Mechanical properties of friction induced nanocrystalline pearlitic steel.

Nanocrystalline structured variants of commercially available alloys have shown potential for boosting the mechanical properties of these materials, leading to a reduction in waste and thereby retaining feasible supply chains. One approach towards achieving these nanostructures resides in frictional treatments on manufactured parts, leading to differential refinement of the surface structure as compared to the bulk material. In this work the machining method is considered to be a testing platform for the formation and study of frictional nanostructured steel, assembly of which is stabilized by fast cooling of the produced chip. Analysis of the mechanical properties has shown extraordinary results at the surface, over 2000 MPa of strength on AISI1045 steel, more than three times the strength of the base material, demonstrating at the same time a reduction of 15% in the elastic modulus. The microscopic analysis suggests a reassembly of the elements in a new lattice of carbon supersaturated nano-ferrite.

A region within the third extracellular loop of rat Aquaporin 6 precludes trafficking to plasma membrane in a heterologous cell line.

The inability to over-express Aquaporin 6 (AQP6) in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail using crystallographic approaches. Using an Aquaporin 3-tGFP Reporter (AGR) system we have identified a region within loop C of AQP6 that is responsible for severely hampering plasma membrane expression. Serine substitution corroborated that amino acids present within AQP6194-213 of AQP6 loop C contribute to intracellular endoplasmic reticulum (ER) retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous expression systems.

[Neurological complications of nitrous oxide consumption. A case report]. / Complicaciones neurológicas del consumo de óxido nitroso. A propósito de un caso.

TITLE: Complicaciones neurológicas del consumo de óxido nitroso. A propósito de un caso.

An uncharacterized region within the N-terminus of mouse TMC1 precludes trafficking to plasma membrane in a heterologous cell line.

Mechanotransduction by hair cell stereocilia lies at the heart of sound detection in vertebrates. Considerable effort has been put forth to identify proteins that comprise the hair cell mechanotransduction apparatus. TMC1, a member of the transmembrane channel-like (TMC) family, was identified as a core protein of the mechanotransduction complex in hair cells. However, the inability of TMC1 to traffic through the endoplasmic reticulum in heterologous cellular systems has hindered efforts to characterize its function and fully identify its role in mechanotransduction. We developed a novel approach that allowed for the detection of uncharacterized protein regions, which preclude trafficking to the plasma membrane (PM) in heterologous cells. Tagging N-terminal fragments of TMC1 with Aquaporin 3 (AQP3) and GFP fusion reporter, which intrinsically label PM in HEK293 cells, indicated that residues at the edges of amino acid sequence 138-168 invoke intracellular localization and/or degradation. This signal is able to preclude surface localization of PM protein AQP3 in HEK293 cells. Substitutions of the residues by alanine or serine corroborated that the information determining the intracellular retention is present within amino acid sequence 138-168 of TMC1 N-terminus. This novel signal may preclude the proper trafficking of TMC1 to the PM in heterologous cells.

Generation of Flp-intm-ready DG44 and Lec 3.2.8.1 CHO cell lines for quick and easy constitutive protein expression.

The well-characterized cell line Chinese hamster ovary (CHO) has been used to produce numerous biopharmaceuticals and is an important tool for basic research. However, introducing foreign DNA into specially modified CHO cells such as DG44 and Lec 3.2.8.1 can sometimes be an arduous process. Here we show that the Flp-intm plasmid can be modified to produce a fluorescent tracer protein tag (mCherrytm) as a fusion reporter, to allow for the rapid selection of single-cell sorted, isogenic Flp-intm-ready DG44 and Lec 3.2.8.1 cell lines. These two cell lines are stable and viable and may be useful for applications such as antibody production and crystallographic studies. Here we provide key details on how the modified pFRT/CherryZeo plasmid may be used to incorporate Flp-intm technology into virtually any desired target cell line in a fast, safe and reliable manner.

