomarkers for the Detection of Recurrent CIN2/3 during Post-Treatment Surveillance.

PURPOSE: Post-treatment follow-up in women with cervical pre-cancers (CIN3) is mandatory due to relapse in up to 10% of patients. Standard follow-up based on hrHPV-DNA/cytology co-testing has high sensitivity but limited specificity. The aim of our prospective, multicenter, observational study was to test the hypothesis that an individualized viral-cellular-junction test (vcj-PCR) combined with cytology has a lower false positive rate for the prediction of recurrence compared to standard co-testing. METHODS: Pre-surgical cervical swabs served for the identification of HPV16/18 DNA integration sites by next-generation-sequencing (NGS). Samples taken at 6, 12 and 24 months post-surgery were evaluated by cytology, hrHPV-DNA and the patients' individual HPV-integration sites (vcj-PCR on the basis of NGS). RESULTS: Integration sites were detected in 48 of 445 patients (10.8%), 39 of them had valid follow-up data. The false positive rate was 18.2% (95% CI 8.6-34.4%) for standard hrHPV/cytology at six months compared to 12.1% (95% CI 4.8-27.3%) for vcj-PCR/cytology, respectively (McNemar p = 0.50). Six patients developed recurrences (1 CIN2, 5 CIN3) during follow-up. Standard co-testing detected all, whereas vcj-PCR/cytology detected only five patients with recurrences. Data of 269 patients without evidence of HPV16/18 integration were subject to post-hoc analyses. Standard co-testing revealed a false positive rate of 15.7% (95% CI 11.7-20.7%) and predicted ten of fourteen recurrences at six months. CONCLUSIONS: Although highly specific on its own vcj-PCR could not detect all recurrent CIN2/3. Possible reasons for this unexpected result may be multifocal lesions, intratumoral heterogeneity with respect to HPV integration and/or incident CIN.

SMAD4: a predictive marker of PDAC cell permissiveness for oncolytic infection with parvovirus H-1PV.

Pancreatic ductal adenocarcinoma (PDAC) represents the eighth frequent solid tumor and fourth leading cause of cancer death. Because current treatments against PDAC are still unsatisfactory, new anticancer strategies are required, including oncolytic viruses. Among these, autonomous parvoviruses (PV), like MVMp (minute virus of mice) and H-1PV are being explored as candidates for cancer gene therapy. Human PDAC cell lines were identified to display various susceptibilities to an infection with H-1PV. The correlation between the integrity of the transcription factor SMAD4, mutated in 50% of all PDAC, and H-1PV permissiveness was particularly striking. Indeed, mutation or deletion of SMAD4 dramatically reduced the activity of the P4 promoter and, consequently, the accumulation of the pivotal NS1 protein. By means of DNA affinity immunoblotting, novel binding sites for SMAD4 and c-JUN transcription factors could be identified in the P4 promoter of H-1PV. The overexpression of wild-type SMAD4 in deficient cell lines (AsPC-1, Capan-1) stimulated the activity of the P4 promoter, whereas interference of endogenous SMAD4 function with a dominant-negative mutant decreased the viral promoter activity in wild-type SMAD4-expressing cells (Panc-1, MiaPaCa-2) reducing progeny virus production. In conclusion, the importance of members of the SMAD family for H-1PV early promoter P4 activity should guide us to select SMAD4-positive PDACs, which may be possible targets for an H-1PV-based cancer therapy.

p16(INK4a) overexpression predicts translational active human papillomavirus infection in tonsillar cancer.

The causal role of human papillomaviruses (HPV) in squamous cell carcinogenesis of tonsillar cancers (TSCC) depends on the activity of the viral oncoproteins E6 and E7, leading to inactivation of the cellular tumor suppressor p53 and the retinoblastoma gene product pRb. Because of the negative feedback mechanisms, the pRb inactivation causes an increase of the inhibitor of the cyclin-dependent kinases p16(INK4a). In 39 TSCC specimens, genotyping based on the amplification of HPV DNA was carried out using PCR by applying HPV type-specific oligonucleotides. Subsequently, amplicons were hybridised with fluorescence-labeled complementary probes using the Southern blot technology. For HPV E6/E7 mRNA expression, Northern hybridization and RT-PCR were performed, and for p16(INK4a) detection, immunohistochemistry was performed. With 21/39 (53%) HPV-positives, the detection rate is within the range that can be expected in TSCC. The E6/E7 oncogene mRNA was detectable in 11 cases, 10 of which showed positive signals after p16(INK4a) staining. Albeit the small study group was investigated, the correlation of the HPV DNA status with the p16(INK4a) expression was of statistical significance (p = 0.02). Kaplan-Meier estimations revealed better survival outcome for patients with HPV-positive tumors with detectable E6/E7 mRNA and p16(INK4a) overexpression (p = 0.02, median observation time 29 months). As mRNA expression tests are not routinely available in many clinical diagnostic laboratories, and based on the high correlation of p16(INK4a) staining with HPV E6/E7 mRNA expression, in conclusion we suggest for a deeper exploration for the use of p16(INK4a) as a surrogate marker with the potential to impact the standard of care of HPV DNA-positive head and neck carcinomas.

Sortilin is essential for proNGF-induced neuronal cell death.

Sortilin (approximately 95 kDa) is a member of the recently discovered family of Vps10p-domain receptors, and is expressed in a variety of tissues, notably brain, spinal cord and muscle. It acts as a receptor for neurotensin, but predominates in regions of the nervous system that neither synthesize nor respond to this neuropeptide, suggesting that sortilin has additional roles. Sortilin is expressed during embryogenesis in areas where nerve growth factor (NGF) and its precursor, proNGF, have well-characterized effects. These neurotrophins can be released by neuronal tissues, and they regulate neuronal development through cell survival and cell death signalling. NGF regulates cell survival and cell death via binding to two different receptors, TrkA and p75NTR (ref. 10). In contrast, proNGF selectively induces apoptosis through p75NTR but not TrkA. However, not all p75NTR-expressing cells respond to proNGF, suggesting that additional membrane proteins are required for the induction of cell death. Here we report that proNGF creates a signalling complex by simultaneously binding to p75NTR and sortilin. Thus sortilin acts as a co-receptor and molecular switch governing the p75NTR-mediated pro-apoptotic signal induced by proNGF.