NH2-truncated human tau induces deregulated mitophagy in neurons by aberrant recruitment of Parkin and UCHL-1: implications in Alzheimer's disease.

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.

AD-linked, toxic NH2 human tau affects the quality control of mitochondria in neurons.

Functional as well as structural alterations in mitochondria size, shape and distribution are precipitating, early events in progression of Alzheimer's Disease (AD). We reported that a 20-22kDa NH2-tau fragment (aka NH2htau), mapping between 26 and 230 amino acids of the longest human tau isoform, is detected in cellular and animal AD models and is neurotoxic in hippocampal neurons. The NH2htau -but not the physiological full-length protein- interacts with Aß at human AD synapses and cooperates with it in inhibiting the mitochondrial ANT-1-dependent ADP/ATP exchange. Here we show that the NH2htau also adversely affects the interplay between the mitochondria dynamics and their selective autophagic clearance. Fragmentation and perinuclear mislocalization of mitochondria with smaller size and density are early found in dying NH2htau-expressing neurons. The specific effect of NH2htau on quality control of mitochondria is accompanied by (i) net reduction in their mass in correlation with a general Parkin-mediated remodeling of membrane proteome; (ii) their extensive association with LC3 and LAMP1 autophagic markers; (iii) bioenergetic deficits and (iv) in vitro synaptic pathology. These results suggest that NH2htau can compromise the mitochondrial biology thereby contributing to AD synaptic deficits not only by ANT-1 inactivation but also, indirectly, by impairing the quality control mechanism of these organelles.

Prevalence of human papillomavirus infection in Italian and immigrant women.

Human papillomavirus (HPV) is the most common sexually transmitted agent worldwide. Prevalence varies according to the geographic regions, and is highest in developing countries. Geographic differences exist also in the detection rate of oncogenic types in malignant cervical lesions. In this study, the prevalence of HPV infection as well as the spectrum of HPV types was evaluated in Italian and immigrant women of the urban area of Rome. Several risk factors (age at first intercourse, number of partners, smoking, pregnancy, age at first pregnancy, contraception, education, and menarche) were taken into consideration. Overall, there was a high prevalence of HPV infection in the two groups studied. No significant differences were observed in the spectrum of HPV types detected. HPV 16 and 18 were the types detected more frequently in both groups. Interestingly, HPV 54 and 70 were found only in the immigrants. Whether this finding reflects a recent introduction of these HPV types in the population studied remains to be established. Monitoring of HPV types in the population is advisable, especially in countries like Italy which is a destination and a gateway for immigrants directed towards north and central Europe. The introduction of high risk HPV variants may have a clinical impact and affect the diagnostic procedures.

Tyrosine kinase nerve growth factor receptor switches from prosurvival to proapoptotic activity via Abeta-mediated phosphorylation.

The present study shows that increased Abeta production in hippocampal neurons, due to a failure of NGF signal, induces an unexpected phosphorylation of tyrosine kinase receptor A (TrkA), followed by activation of the phospholipase C gamma (PLCgamma) pathway and neuronal death. Such phosphorylation seems causally connected with 2 kinases known be involved in amyloidogenesis, Src and CDK5, and associated with alpha and gamma secretase-mediated p75 processing. Pharmacologic inhibition of TrkA phosphorylation and partial silencing of TrkA and/or p75 receptors prevent PLCgamma activation and protect neurons from death. Concomitantly with these events, TrkA, p75, Abeta peptides, and PS1 protein coimmunoprecipitate, suggesting their direct interplay in the subsequent onset of apoptotic death. Together, these findings depict a cellular mechanism whereby the same cellular transducing system may invert its intracellular message from trophic and antiapoptotic to a death signaling, which could also have relevance in the onset of Alzheimer's disease.

Lipofilling in skin affected by radiodermatitis: clinical and ultrasound aspects. Case report.

