The reactivity of cyclopropyl cyanide in titan's atmosphere: a possible pre-biotic mechanism.

Cyclopropyl cyanide and other simple nitriles detected in Titan's atmosphere could be precursors leading to the formation of organic macromolecules in the atmosphere of Saturn's largest satellite. Proposing a thermodynamically possible mechanism that explains their formation and supports experimental results represents a difficult challenge. Experiments done in the Atomic and Molecular Physics Laboratory at the University of Trento (AMPL) have studied the ion-molecule reaction between cyclopropyl cyanide and its protonated form, with reaction products being characterized by mass spectrometry. In addition to the expected ion-molecule adduct stabilized by non-covalent long-range interactions, in this work we prove that another distinct species having the same mass to charge ratio (m/z) of 135 is also produced. Moreover, from a previous study of the neutral cyclopropyl cyanide potential energy surface (PES) which shows a partial biradical character it has been possible to characterize the formation through the bimolecular reaction of a new covalent cyclic organic molecule. Calculations have been carried out at the ab initio Møller-Plesset (MP2) level of theory, ensuring the connectivity of the stationary points by using the intrinsic reaction coordinate (IRC) procedure. In order to characterize the reaction transition state, multireference calculations were done using a complete active space involving six electrons and six molecular orbitals [CAS (6 e-, 6 m.o.)]. This study opens the possibility of exploring the formation of new organic molecules by gaseous phase ion-molecule interaction schemes, with such molecules having relevance in interstellar space and in astrobiology (and may be involved in prebiotic molecular evolution).

Onabotulinumtoxin A for prophylaxis in chronic migraine: preliminary data from Headache Regional Centre of Aosta Valley.

Chronic migraine (CM) is a complex neurological disorder associated with substantial disability that affects approximately 2 % of general population. Onabotulinumtoxin A is employed for patients suffering from CM refractory to common therapeutic prophylaxis. Since May 2013, we have selected 22 patients referring to our headache centre with a history of CM which meets the diagnostic criteria of ICHD-3 beta (2013). The patients have been treated with onabotulinumtoxin A injection in 31 sites according to the protocol of the PREEMPT study at the total dosage of 155 U/treatment every 3 months. So far, eight patients have been subjected to three treatment sessions, five patients to two treatments and nine patients to one treatment. Three patients dropped for low compliance, but there were no serious adverse events. The frequency of headache days, the intensity of headache and the headache disability have been measured using headache diary, migraine disability assessment (MIDAS) questionnaire and headache impact test (HIT)-6 score. Data concerning the 13 patients who have been submitted to at least two treatment sessions have already shown a decrease of headache days of 20.64 % after the first treatment; MIDAS and HIT-6 scores have been significantly improved with a reduction of the scores, respectively, of 38.45 % for MIDAS and of 6.95 % for HIT-6. These are preliminary results because the observation time, the number of treatment sessions and the number of patients treated are still few.

Early application of percutaneous vertebroplasty reduces pain without affecting peripheral blood stem cell (PBSC) collection and transplant in newly diagnosed multiple myeloma (MM) patients.

Vertebral fractures occur in over 60% of newly diagnosed multiple myeloma (MM) patients and can cause pain, disability and poor quality of life. Antimyeloma therapy can lead to symptoms improvement, but these effects can take time to be perceived. Application of radiotherapy prior to peripheral blood stem cells (PBSC) mobilisation can impair stem cell collection. Percutaneous vertebroplasty has been proposed as a suitable option to rapidly relieve bone pain from vertebral fractures in MM patients, but, little is known about the effects of this procedure on subsequent PBSC mobilisation, collection and transplant. Eighteen patients (10M/8F, median age 64.5 years) with untreated MM and painful vertebral lesions underwent vertebroplasty prior to proceed to the planned transplant program at our Institution. Forty-one procedures were performed at C2-L5 levels, eight patients were treated at ≥2 levels. Ninety-five per cent of the cases obtained a complete or optimal pain control. All the patients successfully mobilised PBSC (median CD34+ cells = 10.8 × 10(6) /kg) and underwent autologous PBSC transplant; both polymorphonucleates and platelets recovery averaged 11 days. Our data seem to suggest that percutaneous vertebroplasty is useful in newly diagnosed MM patients with painful vertebral fractures as it allows rapid and durable achievement of pain control, without interfering with further treatment.