¿3 + 4 = 6? Implicaciones de la estratificación del cáncer de próstata localizado Gleason 7 por número y porcentaje de cilindros positivos de biopsia en la selección de pacientes para vigilancia activa / 3+4 = 6? Implications of the stratification of localised Gleason 7 prostate cancer by number and percentage of positive biopsy cores in selecting patients for active surveillance

Objetivo: Investigar si el número y el porcentaje de cilindros positivos de la biopsia identifica un subgrupo de cáncer de próstata (CaP) Gleason 3 + 4 de comportamiento biológico similar a los Gleason 3 + 3. Material y método: Estudio observacional, tras prostatectomía radical, de una cohorte de 799 pacientes con CaP localizado de riesgo bajo (n = 582; Gleason 6; PSA < 10ng/ml y cT1c-2a) e intermedio favorable (n = 217; Gleason 3 + 4; PSA ≤ 10 ng/ml y pT2abc). Los tumores Gleason 3 + 4 se estratificaron por número (≤ 3 vs .> 3) y porcentaje de cilindros positivos (≤ 33% vs. > 33%). Se analizó su asociación con el riesgo de recidiva bioquímica (ReB) y mortalidad cáncer específica (MCE). Se realizaron diferentes modelos predictivos mediante regresión de Cox y se estimó (C-index) y comparó su capacidad predictiva. Resultados: Con una mediana de seguimiento de 71 meses, la probabilidad de ReB y de MCE en el grupo de pacientes con tumores Gleason 3+4 y número (≤3) o porcentaje bajo (≤33%) de cilindros positivos no fue significativamente diferente de las de los pacientes con tumores Gleason 6. A 5 y 10 años, no se observaron diferencias significativas en el número de ReB, en la probabilidad de permanecer libre de ReB, en el número de muertes por CaP ni en la probabilidad de muerte por CaP entre ambos grupos. Por el contrario, los pacientes con tumores Gleason 3+4 y >33% de cilindros positivos presentaron mayor número de muertes por CaP que los pacientes con tumores Gleason 6 y, a 10 años, la probabilidad de MCE fue significativamente mayor. Este subgrupo de tumores presentó un riesgo significativamente mayor de ReB (RR = 1,6; p = 0,02) respecto a los Gleason 6 y, sobre todo, de MCE (RR = 5,8; p = <0,01). El modelo con Gleason 3 + 4 estratificado por porcentaje de cilindros positivos mejoró significativamente la capacidad predictiva de ReB y MCE. Conclusiones: Un número<3 cilindros y un porcentaje < 33% de cilindros positivos identifica un subgrupo de tumores Gleason 3 + 4 con comportamiento biológico similar a los Gleason 6. A 10 años, no se observaron diferencias en el riesgo de ReB y MCE entre ambos grupos. Estos resultados aportan pruebas que apoyan a la vigilancia activa como alternativa para tumores Gleason 3 + 4 y baja extensión tumoral en biopsia

Objective: To determine whether the number and percentage of positive biopsy cores identify a Gleason 3 + 4 prostate cancer (PC) subgroup of similar biologic behaviour to Gleason 3 + 3. Material and method: An observational post-radical prostatectomy study was conducted of a cohort of 799 patients with localised low-risk (n = 582, Gleason 6, PSA < 10ng/ml and cT1c-2a) and favourable intermediate PC (n = 217, Gleason 3 + 4, PSA ≤ 10 ng/ml and pT2abc). The Gleason 3 + 4 tumours were stratified by number (≤ 3 vs.> 3) and by percentage of positive cores (≤ 33% vs. > 33%). We analysed the tumours’ association with the biochemical recurrence risk (BRR) and cancer-specific mortality (CSM). We conducted various predictive models using Cox regression and estimated (C-index) and compared their predictive capacity. Results: With a median follow-up of 71 months, the BRR and CSM of the patient group with Gleason 3 + 4 tumours and a low number (≤ 3) and percentage (≤ 33%) of positive cores were not significantly different from those of the patients with Gleason 6 tumours. At 5 and 10 years, there were no significant differences in the number of biochemical recurrences, the probability of remaining free of biochemical recurrences, the number of deaths by PC or the probability of death by PC between the 2 groups. In contrast, the patients with Gleason 3+4 tumours and more than 33% of positive cores presented more deaths by PC than the patients with Gleason 6 tumours. At 10 years, the probability of CSM was significantly greater. This subgroup of tumours showed a significantly greater BRR (RR, 1.6; P = .02) and CSM (RR, 5.8, P ≤ .01) compared with the Gleason 6 tumours. The model with Gleason 3 + 4 stratified by the percentage of positive cores significantly improved the predictive capacity of BRR and CSM. Conclusions: Fewer than 3 cores and a percentage < 33% of positive cores identifies a subgroup of Gleason 3 + 4 tumours with biological behaviour similar to Gleason 6 tumours. At 10 years, there were no differences in BRR and CSM between the 2 groups. These results provide evidence supporting active surveillance as an alternative for Gleason 3 + 4 tumours and low tumour extension in biopsy