In recent years, lipofilling has established itself as one of the most effective and least invasive techniques to treat connective dystrophy subsequent to radiotherapy. We report the case of a patient diagnosed with intraductal carcinoma of the right breast in 1996, at the age of 41. The patient underwent quadrantectomy with ipsilateral axillary lymph node dissection and adjuvant chemotherapy and radiotherapy. Four years later, a recurrence led the patient to undergo a subcutaneous mastectomy and immediate reconstruction, involving the submuscular insertion of a permanent implant. In 2007 the patient suffered both radiodermatitis and capsular contracture around the implant, causing constant pain and significant functional limitation. She first took a leukotriene inhibitor (Zafirlukast, 20 mg daily for 8 months) to reduce the capsular contracture. She then underwent lipofilling (Coleman's technique) of the area affected by radiodermatitis, in which the skin was considerably thinned and visibly ischemic. A second session followed four months later. Clinical, photographic and ultrasound examination revealed clear and lasting thickening of the superficial tissues, increased coverage of the implant, and reduced skin discoloration and tension.

Management and follow-up of a patient with Familial Atypical Multiple Mole-Melanoma (FAMMM) Syndrome.

INTRODUCTION: Familial Atypical Multiple Mole-Melanoma Syndrome (FAMMM) is an autosomal dominant genodermatosis characterized by the presence of a high number of dysplastic nevi and family history of melanoma or pancreatic cancer. Melanomas in FAMMM patients tend to occur at a younger age, although they are clinically similar to sporadic melanomas in terms of overall survival. CASE REPORT: A 45 year-old woman with a family history of melanoma, a type II phototype and numerous (>100) nevi was admitted to our Department of Dermatology and Plastic Surgery. Over the past years, the patient underwent several surgical operations to remove pigmented lesions and two are dysplastic nevi. Since 1995, she underwent surgery to remove four melanomas. She is followed for skin examinations including dermoscopy. CONCLUSION: Identifying high-risk patients for melanoma represents a primary objective for the specialists that are involved in the management of this disease, especially in order to enact all the necessary surveillance and follow-up strategies.

Spanish radiology residents' views on breast imaging.

OBJECTIVE: To evaluate radiology residents' opinions about breast imaging and the possibility of choosing this subspecialty after completing their residency. MATERIAL AND METHODS: We elaborated a 15-question survey aimed at radiology residents in Spain. The survey was approved by the Spanish Society of Breast Imaging (SEDIM) and the Spanish Society of Medical Radiology (SERAM), and it was disseminated by the SERAM through links to Google Forms via social networks and emails. Responses sent between February 21, 2020 and July 31, 2020 were accepted. RESULTS: A total of 72 residents responded to the survey (7.83% response rate); 69.44% of these were third- or fourth-year residents. Of the respondents, 73.61% knew about the SEDIM, and 18.06% knew about the European Society of Breast Imaging. The duration of training programs was three months for 70.83% of respondents. In 7.84% of the responses, residents stated that their supervision was less than 50%, and 70.59% of the residents stated that the rotation exceeded their expectations. One-third of the respondents would consider a fellowship in breast imaging. In all hospitals, residents did diagnostic mammography and breast ultrasound; not all did interventional procedures. Aspects of breast imaging that were rated negatively included the lack of CT studies and the possible legal repercussions of errors. Aspects that were rated positively were dynamics, interventionism, and the role of the radiologist in the process of care for patients with breast cancer. CONCLUSIONS: Most residents considered that their rotations in breast imaging exceeded their expectations; however, only a small percentage of residents would consider specializing in the field.

A retrospective study on two cohorts of immunocompetent women treated with nonavalent HPV vaccine vs. Ellagic acid complex: outcome of the evolution of persistent cervical HPV infection.

OBJECTIVE: The nonavalent HPV vaccine has demonstrated its efficacy in women and men who already suffer from HPV genital lesions, with little chances to clear the infection. The efficacy of new therapeutic or complementary alternatives as Ellagic acid plus Annona Muricata (Ellagic acid complex) has emerged recently. Our retrospective study compares the evolution of persistent cervical HPV infection in two cohorts of immunocompetent women after the administration of nonavalent vaccine or Ellagic acid complex. PATIENTS AND METHODS: At Tor Vergata University Hospital, Rome, forty women in childbearing age, suffering from persistent cervical HPV infection, were enrolled in two study's groups: nonavalent HPV vaccine (20 women) vs. Ellagic acid complex tablets (20 who refused the vaccine). Cytological features, HPV DNA genotypes and mRNA oncogenic genes E6/E7 presence and clearance were analyzed and confronted between the groups. RESULTS: Demographics and clinical features of the cohorts were comparable. Evaluation of Pap smear, HPV DNA test and mRNA genes E6/E7, were performed at baseline (T0) and after 6 months (T1) and 12 months (T2) from the last dose of vaccine/tablet. At T1 and T2, Ellagic acid complex group showed a statistical reduction of abnormalities in Pap smears (p = 0.018 and 0.006, respectively), probably due to its direct anti-inflammatory, antioxidative and antiviral activities. At T1, vaccinated group showed a higher rate of HPV clearance (p = 0.001), instead Ellagic acid complex group didn't report significative differences. At T2, respect to T0, both groups showed an increase in percentage of negative HPV DNA detection, although more marked for vaccinated group respect to Ellagic acid complex group (p = 0.039 and 0.062 respectively). Regarding mRNA E6/E7 clearance, at T1 and T2, the group of vaccinated women showed a higher negativization respect to the other group (p= 0.077 and 0.042, respectively). CONCLUSIONS: Despite the limited sample of women enrolled for the present study, the results confirmed the clinical usefulness of HPV vaccination as adjuvant agent for the immune system of women affected by persistent HPV infection. Moreover, in women who refused to be vaccinated, the administration of a biocompound like Ellagic acid plus Annona Muricata, represented an interesting clinical strategy in terms of increasing chance of HPV viral clearance.

