Influence of KRAS mutations on clinical outcome in patients with curatively resected stage III colon cancer treated with adjuvant chemotherapy.

OBJECTIVE: To profile and correlate KRAS mutations with outcome in stage III colon cancer (CC) patients who underwent adjuvant chemotherapy following curative resection surgery. PATIENTS AND METHODS: In this retrospective study, eligible patients were those with resected stage III CC who underwent 6-months adjuvant chemotherapy, either with fluoropyrimidine monotherapy (FP) or with oxaliplatin-based regimens (O-FP). Disease-free survival (DFS) and overall survival (OS) were analyzed and computed using the Kaplan-Meier method and the log-rank test. RESULTS: The study population included 148 patients (n=65 FP and n=83 O-FP). We identified KRAS mutations in 41/148 (27%) patients, of which 18 (44%) received FP and 23 (56%) O-FP. Five-year DFS and OS were significantly higher in patients with KRAS wild-type vs. mutant [DFS: 78 vs. 56%, HR: 0.47 (95% CI: 0.25; 0.87), p=0.01; OS: 73 vs. 68%, HR: 0.44 (95% CI: 0.21; 0.88), p=0.01]. In patients treated with FP, the 5-year DFS and OS was significantly improved in the KRAS wild-type vs. mutant group, respectively [DFS: 80 vs. 43%, HR: 2.88 (95% CI: 0.67; 3.76), p=0.014; OS: 85 vs. 68%, HR: 0.27 (95% CI: 0.10; 0.73), p=0.005]. Conversely, 5-year DFS and OS were not statistically different for patients with KRAS wild-type vs. mutations treated with O-FP, respectively [DFS: 78 vs. 65%, HR: 1.59 (95% CI: 0.67; 3.76), p=0.281; OS: 80 vs. 75%, HR: 0.73 (95% CI: 0.55; 2.12), p=0.57)]. CONCLUSIONS: Our results suggest that curatively resected stage III CC patients exhibiting wild-type KRAS status might benefit from FP alone. Conversely, an oxaliplatin-containing regimen should be recommended in KRAS mutated patients.

Hormonal and metabolic gender differences in a cohort of myotonic dystrophy type 1 subjects: a retrospective, case-control study.

PURPOSE: Myotonic dystrophy type 1 (DM1) is a genetic disorder caused by CTG expansion in the DMPK gene. The aim was to investigate the endocrine and metabolic aspects of DM1. PATIENTS AND METHODS: Retrospective, case-control study. We compared pituitary, thyroid, adrenal, gonadal and liver function and glycolipid metabolism of 63 DM1 patients against 100 control subjects. Given age-related differences, 2 further subgroups were created to investigate the pituitary-gonadal axis: < 41 (1a) and ≥ 41 (1b) years old for male subjects and < 46 (2a) and ≥ 46 (2b) years old for female subjects. Testicular and thyroid ultrasounds were also performed in the DM1 group. RESULTS: FT3 and FT4 were significantly lower in DM1 men than controls, while for both males and females, thyroglobulin, ACTH and cortisol were significantly higher in the DM1 group. Gonadotropin levels were significantly higher and inhibin B and DHEA-S levels significantly lower in DM1 patients than controls for both male subgroups. Testosterone and SHBG were significantly higher in controls than in patients for subgroup 1a. Prolactin was significantly higher in patients in subgroups 1b, while testosterone was lower in subgroup 2a than in age-matched female controls. A correlation between the number of CTG repeats and the percentage of male hypogonadal subjects was found. Finally, there was a worse glucose and lipid pattern and significantly higher transaminase and gamma-GT levels in both male and female patients. CONCLUSIONS: The high frequency of endocrine and metabolic abnormalities in DM1 highlights the importance of endocrine monitoring to enable the prompt initiation of a suitable therapy.

Behavioural and physiological indicators of shelter dogs' welfare: reflections on the no-kill policy on free-ranging dogs in Italy revisited on the basis of 15 years of implementation.