Anomalías estructurales del esmalte y afectación estética en escolares de 6-17 años de Cojímar / Structural anomalies of the enamel and aesthetic affection in schools children of 6-17 years of Cojímar

Introducción: las anomalías estructurales del esmalte dentario aparecen en edades tempranas, provocando afectaciones en el desarrollo de los dientes, la oclusión y la estética.Objetivo: describir las anomalías estructurales del esmalte y afectación estética en escolares de 6 a 17 años del área de salud de Cojímar.Métodos: se realizó un estudio descriptivo transversal en el período septiembre 2013 a junio 2014. El universo lo constituyó 1 285 niños de 6 a 17 años, de ambos sexos, que cursan estudios en la enseñanza primaria, secundarias y preuniversitaria del área de salud de Cojímar, municipio La Habana del Este, considerando afectados aquellos que tenían al menos un diente permanente con anomalía estructural del esmalte; se utilizó como medida resumen el porciento y como prueba estadística el Ji cuadrado de Pearson. Los resultados se presentaron en tablas. Resultados: la prevalencia de anomalías del esmalte fue de 39.8por ciento, la opacidad demarcada predominó con el 56.9 por ciento, el grupo incisivo resultó más afectado el 36.1por ciento, la superficie vestibular con el 31.4 por ciento mostró mayores cifras en todos los grupos dentarios, y el 74,8por ciento de los niños consideró tener afectación estética.Conclusiones: las anomalías del esmalte representan a más de la tercera parte de la población, con similitud entre los grupos de edades y ambos sexos, la más común es la opacidad demarcada; los incisivos y la superficie vestibular son los más afectados, lo que establece una relación tácita con la afectación estética dental(AU)

Introduction: the structural anomalies of the dental enamel appear at an early age, causing affectations in the development of the teeth, occlusion and aesthetics. Objective: to describe the structural anomalies of the enamel and aesthetic affectation in children from 6 to 17 years old in Cojimar.Methods: a transversal descriptive study was carried out from September, 2013 to June, 2014. The universe was formed by 1 285 children from 6 to 17 years old, both sexes, who are studying in primary, secondary and pre-university education in the health area of Cojimar, Habana del Este Municipality, considering that those had at least one permanent tooth with structural enamel anomaly were affected, the percentage was used as a summary measure and Pearson´s Chi square as statistical test. The results were presented in tables.Results: the prevalence of enamel anomalies was 39.8 percent, the demarcated opacity prevailed in 56.9 percent , the incisor group was the most affected with a 36.1 percent, the vestibular surface with 31.4 percent showed higher figures in all cases, dental groups, and 74.8 percent of children considered to have aesthetic involvement.Conclusions: the enamel anomalies represent more than a third of the population, with similarity between age groups and both sexes, the most common was the demarcated opacity, the incisors and the vestibular surface were the most affected, which establishes a tacit relationship with dental aesthetic involvement(AU)

Towards the understanding of antibiotic occurrence and transport in groundwater: Findings from the Baix Fluvià alluvial aquifer (NE Catalonia, Spain).

Antibiotics are an increasing focus of interest due to their high detection frequency in the environment. However, their presence in water bodies is not regulated by environmental policies. This field study investigates, for the first time, the occurrence, behavior and fate of a selection of 53 antibiotics, including up to 10 chemical groups, in an alluvial aquifer originated from manure application in an agricultural region using hydrogeological, hydrochemical and isotopic approaches. Up to 11 antibiotics were found in groundwater corresponding to 4 different chemical groups: fluoroquinolones, macrolides, quinolones and sulfonamides. In surface water, only 5 different antibiotics from 2 chemical groups: fluoroquinolones and sulfonamides, were quantified. The most frequent antibiotics were sulfamethoxazole and ciprofloxacin. Concentrations of antibiotics were in the order of ng/L, with maximum concentrations of 300ng/L in groundwater. Hydrochemistry and isotopic data and geostatistics confirmed the spatial trend observed for nitrates, where nitrate concentrations tend to be higher in the margin areas of the study area, and lower concentrations are found nearby the river. On the other hand, no clear continuous spatial concentration trend of antibiotics was observed in the aquifer, supported by the short spatial correlation found in the variograms. This indicates that the physical-chemical properties and processes of each antibiotic (mainly, sorption and degradation), and other environmental issues, such as a patchy diffuse input and the manure antibiotic content itself, play an important role in their spatial distribution in groundwater. A discussion on the estimation of the antibiotic sorption parameter reveals the difficulties of describing such phenomena. Furthermore, retardation factors will extend over several orders of magnitude, which highly affects the movement of individual antibiotics within the aquifer. To summarize, this study points out the difficulties associated with antibiotic research in groundwater in order to define water resources quality management strategies and environmental regulations.