EELS: Autonomous snake-like robot with task and motion planning capabilities for ice world exploration.

Ice worlds are at the forefront of astrobiological interest because of the evidence of subsurface oceans. Enceladus in particular is unique among the icy moons because there are known vent systems that are likely connected to a subsurface ocean, through which the ocean water is ejected to space. An existing study has shown that sending small robots into the vents and directly sampling the ocean water is likely possible. To enable such a mission, NASA's Jet Propulsion Laboratory is developing a snake-like robot called Exobiology Extant Life Surveyor (EELS) that can navigate Enceladus' extreme surface and descend an erupting vent to capture unaltered liquid samples and potentially reach the ocean. However, navigating to and through Enceladus' environment is challenging: Because of the limitations of existing orbital reconnaissance, there is substantial uncertainty with respect to its geometry and the physical properties of the surface/vents; communication is limited, which requires highly autonomous robots to execute the mission with limited human supervision. Here, we provide an overview of the EELS project and its development effort to create a risk-aware autonomous robot to navigate these extreme ice terrains/environments. We describe the robot's architecture and the technical challenges to navigate and sense the icy environment safely and effectively. We focus on the challenges related to surface mobility, task and motion planning under uncertainty, and risk quantification. We provide initial results on mobility and risk-aware task and motion planning from field tests and simulated scenarios.

MR-defecography in obstructed defecation syndrome (ODS): technique, diagnostic criteria and grading.

BACKGROUND: The aim of this study was to evaluate the use of a magnetic resonance (MR)-based classification system of obstructive defecation syndrome (ODS) to guide physicians in patient management. METHODS: The medical records and imaging series of 105 consecutive patients (90 female, 15 male, aged 21-78 years, mean age 46.1 ± 5.1 years) referred to our center between April 2011 and January 2012 for symptoms of ODS were retrospectively examined. After history taking and a complete clinical examination, patients underwent MR imaging according to a standard protocol using a 0.35 T permanent field, horizontally oriented open-configuration magnet. Static and dynamic MR-defecography was performed using recognized parameters and well-established diagnostic criteria. RESULTS: Sixty-seven out of 105 (64 %) patients found the prone position more comfortable for the evacuation of rectal contrast while 10/105 (9.5 %) were unable to empty their rectum despite repeated attempts. Increased hiatus size, anterior rectocele and focal or extensive defects of the levator ani muscle were the most frequent abnormalities (67.6, 60.0 and 51.4 %, respectively). An MR-based classification was developed based on the combinations of abnormalities found: Grade 1 = functional abnormality, including paradoxical contraction of the puborectalis muscle, without anatomical defect affecting the musculo-fascial structures; Grade 2 = functional defect associated with a minor anatomical defect such as rectocele ≤ 2 cm in size and/or first-degree intussusception; Grade 3 = severe defects confined to the posterior anatomical compartment, including >2 cm rectocele, second- or higher-degree intussusception, full-thickness external rectal prolapse, poor mesorectal posterior fixation, rectal descent >5 cm, levator ani muscle rupture, ballooning of the levator hiatus and focal detachment of the endopelvic fascia; Grade 4 = combined defects of two or three pelvic floor compartments, including cystocele, hysterocele, enlarged urogenital hiatus, fascial tears enterocele or peritoneocele; Grade 5 = changes after failed surgical repair abscess/sinus tracts, rectal pockets, anastomotic strictures, small uncompliant rectum, kinking and/or lateral shift of supra-anastomotic portion and pudendal nerve entrapment. CONCLUSIONS: According to our classification, Grades 1 and 2 may be amenable to conservative therapy; Grade 3 may require surgical intervention by a coloproctologist; Grade 4 would need a combined urogynecological and coloproctological approach; and Grade 5 may require an even more complex multidisciplinary approach. Validation studies are needed to assess whether this MR-based classification system leads to a better management of patients with ODS.

Infrared spectroscopy of copper-resveratrol complexes: a joint experimental and theoretical study.