The Italian National Law 281 of 1991 forbids the euthanatization of free-ranging dogs, unless they have an incurable illness or are proved to be dangerous. Without neglecting the undeniable benefits of the "no-kill" policy, nevertheless it has brought about a chronic overpopulation in shelters and, as a result, higher costs of management and welfare problems since some dogs remain in the shelter for life. In 2004-2008, the Istituto Zooprofilattico Sperimentale of the Lazio and Tuscany regions carried out a survey in the Lazio Region to verify the effects of the Italian National Law 281/91 on free-ranging dog management following 15 years from its implementation. One of the aims of the study was an assessment of the welfare of dogs in a shelter sample (8 shelters out of 47 censused in the Lazio Region). 97 mixed-breed dogs were selected, their behaviour was studied and a blood sample was taken for each dog in order to determine the individual blood concentration of cortisol and the amount of oxidative damage (level of dRoms), as well as the amount of antioxidants to cope with it. Moreover, the total leukocyte count (leukogram) was accomplished. We ran general backward stepwise regression models using "level of antioxidant", "level of dRoms" and "level of serum cortisol" as dependent variables respectively. The results showed that the most important variable that improved the level of welfare of dogs consisted in having the opportunity to regularly go out of the cage for a walk, whereas other variables like gender, size of the cage (small, medium, large), being alone in the cage, and being neutered/entire, had no significant effect on the physiological indicators of welfare. Dogs that enjoyed the regular walk had a higher total antioxidant capacity, and performed a lower frequency of displacing activities and stereotyped behaviour. Moreover, oxidative stress parameters seem to be indicators well matched with behavioural indicators of stress. Thus, for the first time, markers of oxidative status are utilised for the welfare evaluation in the domestic dog. Furthermore, the results of this paper give some suggestion about how small steps can help to improve shelters and, furthermore, this paper intends to solicit the debate on the no-kill policy.

Ultrasound transmission gel as a bolus device for skin irradiation of irregular surfaces: technical note.

PURPOSE: This paper describes an uncommon radiation treatment of the external ear, with ultrasound (US) transmission gel used as bolus device to compensate for the irregularity of the target surface. MATERIALS AND METHODS: Postoperative radiotherapy for cutaneous carcinoma was performed with a single high-energy electron beam directed over the ear auricle. Due to the irregular surface of the target, a "missing tissue" compensator was employed. Daily, after patient positioning, the concha was filled and flattened with US gel, and a dose of 54 Gy in 27 fractions was delivered. RESULTS: Water equivalence of the gel was verified by comparing the gel's computed tomography (CT) number [Hounsfield units (HU)] and density with the corresponding values for water and another commercial bolus device. Whereas ultrasound gel and water had comparable values (HU: 0; density 1 g/cm(3) for both), the corresponding values for the commercial device were slightly higher (HU: 80; density 1.02 g/cm(3)). CONCLUSIONS: Ultrasound gel proved to be an easy, fast and cheap compensating tool. Its water equivalence allows it to be used as an alternative to water, though easier to position and with lower risk of displacement. Thus, it is recommendable as a practical tool for most irregular sites. Further investigations are warranted to validate this solution in more complex irradiation techniques.

15-week long school-based nutritional education program to promote Italian primary schoolchildren's fruit and vegetable intake.

A short-version (15-week long) of school-based nutrition education program called "Bring Fruit to School" to enhance primary schoolchildren's fruit and vegetable (F&V) intake was evaluated. We recruited 199 primary schoolchildren from 8 classes (grade 2-5). The primary end-point was an increase in the children's F&V intake. The intervention period lasted 15 weeks, and was divided into three phases: weeks 1-5, weeks 6-10 and weeks 11-15. The F&V intake of the schoolchildren was also monitored in a subsequent follow-up period (16th-32nd week). By week 15, 92 (46.2%, P < .001) schoolchildren increase fruit intake and 91 (45.7%, P < .001) increase vegetable intake. The F&V intake increased during the intervention period and was constant in follow-up. The BFtS program is an effective means to promote primary schoolchildren's F&V intake, and was implemented in Italy by the Food and Nutrition Service of Foggia's Local Health Authority. It could be extended to many other countries by local nutrition agencies, such as associations for nutrition.