3+4 = 6? Implications of the stratification of localised Gleason 7 prostate cancer by number and percentage of positive biopsy cores in selecting patients for active surveillance. / ¿3 + 4 = 6? Implicaciones de la estratificación del cáncer de próstata localizado Gleason 7 por número y porcentaje de cilindros positivos de biopsia en la selección de pacientes para vigilancia activa.

OBJECTIVE: To determine whether the number and percentage of positive biopsy cores identify a Gleason 3+4 prostate cancer (PC) subgroup of similar biologic behaviour to Gleason 3+3. MATERIAL AND METHOD: An observational post-radical prostatectomy study was conducted of a cohort of 799 patients with localised low-risk (n=582, Gleason 6, PSA <10ng/ml and cT1c-2a) and favourable intermediate PC (n=217, Gleason 3+4, PSA ≤10 ng/ml and pT2abc). The Gleason 3+4 tumours were stratified by number (≤3 vs.>3) and by percentage of positive cores (≤33% vs. >33%). We analysed the tumours' association with the biochemical recurrence risk (BRR) and cancer-specific mortality (CSM). We conducted various predictive models using Cox regression and estimated (C-index) and compared their predictive capacity. RESULTS: With a median follow-up of 71 months, the BRR and CSM of the patient group with Gleason 3+4 tumours and a low number (≤3) and percentage (≤33%) of positive cores were not significantly different from those of the patients with Gleason 6 tumours. At 5 and 10 years, there were no significant differences in the number of biochemical recurrences, the probability of remaining free of biochemical recurrences, the number of deaths by PC or the probability of death by PC between the 2 groups. In contrast, the patients with Gleason 3+4 tumours and more than 33% of positive cores presented more deaths by PC than the patients with Gleason 6 tumours. At 10 years, the probability of CSM was significantly greater. This subgroup of tumours showed a significantly greater BRR (RR, 1.6; P=.02) and CSM (RR, 5.8, P≤.01) compared with the Gleason 6 tumours. The model with Gleason 3+4 stratified by the percentage of positive cores significantly improved the predictive capacity of BRR and CSM. CONCLUSIONS: Fewer than 3 cores and a percentage <33% of positive cores identifies a subgroup of Gleason 3+4 tumours with biological behaviour similar to Gleason 6 tumours. At 10 years, there were no differences in BRR and CSM between the 2 groups. These results provide evidence supporting active surveillance as an alternative for Gleason 3+4 tumours and low tumour extension in biopsy.

The key role of aquaporin 3 and aquaporin 10 in the pathogenesis of pompholyx.

Pompholyx remains a chronic skin affliction without a compelling pathophysiological explanation. The disease is characterized by the sudden onset of vesicles exclusively in the palms and soles which generally resolves. However, the disease may progress and the vesicles may expand and fuse; with chronicity there is deep fissuring. Multiple therapeutic approaches are available, but the disease is often resistant to conventional treatments. Currently, oral alitretinoin is used for patients with resistant chronic disease; however, this therapy is only approved for use in the UK, Europe and Canada. In this paper we wish to put forward a hypothesis: exposure to water and the subsequent steep osmotic gradient imbalance are key factors driving skin dehydration seen in pompholyx patients once the disease becomes chronic. The mechanistic explanation for the epidermal fissuring might lie in the over-expression across the mid and upper epidermis, including the stratum corneum, of two water/glycerol channel proteins aquaporin 3 and aquaporin 10, expressed in the keratinocytes of afflicted pompholyx patients. The over-expression of these two aquaporins may bridge the abundantly hydrated dermis and basal epidermis to the outer environment allowing cutaneous water and glycerol to flow outward. The beneficial effects reported in alitretinoin-treated patients with chronic hand eczemas may be due potential regulation of aquaporin 3 and aquaporin 10 by alitretinoin.