Infrared multiple-photon dissociation spectroscopy has been used to record vibrational spectra of charged copper-resveratrol complexes in the 3500-3700 cm(-1) and 1100-1900 cm(-1) regions. Minimum energy structures have been determined by density functional theory calculations using plane waves and pseudopotentials. In particular, the copper(I)-resveratrol complex presents a tetra-coordinated metal bound with two carbon atoms of the alkenyl moiety and two closest carbons of the adjoining resorcinol ring. For these geometries vibrational spectra have been calculated by using linear response theory. The good agreement between experimental and calculated IR spectra for the selected species confirms the overall reliability of the proposed geometries.

Idiosyncratic control of the center of mass in expert climbers.

Here, we studied posture and movement coordination adopted by expert climbers. The investigation of such expertise might be of particular interest to gain understanding about the mechanisms underlying the biomechanical control of vertical quadrupedal locomotion. A novel custom setup was developed to analyze the motion of the center-of-mass (COM) and complementary information about the dynamic distribution of vertical reaction forces under the feet during climbing in nine elite climbers (EC) and nine control subjects. Two adaptive features were found in EC. First, unexpectedly they tended to maintain larger COM distances from the wall relative to controls, during both the static and dynamic phases of vertical motion (by ∼5 cm in both cases). Second, while the control subjects tended to restrain the lateral motion of the COM, all EC demonstrated systematic COM oscillations (∼1.3 times larger) associated with a significant alternating dynamic redistribution of the body weight between the limbs during the double support phase. The latter phenomenon likely reflects an adopted basic climbing strategy in experts. Furthermore, a convergence of the optimal solution towards a more diagonal climbing strategy in EC may shed light on the origin of the diagonal gait in primates and early hominids habituated to quadrupedal vertical locomotion.

Energy cost and efficiency of ski mountaineering. A laboratory study.

AIM: The purpose of this study is to estimate the energy cost of ski mountaineering at different speeds under laboratory conditions. METHODS: By using roller skis on a motorized treadmill we have estimated the energy cost and biomechanics parameters of ski mountaineering as a function of climbing speed at the gradient of 21%. RESULTS: The metabolic energy spent for unit mass and distance, C, shows a broad minimum of about 10.6±0.2 J kg-1m-1 at roughly 3.5 km h-1. In addition we find a size-dependent effect: tall subjects spend less metabolic energy for unit mass and distance than small subjects at the same speed. CONCLUSION: The value of C measured in laboratory agrees with that obtained in the field at the preferred speed. This shows that skiers self select a speed that minimizes their metabolic cost. The dependence of C on the subject's size is explained by a simple model of the skier's dynamics. In addition we have calculated the ratio between mechanical work and metabolic energy, which may give some hints on the efficiency as a function of the speed. It turns out that efficiency increases with the speed up to a maximum located at around 4.5 km/h.

Eosinophilic esophagitis: an Italian experience.

BACKGROUND: Eosinophilic esophagitis is an esophageal disorder characterized by esophageal and/or upper gastrointestinal tract symptoms, and by dense esophageal eosinophilia associated with a normal gastric and duodenal mucosa. Prevalently reported in children, eosinophilic esophagitis has recently been reported with increased frequency also in adults. AIMS: The purpose of this study was to report our experience with eosinophilic esophagitis in Italy, since there are only very few series of such patients in our country. PATIENTS AND METHODS: We retrospectively reviewed the histological data of consecutive patients with a diagnosis of esophagitis or reflux disease in the period September 2004-September 2008. Eosinophils were counted where they appeared most numerous in the biopsy, with a cutoff > 15 eosinophils in more than one high-power field as diagnostic of eosinophilic esophagitis. Patients were excluded if gastric or duodenal biopsies showed a prominent eosinophilic infiltrate. RESULTS: Twenty two patients (14 adults, 8 children, age range 2-59 years) were identified according to the above criteria. The average eosinophil count was 86/ high-power field (range 31-150), associated with other pathologic features (eosinophilic microabscesses eosinophil degranulation, basal zone hyperplasia, papillary elongation). The main clinical complaints were dysphagia, food impaction, and heartburn, and endoscopic findings consisted of mucosal thickening and inelasticity, longitudinal shearing, rings, and white specks, without difference between adults and children for both clinical and endoscopic variables. CONCLUSIONS: Eosinophilic esophagitis is not rare in Italy, and displays clinical, endoscopic, and pathologic features similar to those described in other countries.