Spanish radiology residents' views on breast imaging. / Visión del residente de radiodiagnóstico en España sobre la radiología mamaria.

OBJECTIVE: To evaluate radiology residents' opinions about breast imaging and the possibility of choosing this subspecialty after completing their residency. MATERIAL AND METHODS: We elaborated a 15-question survey aimed at radiology residents in Spain. The survey was approved by the Spanish Society of Breast Imaging (SEDIM) and the Spanish Society of Medical Radiology (SERAM), and it was disseminated by the SERAM through links to Google Forms via social networks and emails. Responses sent between February 21, 2020 and July 31, 2020 were accepted. RESULTS: A total of 72 residents responded to the survey (7.83% response rate); 69.44% of these were third- or fourth-year residents. Of the respondents, 73.61% knew about the SEDIM, and 18.06% knew about the European Society of Breast Imaging. The duration of training programs was three months for 70.83% of respondents. In 7.84% of the responses, residents stated that their supervision was less than 50%, and 70.59% of the residents stated that the rotation exceeded their expectations. One-third of the respondents would consider a fellowship in breast imaging. In all hospitals, residents did diagnostic mammography and breast ultrasound; not all did interventional procedures. Aspects of breast imaging that were rated negatively included the lack of CT studies and the possible legal repercussions of errors. Aspects that were rated positively were dynamics, interventionism, and the role of the radiologist in the process of care for patients with breast cancer. CONCLUSIONS: Most residents considered that their rotations in breast imaging exceeded their expectations; however, only a small percentage of residents would consider specializing in the field.

Cognitive Decline and Modulation of Alzheimer's Disease-Related Genes After Inhibition of MicroRNA-101 in Mouse Hippocampal Neurons.

MicroRNAs have emerged as regulators of brain development and function. Reduction of miR-101 expression has been reported in rodent hippocampus during ageing, in the brain of Alzheimer's disease (AD) patients and in AD animal models. In this study, we investigated the behavioral and molecular consequences of inhibition of endogenous miR-101 in 4-5-month-old C57BL/6J mice, infused with lentiviral particles expressing a miR-101 sponge (pLSyn-miR-101 sponge) in the CA1 field of the hippocampus. The sponge-infected mouse model showed cognitive impairment. The pLSyn-miR-101 sponge-infected mice were unable to discriminate either a novel object location or a novel object as assessed by object place recognition (OPR) and novel object recognition (NOR) tasks, respectively. Moreover, the sponge-infected mice evaluated for contextual memory in inhibitory avoidance task showed shorter retention latency compared to control pLSyn mice. These cognitive impairment features were associated with increased hippocampal expression of relevant miR-101 target genes, amyloid precursor protein (APP), RanBP9 and Rab5 and overproduction of amyloid beta (Aß) 42 levels, the more toxic species of Aß peptide. Notably, phosphorylation-dependent AMP-activated protein kinase (AMPK) hyperactivation is associated with AD pathology and age-dependent memory decline, and we found AMPK hyperphosphorylation in the hippocampus of pLSyn-miR-101 sponge mice. This study demonstrates that mimicking age-associated loss of miR-101 in hippocampal neurons induces cognitive decline and modulation of AD-related genes in mice.