The effects of transcranial direct current stimulation with visual illusion in neuropathic pain due to spinal cord injury: an evoked potentials and quantitative thermal testing study.

BACKGROUND: Neuropathic pain (NP) is common in spinal cord injury (SCI) patients. One of its manifestations is a lowering of pain perception threshold in quantitative thermal testing (QTT) in dermatomes rostral to the injury level. Transcranial direct current stimulation (tDCS) combined with visual illusion (VI) improves pain in SCI patients. We studied whether pain relief with tDCS + VI intervention is accompanied by a change in contact heat- evoked potentials (CHEPs) or in QTT. METHODS: We examined 18 patients with SCI and NP before and after 2 weeks of daily tDCS + VI intervention. Twenty SCI patients without NP and 14 healthy subjects served as controls. We assessed NP intensity using a numerical rating scale (NRS) and determined heat and pain thresholds with thermal probes. CHEPs were recorded to stimuli applied at C4 level, and subjects rated their perception of evoked pain using NRS during CHEPs. RESULTS: Thirteen patients reported a mean decrease of 50% in the NRS for NP after tDCS + VI. Evoked pain perception was significantly higher than in the other two groups, and reduced significantly together with CHEPs amplitude after tDCS + VI with respect to baseline. Pain perception threshold was significantly lower than in the other two groups before tDCS + VI intervention, and increased significantly afterwards. CONCLUSION: Two weeks of tDCS + VI induced significant changes in CHEPs, evoked pain and heat pain threshold in SCI patients with NP. These neurophysiological tests might be objective biomarkers of treatment effects for NP in patients with SCI.

Valoración de la satisfacción de los padres en una unidad neonatal / Assessment of parental satisfaction in a neonatal unit

Introducción: La satisfacción de los padres en una unidad neonatal (UN) debe ser evaluada como elemento de calidad asistencial. Objetivos: 1/Determinar cómo influye el nivel de gravedad sobre la satisfacción de los padres. 2/Comparar los resultados antes y después de un traslado de centro. Material y métodos: Se aplicó una encuesta de satisfacción a los padres de los neonatos (RN) ingresados en la UN. Se recogieron datos de 87 de los RN ingresados durante 10 meses, distribuidos en 2 categorías según gravedad. Se valoró la satisfacción de los padres sobre aspectos humanos y físicos en la UN, estudiando si esta se afectó por la gravedad del RN. Se valoró el impacto del traslado sobre la satisfacción. Se realizaron análisis estadísticos descriptivos y modelos multivariantes. Resultados: Se observó una polarización entre los aspectos humanos y los relacionados con el medio. Fueron estadísticamente significativos la valoración del horario de acceso de los padres y la crítica sobre mobiliario, luz y espacio físico. Los padres de los RN más graves valoraron mejor el horario pero peor el entorno. La percepción de dolor en los procedimientos es mayor en niños más graves y un 66% de los padres preferiría estar presente en los mismos. Comparando entre la UN antigua y la nueva, el único parámetro significativo fue la percepción sobre la información suministrada, peor valorada en el nuevo hospital. La lactancia materna mejoró significativamente. La satisfacción general fue elevada. Conclusiones: Los padres de niños graves valoran la accesibilidad y son críticos con la comodidad. El horario de entrada de los padres y la ausencia de luz y ruido son predictores significativos de satisfacción en niños con estancias prolongadas. La percepción del acceso a la información empeoró con el traslado, probablemente por falta de adaptación al medio(AU)

Introduction: Parental satisfaction in a neonatal unit needs to be checked as a quality element in health care. Objectives: 1/To measure how the severity of the newborn condition influences parental satisfaction. 2/To compare the results before and after moving to a new building. Methods: Parental satisfaction in 87 of the newborns admitted to our neonatal unit over a 10 month period was documented using a questionnaire survey. The newborns were divided into 2 categories, depending on the severity of their condition. Parental satisfaction as regards human and environmental issues was checked as to whether it was affected by the severity of the illness. The impact of the move to a new building on satisfaction was also analysed. Statistical descriptive studies and multivariate models were used. Results: A polarised tendency was seen between human and environmental aspects. The following issues were statistically significant: visiting hours for parents, and criticisms about furniture, light and physical space. Parents of seriously ill children appreciated the visiting hours but judged the environment negatively. Pain perception in procedures was higher in serious illnesses, and 66% of parents would rather be present during those procedures. When comparing both hospitals, the only significant value regarding the new centre, was the lower perception of information given by the staff. Breastfeeding improved significantly. General satisfaction was high. Conclusions: Parents of seriously ill newborns are mainly concerned about access hours and comfort. Parent visiting hours and lack of noise and light are significant parameters predicting satisfaction in children with long stays. Perception of access to information got worse in the new hospital, probably due to lack of staff adaptation(AU)