MYC translocation-negative classical Burkitt lymphoma cases: an alternative pathogenetic mechanism involving miRNA deregulation.

The molecular feature of Burkitt lymphoma (BL) is the translocation that places c-Myc under the control of immunoglobulin gene regulatory elements. However, there is accumulating evidence that some cases may lack an identifiable MYC translocation. In addition, during the EUROFISH project, aiming at the standardization of FISH procedures in lymphoma diagnosis, we found that five cases out of 35 classic endemic BLs were negative for MYC translocations by using a split-signal as well as a dual-fusion probe. Here we investigated the expression pattern of miRNAs predicted to target c-Myc, in BL cases, to clarify whether alternative pathogenetic mechanisms may be responsible for lymphomagenesis in cases lacking the MYC translocation. miRNAs are a class of small RNAs that are able to regulate gene expression at the post-transcriptional level. Several studies have reported their involvement in cancer and their association with fragile sites in the genome. They have also been shown to control cell growth, differentiation, and apoptosis, suggesting that these molecules could act as tumour suppressors or oncogenes. Our results demonstrated a modulation of specific miRNAs. In particular, down-regulation of hsa-let-7c was observed in BL cases, compared to normal controls. More interestingly, hsa-mir-34b was found to be down-regulated only in BL cases that were negative for MYC translocation, suggesting that this event might be responsible for c-Myc deregulation in such cases. This hypothesis was further confirmed by our in vitro experiments, which demonstrated that increasing doses of synthetic hsa-mir-34b were able to modulate c-Myc expression. These results indicate for the first time that hsa-mir-34b may influence c-Myc expression in Burkitt lymphoma as the more common aberrant control exercised by the immunoglobulin enhancer locus.

Guided-ion-beam and ab initio study of the Li+, K+, and Rb+ association reactions with gas-phase butanone and cyclohexanone in their ground electronic states.

The association reactions between Li(+), K(+), and Rb(+) (M) and butanone and cyclohexanone molecules under single collision conditions have been studied using a radiofrequency-guided ion-beam apparatus, characterizing the adducts by mass spectrometry. The excitation function for the [M-(molecule)](+) adducts (in arbitrary units) has been obtained at low collision energies in the 0.10 eV up to a few eV range in the center of mass frame. The measured relative cross sections decrease when collision energy increases, showing the expected energy dependence for adduct formation. The energetics and structure of the different adducts have been calculated ab initio at the MP2(full) level, showing that the M(+)-molecule interaction takes place through the carbonyl oxygen atom, as an example of a nontypical covalent chemical bond. The cross-section energy dependence and the role of radiative cooling rates allowing the stabilization of the collision complexes are also discussed.

An experimental guided-ion-beam and ab initio study of the ion-molecule gas-phase reactions between Li+ ions and iso-C3H7Cl in their ground electronic state.

Reactive collisions between Li(+) ions and i-C(3)H(7)Cl molecules have been studied in the 0.20-12.00 eV center-of-mass energy range using an octopole radio frequency guided-ion beam apparatus recently developed in our laboratory. At low collision energies, dehydrohalogenation reactions giving rise to Li(C(3)H(6))(+) and Li(HCl)(+) are the main reaction channels, while at higher ones C(3)H(7)(+) and C(2)H(3)(+) become dominant, all their reactive cross sections having been measured as a function of the collision energy. To obtain information about the potential energy surfaces (PESs) on which the reactive processes take place, ab initio calculations at the MP2 level have been performed. For dehydrohalogenations, the reactive ground singlet PES shows ion-molecule adduct formation in both the reactant and product sides of the surface. Following the minimum energy path connecting both minima, an unstable intermediate and the corresponding barriers, both lying below the reactant's energy, have been characterized. The entrance channel ion-molecule adduct is also involved in the formation of C(3)H(7)(+), which then generates C(2)H(3)(+) via an CH(4) unimolecular elimination. A qualitative interpretation of the experimental results based on ab initio calculations is also included.

The energy cost of ski mountaineering: effects of speed and ankle loading.