Identification of a caspase-derived N-terminal tau fragment in cellular and animal Alzheimer's disease models.

Biochemical modifications of tau proteins have been proposed to be among the earliest neurobiological changes in Alzheimer's disease (AD) and correlate better with cognitive symptoms than do beta-amyloid plaques. We have recently reported that adenovirus-mediated overexpression of the NH2 26-230aa tau fragment evokes a potent NMDA-mediated neurotoxic effect in primary neuronal cultures. In order to assess whether such N-terminal tau fragment(s) are indeed produced during apoptosis or neurodegeneration in vivo, we attempted to ascertain their presence in cell and animal models using an anti-tau antibody directed against the N-terminal sequence of human protein located downstream of the caspase(s)-cleavage site DRKD(25)-QGGYTMHQDQ. We provide biochemical evidence that a caspase(s)-cleaved NH2-terminal tau fragment of 20-22 kDa, consistent with the size of the NH2 26-230aa neurotoxic fragment of tau, is generated in vitro in differentiated human SH-SY5Y cells undergoing apoptosis by BDNF withdrawal or following treatment with staurosporine. In addition this NH2-terminally cleaved tau fragment, whose expression correlates with a significant up-regulation of caspase(s) activity, is also specifically detected in vivo in the hippocampus of 15 month-old AD11 transgenic mice, a model in which a progressive AD-like neurodegeneration is induced by the expression of transgenic anti-NGF antibodies. The results support the idea that aberrant activation of caspase(s), following apoptotic stimuli or neurodegeneration insults, may produce one or more toxic NH2 tau fragments, that further contribute to propagate and increase cellular dysfunctions in AD.

Viral hepatitis, HIV, human herpes virus and Treponema pallidum infection in haemodialysis patients from Kosovo, 2005.

The serological status of hepatitis viruses and other infectious diseases in the 66 dialysed patients of one haemodialysis unit in Kosovo were studied, comparing the data with a large group of blood donors and out-patients. All dialysed patients were hepatitis A virus (HAV) positive. Prevalence of hepatitis B surface antigen (HBsAg), hepatitis B surface antibodies (anti-HBs), and hepatitis B core antibodies (anti-HBc) was 14 of 66, 21% (95% confidence interval (CI): 12-33%), 5 of 66, 8% (95%CI: 5-22%), and 50 of 66, 76% (95%CI: 64-85%), respectively. Antibodies to hepatitis C virus (anti-HCV) prevalence was 57 of 66, 86% (95%CI: 76-94%). No human immunodeficiency virus (HIV) positive case was found. Prevalence of past herpes simplex virus type 2 (HSV-2) infection was 29% (95%CI: 18-41%). Two patients (3%, 95%CI: 0-10%) were positive for Treponema pallidum and 18% (95%CI: 10-30%) were human herpesvirus 8 (HHV-8) antibody positive. Four hundred and fifty-two subjects were recruited for comparison. Markers of past HAV infection was associated with haemodialysis (Fisher s exact test p-value=0.037). Dialysed patients were at a higher risk of being HBsAg positive than others: the sex- and age-adjusted odds ratio (OR) was 5.18 (95%CI: 1.87-14.32). Anti-HBc positivity was strongly associated with haemodialysis: the sex- and age-adjusted OR was 6.43 (95%CI: 3.22-12-85). Anti-HCV positivity was 86% and 1% in presence and absence of haemodialysis, respectively. The Fisher s exact test for association proved a strong association between haemodialysis and HCV (p-value<0.0001). The OR for association between haemodialysis and HSV-2 positivity was 3.20 (95%CI: 1.46-7.00). Significant associations were also observed between haemodialysis status and antibodies to Treponema pallidum (Fisher s exact test p-value=0.044). In Kosovo, the prevalence of viral hepatitis infection and other viral infections and Treponema pallidum among dialysed patients is high, indicating major ongoing nosocomial transmission.

[New therapeutic strategies for the treatment of difficult wounds]. / Nuove strategie terapeutiche nel trattamento delle ferite difficili.