Frequency of mutations in the genes associated with hereditary sensory and autonomic neuropathy in a UK cohort.

The hereditary sensory and autonomic neuropathies (HSAN, also known as the hereditary sensory neuropathies) are a clinically and genetically heterogeneous group of disorders, characterised by a progressive sensory neuropathy often complicated by ulcers and amputations, with variable motor and autonomic involvement. To date, mutations in twelve genes have been identified as causing HSAN. To study the frequency of mutations in these genes and the associated phenotypes, we screened 140 index patients in our inherited neuropathy cohort with a clinical diagnosis of HSAN for mutations in the coding regions of SPTLC1, RAB7, WNK1/HSN2, FAM134B, NTRK1 (TRKA) and NGFB. We identified 25 index patients with mutations in six genes associated with HSAN (SPTLC1, RAB7, WNK1/HSN2, FAM134B, NTRK1 and NGFB); 20 of which appear to be pathogenic giving an overall mutation frequency of 14.3%. Mutations in the known genes for HSAN are rare suggesting that further HSAN genes are yet to be identified. The p.Cys133Trp mutation in SPTLC1 is the most common cause of HSAN in the UK population and should be screened first in all patients with sporadic or autosomal dominant HSAN.

[Assessment of parental satisfaction in a neonatal unit]. / Valoración de la satisfacción de los padres en una unidad neonatal.

INTRODUCTION: Parental satisfaction in a neonatal unit needs to be checked as a quality element in health care. OBJECTIVES: 1/To measure how the severity of the newborn condition influences parental satisfaction. 2/To compare the results before and after moving to a new building. METHODS: Parental satisfaction in 87 of the newborns admitted to our neonatal unit over a 10 month period was documented using a questionnaire survey. The newborns were divided into 2 categories, depending on the severity of their condition. Parental satisfaction as regards human and environmental issues was checked as to whether it was affected by the severity of the illness. The impact of the move to a new building on satisfaction was also analysed. Statistical descriptive studies and multivariate models were used. RESULTS: A polarised tendency was seen between human and environmental aspects. The following issues were statistically significant: visiting hours for parents, and criticisms about furniture, light and physical space. Parents of seriously ill children appreciated the visiting hours but judged the environment negatively. Pain perception in procedures was higher in serious illnesses, and 66% of parents would rather be present during those procedures. When comparing both hospitals, the only significant value regarding the new centre, was the lower perception of information given by the staff. Breastfeeding improved significantly. General satisfaction was high. CONCLUSIONS: Parents of seriously ill newborns are mainly concerned about access hours and comfort. Parent visiting hours and lack of noise and light are significant parameters predicting satisfaction in children with long stays. Perception of access to information got worse in the new hospital, probably due to lack of staff adaptation.

Expression of CCR8 is increased in asthma.