AIM: The purpose of this study is to determine the energy cost (EC) of ski mountaineering and its variation with speed and ankle loading. METHODS: Seven male skiers volunteered to participate in this study. Field tests (500 m, gradient 21%) were executed on packed snow at an altitude of about 1600 m. Measurements were carried out breath by breath by a portable gas analyzer. Energy cost of uphill skiing was calculated from the steady state VO(2). In the speed protocol each subject was asked to repeat the same route at three different speed levels. In the weight protocol, subjects were instructed to maintain the preferred speed for three trials on the same track while wearing different weight bands on their ankle. RESULTS: At the self-selected speed of 1.07+/-0.05 m s(-1) and without extra load beside the normal equipment, the mean value of EC on packed snow is 10.6+/-0.4 J kg(-1m)(-1). A percentage variation of the speed (%speed) produces a corresponding percentage variation of the energy cost %EC = 0.32 x %speed. The %EC as a function of the percentage of added load, %weight, with respect to the total weight of the subject, including ski, bindings, and boots is given by %EC = 1.71 x %weight. CONCLUSIONS: Data obtained in the present study constitute the first quantitative description of EC for ski mountaineering and result higher than for walking or snowshoeing. Effects due to ankle loading appear negligible for recreational skiers, while they should be taken into account in agonistic competition.

Expert Panel Consensus Statement for Proper Evaluation of First Relapse in Multiple Myeloma.

PURPOSE OF REVIEW: A working group of six expert physicians convened to assess the spectrum of multiple myeloma relapse presentations, discussed the features that can define the disease as aggressive and not aggressive, and established whether this information could help in selecting treatment together with the characteristics of disease and of patients and type of prior therapy. RECENT FINDINGS: The working group agreed that relapse should be distinguished between biochemical and clinical according to IMWG. Moreover, the expert panel defined "aggressive disease" as a clinical condition that requires therapy able to induce a rapid and as deep as possible response to release symptoms and to avoid impending danger of new events. According to this definition, relapse was considered aggressive if it presents with at least one of the following features: doubling of M protein rate over 2 months, renal insufficiency, hypercalcemia, extramedullary disease, elevated LDH, high plasma cell proliferative index, presence of plasma cells in peripheral blood, or skeletal-related complications. Moreover, the panel agreed that this classification can be useful to choose therapy in first relapse together with other patient, disease, and prior therapy characteristics. So, this item was included in a new therapeutic algorithm. The treatment choice in MM at relapse is wider than in the past with the availability of many new therapeutic regimens leading to increased diversity of approaches and relevant risk of inappropriate treatment decisions. A practical classification of relapses into aggressive or non-aggressive, included in a decisional algorithm on MM management at first relapse, could help to make the appropriate treatment decisions.

Renal involvement in systemic amyloidosis: an Italian collaborative study on survival and renal outcome.

BACKGROUND: Few data are available from large population-based studies on survival and renal outcome of patients with renal involvement and different types of systemic amyloidosis. METHODS: Two hundred and ninety of over 373 patients affected from systemic amyloidosis with renal involvement diagnosed in Italy between January 1995 and December 2000 were followed from diagnosis to death or until the last available clinical control. Eighty-three patients were excluded from analysis either because the amyloid type remained undetermined or they were lost at follow-up. Clinical and laboratory information was collected according to the different types of amyloidosis using a specific form which included renal function with 24 h proteinuria at diagnosis and at the end of follow-up, the type and the date of onset of dialysis and the kind of treatment they underwent. RESULTS: The median time of follow-up was 24 months in primary (AL) amyloidosis (range: 1-88 months), 16 months in AL with associated multiple myeloma (MM + AL: range 1-76 months), 30 months in reactive (AA) amyloidosis (range: 1-99 months) and 52 months in patients with familial forms (AF: range 14-82 months). Patients with AL showed a significantly shorter survival than AA. Despite no significant differences of renal outcome or survival on dialysis being observed between the two groups, a lower renal survival with a higher number of patients who progressed to end-stage renal disease (ESRD) was observed in patients with AA. Overall survival was markedly improved in patients with AL who underwent a specific therapy (conventional chemotherapy or autologous stem cell transplantation (ASCT)) even in the absence of a positive kidney response. Multivariate analysis showed cardiac involvement and specific therapy to significantly influence survival in AL whereas age, serum creatinine (sCr) and heart involvement significantly affected survival in AA. In both groups, sCr and heart involvement were the most relevant predictors for renal outcome, together with urinary protein excretion, in patients with AA. CONCLUSIONS: Our results show a worse survival in AL due to the higher prevalence of heart involvement in this group and emphasize that a specific therapy significantly prolongs survival and slows the progression of renal disease in patients with AL. We suggest that a late nephrological referral is likely the cause of the higher sCr found at presentation in patients with AA and probably accounts for the lower renal survival observed in the short term in these patients. At the time being, renal transplantation and ASCT are still rare therapeutic options for renal patients affected from systemic amyloidosis.