Synthesis, characterization and hybridization studies of an alternate nucleo-epsilon/gamma-peptide: complexes formation with natural nucleic acids.

In order to develop new oligodeoxyribonucleotide (ODN) analogs to be used in biotechnological applications, we report here the synthesis, characterization and nucleic acid binding studies of novel nucleopeptides, that we called epsilon-lys/gamma-dabPNAs, containing a backbone of alternated L-diaminobutyric acid and L-lysine moieties. Exploring the hybridization properties of the new ODN analog, we found, by circular dichroism and UV spectroscopies, that a homothymine epsilon-lys/gamma-dabPNA hexamer binds both DNA and RNA of complementary sequence. Furthermore, human serum stability assays on the alternate nucleopeptide evidenced a noteworthy degradation resistance. These results encourage us to deepen the knowledge of this analog, in order to evaluate its possible use in antigene/antisense or diagnostic applications.

Evidences of complex formation between DABA-based nucleo-gamma-peptides with alternate configuration backbone.

In the present work, we report the synthesis and the characterization of dab PNA hexamers with diaminobutyric acid backbone of D- or/and L-configuration. In particular, the four nucleo-amino acids we synthesized, D- and L-diaminobutyryl adenines and D- and L-diaminobutyryl thymines, were used in various combinations to assemble the following oligomers: H-G-(t( L-dab))(6)-K-NH(2), H-G-(t( D-dab))(6)-K-NH(2), H-G-(a( L-dab))(6)-K-NH(2), H-G-(t( L-dab)-t( D-dab))(3)-K-NH(2), H-G-(a( L-dab)-a( D-dab))(3)-K-NH(2), H-G-(a( L-dab)-t( D-dab))(3)-K-NH(2). By using CD and UV spectroscopies, we investigated the ability of complementary dab PNA strands to bind to each other. We found that binding occurs only between oligomers with backbone of alternate configuration [(t( L-dab)-t( D-dab))(3)/(a( L-dab)-a( D-dab))(3) and (a( L-dab)-t( D-dab))(3)/(a( L-dab)-t( D-dab))(3)] and implies cooperative hydrogen bonds and base stacking. Furthermore, interesting properties relative to the self-complementary oligomer (a( L-dab)-t( D-dab))(3) forming palindromic complexes emerged from preliminary dynamic light-scattering experiments that suggested the formation of multimeric aggregates. These results, together with the high serum stability of the DABA-based oligomers, as shown by HPLC analysis, encourage us to further study dab PNAs as new self-recognizing bio-inspired polymers, to develop new nanomaterials in biotechnological and biomedical applications.

Solid phase synthesis and RNA-binding studies of a serum-resistant nucleo-epsilon-peptide.

In the present work we report the synthesis of a new Fmoc-protected L-lysine-based nucleo-amino acid suitable for the solid phase assembly and its oligomerisation to the corresponding nucleo-epsilon-peptide that we called epsilon-lysPNA. The ability to bind complementary RNA and the stability in serum of this synthetic nucleo-epsilon-peptide were studied to explore its possible use in antisense or diagnostic applications. Our interest to the presented oligonucleotide analogue was also supported by the importance of epsilon-peptides and other epsilon-amino acid-containing compounds in natural products with biological activity such as the poly-epsilon-lysines produced by Streptomyces albulus that possess a highly selective antimicrobial activity. Another aspect we intended to evaluate by this work is the possible prebiotic implication of these nucleopeptides, since epsilon-peptides, and not alpha-peptides, were mainly obtained among the other thermal prebiotic polypeptides in pyrocondensation of lysine, a diamino acid also detected in Mighei meteorite. Besides this intriguing question, all the remarkable properties emerged from the present investigation on epsilon-lysPNAs encourage, without doubts, interest in the therapeutic and diagnostic potential of these bioinspired nucleopeptides.