BACKGROUND: Chemokines and their receptors could play key roles in the recruitment of T cells to the asthmatic lung. CCR8 is preferentially expressed on T-helper type 2 cells, and is thought to play a role in the pathogenesis of human asthma. OBJECTIVE: Determine the expression of CCR8 on T cells in blood, bronchoalveolar lavage (BAL) and bronchial mucosa from asthmatics and normal subjects. METHODS: CCR8 expression in blood and BAL from asthma and normal subjects was studied using flow cytometry. CCR8 expression on IFN-gamma+ and IL-4+/IL-13+ blood and BAL T cells was studied following stimulation with Phorbol-Myristate-Acetate and Calcium Ionophore. Paraffin-embedded bronchial biopsies were used to study CCR8 in bronchial epithelium. RESULTS: The percentage of CD3+ cells expressing CCR8 in the blood was higher in asthmatics (4.7+/-0.4%) compared with normal subjects (3.0+/-0.4%; P<0.01). There was an approximately sixfold enrichment of CCR8 on IL-4+/IL-13+ cells compared with IFN-gamma+ T cells (P<0.001) in both asthmatic and normal subjects in both blood and BAL. Significantly more BAL T cells expressed CCR8 in asthmatic (8.6+/-0.8%) compared with normal subjects (3.9+/-0.7%) (P<0.01). In paired blood-BAL samples from asthmatics, significantly more CCR8+CD3+ T cells were present in BAL (9.0+/-0.9%) than in blood (5.6+/-0.9%; P<0.05). There were more CCR8-positive cells in bronchial biopsies from asthmatic (93+/-11 cells/mm2) compared with normal subjects (30+/-16 cells/mm2) (P<0.05). The ligand CCL1 was increased in the BAL of asthmatics compared with normal subjects (35+/-6 vs. 12.9+/-7 pg/mL; P<0.05). CONCLUSION: There may be a role for CCR8 in the recruitment of T cells to the lung in asthmatics.

Telomere dysfunction and chromosome structure modulate the contribution of individual chromosomes in abnormal nuclear morphologies.

The cytokinesis-block micronucleus assay has emerged as a biomarker of chromosome damage relevant to cancer. Although it was initially developed to measure micronuclei, it is also useful for measuring nucleoplasmic bridges and nuclear buds. Abnormal nuclear morphologies are frequently observed in malignant tissues and short-term tumour cell cultures. Changes in chromosome structure and number resulting from chromosome instability are important factors in oncogenesis. Telomeres have become key players in the initiation of chromosome instability related to carcinogenesis by means of breakage-fusion-bridge cycles. To better understand the connection between telomere dysfunction and the appearance of abnormal nuclear morphologies, we have characterised the presence of micronuclei, nucleoplasmic bridges and nuclear buds in human mammary primary epithelial cells. These cells can proliferate beyond the Hayflick limit by spontaneously losing expression of the p16(INK4a) protein. Progressive telomere shortening leads to the loss of the capping function, and the appearance of end-to-end chromosome fusions that can enter into breakage-fusion-bridge cycles generating massive chromosomal instability. In human mammary epithelial cells, different types of abnormal nuclear morphologies were observed, however only nucleoplasmatic bridges and buds increased significantly with population doublings. Fluorescent in situ hybridisation using centromeric and painting specific probes for chromosomes with eroded telomeres has revealed that these chromosomes are preferentially included in the different types of abnormal nuclear morphologies observed, thus reflecting their common origin. Accordingly, real-time imaging of cell divisions enabled us to determine that anaphase bridge resolution was mainly through chromatin breakage and the formation of symmetric buds in daughter nuclei. Few micronuclei emerged in this cell system thus validating the scoring of nucleoplasmic bridges and nuclear buds for measuring chromosome instability in telomere-dysfunction cell environments.

The number of dysfunctional telomeres in a cell: one amplifies; more than one translocate.

Chromosomal instability is increasingly appreciated as a key component of tumorigenesis in humans. A combination of abnormal telomere shortening and cell-cycle checkpoint deficiency has been proposed as the initial lesions causing destabilizing chromatin bridges in proliferative cells. We examined the participation of the different types of end-to-end fusions in generating instable karyotypes in non-transformed human breast epithelial cells. We concluded that short dysfunctional telomeres represent an initiating substrate for post-replicative rejoining of sister chromatids and are likely to make an important contribution to the formation of chromosomal rearrangements and the amplification of chromosome arm segments in breast epithelial cells. We propose that there is a chronological order in the participation of the different types of end-to-end fusions in the generation of chromosomal instability. Thus, intrachromosomal post-replicative joining would proceed mainly in the early stages after overcoming growth arrest, when telomere dysfunction is limited and affects only one chromosome end in a cell. The absence of a second substrate for end joining will conduct the cell with the uncapped chromosome to replicate its DNA and fuse the uncapped sister chromatids after replication. Later, since telomeres shorten progressively with each DNA replication round, the uncapping will affect many more chromosome ends, and fusions between the uncapped ends from different chromosomes will be produced. While the fusion of sister chromatids will produce chromosome segment amplification and terminal deletions in the daughter cells, interchromosomal fusion will produce unbalanced rearrangements other than chromosome segment amplifications.