Epidemiological, clinical and viral determinants of the increased prevalence of high-risk human papillomavirus (HPV) infections in elderly women.

BACKGROUND: Population-based studies have reported a second peak of human papillomavirus (HPV) prevalence among women > 55 years, but reasons for this U-shaped HPV prevalence curve are poorly understood. OBJECTIVES: To analyse determinants of high-risk HPV (HR-HPV) infections among postmenopausal women. STUDY DESIGN AND METHODS: A cohort of 3,187 women was stratified into three age categories: i) youngest age group < 25 years (n = 1.103); ii) women between 26-55 years (n = 2.004), and iii) women > 55 years (n = 80), analysed for epidemiological, clinical and virological determinants of their HR-HPV infections. Real-time PCR was used for HPV genotyping, analysis of viral loads for HPV16, 18/45, 31, 33/52/58, 35 and 39, and load of integrated HPV16. RESULTS: Age-standardised prevalence of HR-HPV infections showed a second peak among women > 55 years, with a perfect U-shaped curve (R2 = 0.966). The factors explaining this increased HR-HPV prevalence among older women include: i) cohort effect, ii) higher viral loads for HR-HPV types with cubic model curve (R2 = 0.714) for HPV 16, iii) distinct shift (p = 0.0001) from multiple-type infections to single HR-HPV types, iv) transition from episomal to integrated HPV16 (p = 0.009), v) higher load of integrated HPV16 (p = 0.009), and, vi) higher proportion of incident infections, higher rate of viral persistence, and lower rate of HR-HPV clearance. CONCLUSIONS: These data suggest that in women who fail to eradicate their HR-HPV infection until menopause, selection of integrated viral clone has taken place, driving the process towards progressing disease. Consequent to this, most of the HR-HPV infections in women > 55 years were associated with high-grade CIN or invasive carcinoma.

SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis.

The molecular basis of advanced systemic mastocytosis (SM) is not fully understood and despite novel therapies the prognosis remains dismal. Exome sequencing of an index-patient with mast cell leukemia (MCL) uncovered biallelic loss-of-function mutations in the SETD2 histone methyltransferase gene. Copy-neutral loss-of-heterozygosity at 3p21.3 (where SETD2 maps) was subsequently found in SM patients and prompted us to undertake an in-depth analysis of SETD2 copy number, mutation status, transcript expression and methylation levels, as well as functional studies in the HMC-1 cell line and in a validation cohort of 57 additional cases with SM, including MCL, aggressive SM and indolent SM. Reduced or no SETD2 protein expression-and consequently, H3K36 trimethylation-was found in all cases and inversely correlated with disease aggressiveness. Proteasome inhibition rescued SETD2 expression and H3K36 trimethylation and resulted in marked accumulation of ubiquitinated SETD2 in SETD2-deficient patients but not in patients with near-normal SETD2 expression. Bortezomib and, to a lesser extent, AZD1775 alone or in combination with midostaurin induced apoptosis and reduced clonogenic growth of HMC-1 cells and of neoplastic mast cells from advanced SM patients. Our findings may have implications for prognostication of SM patients and for the development of improved treatment approaches in advanced SM.

International uniform response criteria for multiple myeloma.