ODN-based drugs for targeting of extracellular proteins.

In this work a novel approach to identify new therapeutic targets consisting of serum proteins which contain an oligonucleotide binding domain is presented.

Bent oligonucleotide duplexes as HMGB1 inhibitors: a comparative study.

In this work we explore the ability of a chimeric LNA/DNA bent duplex, in which the kink is induced by 2 unpaired adenines in the middle of one strand, to bind HMGB1, a protein involved in many inflammatory processes. The LNA/DNA duplex was compared with the corresponding full DNA and PNA/DNA chimera duplexes from a thermodynamic and spectroscopic point of view.

Synthesis, characterization and hybridization studies of new nucleo-gamma-peptides based on diaminobutyric acid.

In the present work, we report the synthesis and the characterization of a new chiral nucleoaminoacid, in which a diaminobutyric moiety is connected to the DNA nucleobase by an amidic bond, and its oligomerization to give the corresponding nucleo-gamma-peptide. The ability of this synthetic polymer to bind complementary DNA was studied in order to explore its possible use in antigene/antisense or diagnostic applications. Our interest in the presented DNA analogue was also supported by the importance of gamma-aminoacid-containing compounds in natural products of biological activity and by the known stability of gamma-peptides to enzymatic degradation. Furthermore, our work could contribute to the study of the role of nucleopeptides as prebiotic material in a PNA world that could successively lead to the actual DNA/RNA/protein world, as recently assumed.

Manufacturing process of anti-thrombin III concentrate: viral safety validation studies and effect of column re-use on viral clearance.

A manufacturing process for the production of Anti-thrombin IIII concentrate is described, which is based primarily on Heparin Sepharose affinity chromatography. The process includes two sequential viral inactivation/removal procedures, applied to the fraction eluted from the column, the first by heating in aqueous solution at 60 degrees C for 10 h and the second by nanofiltration. Using viral validation on a scaled-down process both treatments proved to be effective steps; able to inactivate or remove more than 4 logs of virus, and their combined effect (>8 logs) assured the safety of the final product. Viral validation studies of the Heparin Sepharose chromatographic step demonstrated a consistency of the affinity of the resin for viruses over repeated use (16 runs), thus providing evidence of absence of cross-contamination from one batch to the next. It was concluded that the process of ATIII manufacturing provides a high level of confidence that the product will not transmit viruses.

Autoantibodies to glutamic acid decarboxylase in downbeat nystagmus.

The cause of downbeat nystagmus (DBN) remains undiagnosed in about 40% of patients. This paper reports the presence of antiglutamic acid decarboxylase antibodies (GAD-Ab) in a patient with DBN. Antibodies against GABAergic neurons located in the vestibular complex may induce chemical denervation of the floccular neurons, which normally suppress the peripheral imbalance between vertical semicircular canal systems, thereby causing DBN. Testing for GAD-Ab may be indicated in DBN patients without an identifiable anatomical brain lesion.

Analysis of B19 virus contamination in plasma pools for manufacturing, by using a competitive polymerase chain reaction assay.

BACKGROUND AND OBJECTIVES: The presence of B19 virus in blood poses a risk of transmission of the virus via blood or blood products. Screening processes for manufacturing should be aimed at achieving production plasma pools with B19 virus contamination levels below 104 genome equivalents/ml (geq/ml) in order to prevent transmission of infection through plasma derivatives. MATERIALS AND METHODS: The suitability of a competitor plasmid as an internal analytical standard for the detection of B19 virus in plasma pools was assessed by using a competitive polymerase chain reaction (PCR) assay. Seventy-five plasma pools, each consisting of 960 single donations, were analysed for B19 virus contamination following a lysis treatment. RESULTS: The amount of competitor plasmid in the competitive PCR assay established, with good accuracy, a threshold value for discrimination of the viral load in plasma pools. Analysis of samples from plasma pools showed that 12% of pools were contaminated with B19 virus at levels above the set threshold value. CONCLUSIONS: The competitive PCR assay developed proved to be effective for discrimination of the B19 virus contamination level in screening of plasma pools for manufacturing.