BACKGROUND: The medical-surgical treatment of the difficult wounds represents a socio-sanitary problem in continuous growth, currently involving in our Country around 2,000,000 people. The "difficult wound" is a loss of cutaneous substances, usually due to multifactorial pathogenesis, that do not spontaneously lead to a complete recovery. Numerous studies in the literature have evidenced that the use of the advanced wound dressings allows to reach the best clinical and economic results in the process of recovery of the difficult wounds. The advanced would dressing assures a longer period of permanence on the injury and shorten the time of treatment and, as a consequence, it is required a smaller number of applications in comparison with the traditional medications. The Wound Bed Preparation (WBP) can be defined as the global and coordinate management of the cutaneous injury, enabling to chip off the local barriers to the recovery, or promoting the effectiveness of the innovative therapeutic instruments. The term advanced wound dressing indicates the dressing material having biocompatibility characteristics. The purpose of the advanced wound dressings is the one to create the ideal environment for the cicatrization process and isolate the wound from traumas and external infections. PATIENTS AND METHODS: The "Difficult Wounds" Unit of the Department of Plastic and Reconstructive Surgery of the Policlinico Umberto I in Rome, from January to December 2006, treated 570 patients (308 men and 262 women), whose age was between 2 days and 85 years, affected by ulcers of various nature. Among our cases, 200 patients were selected and randomly separated in two different groups: group A consisting of 100 patients entirely treated with traditional medications; group B composed by 100 patients treated with advanced dressings. Every patient has locally been treated with periodic and specific medications, according to the type of difficult wound, and subsequently they proceeded to find out how to treat the systemic factors causing ulcer. The patients underwent 3 times a week to medications in those cases presenting infection signs and 2 times a week in those cases where no infection signs were shown, for period varying from 1 month up to one year for the chronic forms. RESULTS: The results showed a higher percentage of recovery reached by using the advanced dressings. Group A showed the followings results: the 53% of patients recovered from wounds; the remaining 47% patients did'nt not recover but in 17% cases medications showed to be of some help in the preparation of the vascular bed for the execution of a definitive operation (application of grafts or local edges), while the remaining 30% has shown a scarce improvement of the injury and they are still under treatment. Group B showed the 65% of patients recovered from wounds; as for the remaining 35% not recovered patients, medications represented an auxiliary aid to the preparation of the vascular bed for the execution of a definitive operation (application of grafts or local edges) for the 15% of patients, while the remaining 20%, even if not completely recovered, showed a notable improvement of the injury (reduction of the dimensions and disappearance of the infection and improvement of the patient quality of life). CONCLUSIONS: In synthesis, it emerges that the advanced dressings, if correctly used, offer advantages in terms of clinical effectiveness (rapid recovery from the injury), patient quality of the life and cheapness. It has also to be considered that the difficult wound is often the epiphenomenon of a systemic illness. The difficult wound requires, therefore, a multidisciplinary treatment.

Substance P provides neuroprotection in cerebellar granule cells through Akt and MAPK/Erk activation: evidence for the involvement of the delayed rectifier potassium current.

In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.

Up-regulation of proliferating cell nuclear antigen (PCNA) is closely associated with high-risk human papillomavirus (HPV) and progression of cervical intraepithelial neoplasia (CIN), but does not predict disease outcome in cervical cancer.

OBJECTIVE: Proliferating cell nuclear antigen (PCNA) is essential for DNA replication of mammalian cells and their small DNA tumour viruses. The E7 oncoprotein of high-risk human papillomavirus (HPV) is known to activate PCNA, shown to be up-regulated in CIN and cervical cancer (CC), but still incompletely studied as an intermediate endpoint marker in this disease. MATERIAL AND METHODS: As part of our HPV-PathogenISS study, a series of 150 CCs and 152 CIN lesions were examined using immunohistochemical (IHC) staining for PCNA, and tested for HPV using PCR with three primer sets (MY09/11, GP5+/GP6+, SPF). Follow-up data were available from all SCC patients, and 67 of the CIN lesions had been monitored with serial PCR for HPV after cone treatment. RESULTS: Expression of PCNA increased in parallel with the grade of CIN, with major up-regulation upon transition to CIN3 (OR 21.77; 95%CI 6.59-71.94) (p = 0.0001). Intense PCNA expression was 100% specific indicator of CIN, with 100% PPV, but suffers from low sensitivity (34.8%) and NPV (10.8%). PCNA expression was also significantly associated to HR-HPV with OR 3.02 (95%CI 1.71-5.34) (p = 0.0001), and this association was not confounded by the histological grade (Mantel-Haenszel common OR = 2.03; 95%CI 1.06-3.89) (p = 0.033). Expression of PCNA did not predict clearance/persistence of HR-HPV after treatment of CIN, and it was not a prognostic predictor in CC in univariate or in multivariate analysis. CONCLUSIONS: Up-regulation of PCNA was closely associated with HR-HPV and progressive CIN, most feasibly explained by the abrogation of normal cell cycle control by the E7 ongogene, reverting the p21(Cip1)-mediated inhibition of PCNA. However, the fact that PCNA is also expressed in normal squamous epithelium precludes the use of this marker as a potential screening tool for CC.