New uniform response criteria are required to adequately assess clinical outcomes in myeloma. The European Group for Blood and Bone Marrow Transplant/International Bone Marrow Transplant Registry criteria have been expanded, clarified and updated to provide a new comprehensive evaluation system. Categories for stringent complete response and very good partial response are added. The serum free light-chain assay is included to allow evaluation of patients with oligo-secretory disease. Inconsistencies in prior criteria are clarified making confirmation of response and disease progression easier to perform. Emphasis is placed upon time to event and duration of response as critical end points. The requirements necessary to use overall survival duration as the ultimate end point are discussed. It is anticipated that the International Response Criteria for multiple myeloma will be widely used in future clinical trials of myeloma.

Early integration of high copy HPV16 detectable in women with normal and low grade cervical cytology and histology.

BACKGROUND: Integration of human papillomavirus (HPV) DNA has been considered a late event in cervical carcinogenesis. However, integrated forms of HPV were recently detected in cancer precursor lesions using a new real time polymerase chain reaction (PCR) to detect the deletions at the 3362-3443 region of HPV16 E2 OBJECTIVE: To study the frequency of HPV16 DNA integration in cervical lesions and compare the sensitivity of an additional upstream region of the E2 ORF (2962-3138) in detecting HPV integration. METHODS: Using the TaqMan based PCR, HPV16 positive DNA samples were analysed in 164 cervical scrapings from women participating in a multicentre screening trial. Biopsy confirmation was available in 62 cases. RESULTS: Primers targeting the 3362-3443 region detected the majority of E2 deletions. In only 23% of the samples was the E2 upstream region equal or better target than the 3362-3443 region. Mixed (episomal/integrated) pattern was the most prevalent physical state of HPV16, also present in PAP smears with normal morphology. Pure integrated form was most prevalent in HSIL and cancer lesions, but also detectable in low grade abnormalities (NSIL, ASC-US, LSIL). Women with only integrated HPV16 were almost 10 years older than those with episomal HPV16. Viral load of integrated HPV16 was related to cytological abnormality (p = 0.003) but not to histology. CONCLUSIONS: Integrated HPV16 is present in low grade cervical lesions, mostly mixed with the episomal form. Women with the pure integrated form of HPV16 are older than those with the other forms.

Azidothymidine-induced cytotoxicity and incorporation into DNA in the human colon tumor cell line HCT-8 is enhanced by methotrexate in vitro and in vivo.

We have reported that 5-fluorouracil can increase the cytotoxic and antineoplastic activity of 3'-azido-3'-deoxythymidine (AZT). To further evaluate the antineoplastic utility of AZT we now have assessed its effect in combination with methotrexate (MTX) in the human colon tumor model HCT-8. Incubation of these cells for 5 days in AZT and MTX caused a reduction in the 50% inhibitory concentration of AZT and isobologram analysis revealed additive effects which were reversed by the addition of 50 microM thymidine to the incubation media. This enhanced cytotoxicity appeared not to be related to an effect of AZT on MTX activity; in whole-cell assays the ability of MTX to inhibit de novo dTMP synthesis and deplete intracellular pools of dTTP was not affected by AZT. In contrast, although MTX did not alter AZT triphosphate production, it did affect AZT triphosphate utilization in DNA synthesis. Incubation of cells for 24 h in [3H]AZT alone (5 microM, 3 microCi/ml) resulted in 6.6 pmol AZT incorporated into cellular DNA/10(6) cells. Coincubation of these cells in [3H]AZT (5 microM) plus 5 or 15 nM MTX increased AZT incorporation into DNA to 8.0 and 20.5 pmol/10(6) cells, respectively. Biochemically, this effect appeared to correlate with the concentration-dependent ability of 5 or 15 nM MTX to deplete intracellular dTTP pools, which were reduced by 25 and 49%, respectively. Further evidence of the relationship between intracellular dTTP pools and AZT cytotoxicity was that, in the presence of both MTX and 50 microM thymidine, intracellular dTTP pools remained near normal levels and the incorporation of 5 microM AZT into DNA was not enhanced. Therapeutically, studies conducted in athymic (nude) mice bearing HCT-8 xenografts that received six weekly cycles of MTX (87.5 mg/kg) and AZT (300 mg/kg) revealed that the two-drug regimen exerted superior antineoplastic effects compared to either drug alone (treated versus control approximately 0.9 for AZT or MTX and approximately 0.3 for MTX plus AZT). In addition, the combination did not increase toxicity compared to therapy with MTX alone. These findings are discussed in light of their biochemical and clinical implications.