The treatment of oral aphthous ulceration or erosive lichen planus with topical clobetasol propionate in three preparations: a clinical and pilot study on 54 patients.

BACKGROUND: This study evaluated the clinical use of a corticosteroid in three preparations (topical clobetasol propionate ointment, clobetasol propionate in an oral analgesic base, and clobetasol propionate in an adhesive denture paste). METHODS: Fifty-four patients (34 males and 20 females) with a history of vesiculo-ulcero-erosive oral lesions were selected: 24 with oral erosive lichen planus and 30 with aphthae. The subjects enrolled were randomly divided into three groups, each of 18 patients (10 with aphthae and 8 with lichen planus): the first was treated with topical clobetasol propionate ointment (0.05%) directly on the lesion(s) three times a day; the second with clobetasol propionate in an adhesive denture paste in equal amounts (1:1) two times a day; the third with clobetasol propionate in an oral analgesic base (Orabase-B) in equal amounts (1:1) two times a day. Each subject scored his or her symptoms daily from most severe (7) to none (0) by verbal assessments using a categorical scale. RESULTS: In all cases, the administration of the corticosteroid was effective in producing remission of symptoms in each group of patients. Significant differences (P<0.05) between groups were determined by the Kruskal-Wallis test. The Dunn test was used in order to detect which group differs from the others; clobetasol and adhesive denture paste correlated with an early remission of pain in lichen and apthous lesions. CONCLUSION: The results suggest that topical application of clobetasol in an adhesive denture paste is an effective drug for symptomatic oral vesiculo-erosive and/or ulcerative lesions.

Satellite potentials on EMG: neurophysiologic evidence of axonal transection in MS?

To detect signs of axonal damage in MS, the authors investigated the occurrence in EMG of motor unit action potentials with satellite potentials (SP-MUAP) in the upper limb muscles in 10 consecutive patients with MS with cervical spinal cord demyelinating lesions and 10 control subjects. Subjects' SP-MUAP rate was 0 to 2.5% (median 0%) in the control group, and 0 to 17.5% (median 7.5%) in the MS group (p < 0.01). Motor unit remodeling secondary to axonal transection of spinal motor neurons traversing cervical demyelinating lesions may be hypothesized.

Lack of hypercapnic increase in cerebral blood flow at high blood viscosity in conscious blood-exchanged rats.

BACKGROUND: The hypothesis of a compensatory dilation of cerebral vessels to maintain cerebral blood flow at a high blood viscosity was tested during hypercapnia in the study after replacement of blood by hemoglobin solutions of defined viscosities. If compensatory vasodilation exists at normocapnia at a high blood viscosity, vasodilatory mechanisms may be exhausted when hypercapnia is added, resulting in a lack of increase in cerebral blood flow at hypercapnia. METHODS: In conscious rats, blood was replaced by ultrapurified cross-linked hemoglobin solutions that had defined and shear rate-independent low or high viscosities (low- and high-viscosity groups). Blood viscosity differed threefold between both groups (1.2 vs. 3.6 mP x s). Thereafter, rats inhaled either a normal or an increased concentration of carbon dioxide in air. Cerebral blood flow was determined by the iodo[14C]antipyrine method. RESULTS: During normocapnia, global and local cerebral blood flows did not differ between both groups. With increasing degrees of hypercapnia, global and local cerebral blood flows were gradually elevated in the low-viscosity group (2.8 ml x mmHg(-1) CO2 x 100 g(-1) x min(-1)), whereas they remained unchanged in the high-viscosity group. CONCLUSIONS: Changes in blood viscosity do not result in changes of cerebral blood flow as long as cerebral vessels can compensate for these changes by vasodilation or vasoconstriction. However, such vascular compensatory adjustments may be exhausted in their response to further pathophysiologic conditions in blood vessels that have already been dilated or constricted as a result of changes in blood viscosity.