Nerve growth factor derivative NGF61/100 promotes outgrowth of primary sensory neurons with reduced signs of nociceptive sensitization.

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease. However, the development of an NGF-based therapy is limited by its potent pain activity. We have developed a "painless" derivative form of human NGF (NGF61/100), characterized by identical neurotrophic properties but a reduced nociceptive sensitization activity in vivo. Here we characterized the response of rat dorsal root ganglia neurons (DRG) to the NGF derivative NGF61/100, in comparison to that of control NGF (NGF61), analyzing the expression of noxious pro-nociceptive mediators. NGF61/100 displays a neurotrophic activity on DRG neurons comparable to that of control NGF61, despite a reduced activation of PLCγ, Akt and Erk1/2. NGF61/100 does not differ from NGF61 in its ability to up-regulate Substance P (SP) and Calcitonin Gene Related Peptide (CGRP) expression. However, upon Bradykinin (BK) stimulation, NGF61/100-treated DRG neurons release a much lower amount of SP and CGRP, compared to control NGF61 pre-treated neurons. This effect of painless NGF is explained by the reduced up-regulation of BK receptor 2 (B2R), respect to control NGF61. As a consequence, BK treatment reduced phosphorylation of the transient receptor channel subfamily V member 1 (TRPV1) in NGF61/100-treated cultures and induced a significantly lower intracellular Ca2+ mobilization, responsible for the lower release of noxious mediators. Transcriptomic analysis of DRG neurons treated with NGF61/100 or control NGF allowed identifying a small number of nociceptive-related genes that constitute an "NGF pain fingerprint", whose differential regulation by NGF61/100 provides a strong mechanistic basis for its selective reduced pain sensitizing actions.

Aberrant expression of VEGF-C is related to grade of cervical intraepithelial neoplasia (CIN) and high risk HPV, but does not predict virus clearance after treatment of CIN or prognosis of cervical cancer.

AIMS: Increased angiogenesis leads to invasion in cervical cancer. Vascular endothelial growth factors (VEGFs) are involved in angiogenesis, but molecular links to the most important aetiological agent, human papillomavirus (HPV), need clarifying. MATERIAL/METHODS: Archival samples-150 squamous cell carcinomas (SCCs) and 152 cervical intraepithelial neoplasia (CIN) lesions-were examined immunohistochemically for anti-VEGF-C antibody and for HPV by polymerase chain reaction (PCR). Follow up data were available for all SCC cases, and 67 CIN lesions were monitored with serial PCR to assess HPV clearance/persistence after treatment. RESULTS: High risk (HR) HPV types were closely associated with CIN (odds ratio, 19.12; 95% confidence interval, 2.31 to 157.81) and SCC (27.25; 3.28 to 226.09). There was a linear increase of VEGF-C expression-weak in CIN1 and intense in CIN3 and SCC (20.49; 8.69 to 48.26). VEGF-C upregulation was a sensitive (93.5%; 95% CI, 90.1% to 96.9%) marker of HR-HPV type (4.70; 2.17 to 10.21), but lost its significance in multivariate regression-p16(INK4a) and survivin were equally strong independent predictors of HR-HPV. Aberrant expression of VEGF-C did not predict clearance/persistence of HR-HPV after treatment of CIN. In cervical cancer, VEGF-C had no prognostic value in univariate or multivariate survival analysis. After adjustment for HR-HPV, FIGO stage, age, and tumour grade, only FIGO stage and age remained independent prognostic predictors. CONCLUSIONS: VEGF-C is an early marker of cervical carcinogenesis, with linearly increasing expression starting from low grade CIN. VEGF-C expression is closely related to HR-HPV in cervical lesions, probably because of its p53 independent upregulation by the E6 oncoprotein of HR-HPV.

Down-regulated nucleoside diphosphate kinase nm23-H1 expression is unrelated to high-risk human papillomavirus but associated with progression of cervical intraepithelial neoplasia and unfavourable prognosis in cervical cancer.

OBJECTIVE: One of the factors leading to an invasive phenotype is the nm23 family of metastases-associated genes. Of the six known members, nm23-H1 is the most frequently studied potential anti-metastatic gene in cervical cancer. However, the possible molecular links to oncogenic human papillomavirus (HPV) are completely unexplored as yet. MATERIALS AND METHODS: As a part of the HPV-Pathogen Istituto Superiore di Sanità study, a series of 150 squamous cell carcinomas (SCCs) and 152 cervical intraepithelial neoplasia (CIN) lesions were examined by immunohistochemical staining for nm23-H1, and tested for HPV by polymerase chain reaction (PCR) with three sets of primers (MY09/11, GP5(+)/GP6(+) and short PCR fragment). Follow-up data were available on all patients with SCC, and 67 CIN lesions were monitored by serial PCR for clearance or persistence of HPV after cone treatment. RESULTS: A linear decrease (p = 0.001) was observed in nm23-H1 expression, starting from CIN1 (85% with normal expression), with the most dramatic down regulation on transition from CIN2 (70% normal) to CIN3 (39%) and further to SCC (25%). Reduced expression was associated with CIN3 or cancer at an odds ratio 8.72 (95% confidence interval 4.13 to 18.41). Nm23-H1 was of no use as a marker of the high-risk human papillomavirus (HR-HPV) type, and it did not predict clearance or persistence of HR-HPV after treatment of CIN. Importantly, nm23-H1 expression was a significant prognostic factor in cervical cancer, reduced expression being associated with lower survival (p = 0.022) in univariate analysis. In the multivariate (Cox) regression model, however, only the International Federation of Gynecology and Obstetrics stage (p = 0.001) and age (p = 0.011) remained independent prognostic predictors. CONCLUSIONS: Down-regulated nm23-H1 expression is markedly associated with progression from CIN2 to CIN3, and predicts poor prognosis in cervical cancer. Nm23-H1 down regulation is probably orchestrated by mechanisms independent of HR-HPV oncoproteins and is possibly related to the emergence of a proteolytic phenotype.

Matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor (TIMP-2) are prognostic factors in cervical cancer, related to invasive disease but not to high-risk human papillomavirus (HPV) or virus persistence after treatment of CIN.

OBJECTIVE: Matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor (TIMP-2) are important regulators of cancer invasion and metastasis. Their associations to high-risk (HR) human papillomavirus (HPV) in cervical intra-epithelial neoplasia (CIN) and cervical cancer (CC) are unexplored and their prognostic significance in CC remains controversial. MATERIALS AND METHODS: As part of our HPV-PathogenISS study, a series of 150 CCs and 152 CIN lesions were examined using immunohistochemical (IHC) staining for MMP-2 and TIMP-2 and tested for HPV using PCR with 3 primer sets (MY09/11, GP5+/GP6+, SPF). Follow-up data were available from all squamous cell carcinoma patients and 67 CIN lesions had been monitored with serial PCR for HPV after cone treatment. RESULTS: MMP-2 increased with the grade of CIN, with major up-regulation upon transition to invasive cancer (OR 20.78) (95%CI 7.16-60.23) (p=0.0001). TIMP-2 retained its normal expression until CIN3, with dramatic down-regulation in invasive disease (p=0.0001 for trend). Thus, the MMP2:TIMP-2 ratio increased with progressive CIN, exceeding the value 1.0 only in invasive disease. Both MMP-2 and TIMP-2 are highly specific (TIMP-2; 100%) discriminators of CIN with 100% positive predictive value (TIMP-2), but suffer from low sensitivity and negative predictive value. Neither MMP-2 nor TIMP-2 showed any significant association with HR HPV or virus persistence/clearance. TIMP-2 (but not MMP-2) was a significant predictor of survival in univariate (Kaplan-Meier) analysis (p=0.007), but lost its significance in multivariate (Cox) analysis. CONCLUSION: The activities of MMP-2 and TIMP-2 in cervical carcinogenesis seem to be unrelated to HR-HPV The inverse MMP-2:TIMP-2 ratio is a sign of poor prognosis. A combination of a TIMP-2 assay with another test showing high SE and high NPV (e.g., HCII for HPV) should provide a potential screening tool capable of accurate detection of CIN.