Estrogen regulated transcription of the non-estrogen-regulated hamster uteroglobin gene in MCF-7 cells.

To study the estrogen regulated transcription of the uteroglobin (UG) gene, the founding member of the secretoglobin family widely expressed in many different mammalian species, we re-created functional estrogen response elements (EREs) in the UG gene promoter from a species where UG expression is not regulated by estrogens: the hamster Mesocricetus auratus (Ma), to ascertain if the lack of functional EREs is the real cause of its estrogen insensitivity. Functional EREs in the hamster promoter, including the consensus ERE (cERE), failed to respond to an appropriate estrogen stimulus compared with its estrogen regulated ortholog from the brown hare Lepus capensis (Lc). As the nucleotide sequence is the only difference between genetic constructs from these two species, we suspected that the UG promoter from the hamster probably contains cis-acting genetic elements that negatively impairs the estrogen-regulated transcription mediated by the functional ERE. Accordingly, we prepared chimeric DNA constructs which eventually allowed to identify a region located 29 base pairs (bp) downstream of the ERE as responsible for the lack of estrogen-responsiveness of the Ma-UG gene in the breast cancer cell line MCF-7. This region contains the sequence ACACCCC which has been identified as the core sequence of the Sp/ Krüppel-like factor (KLF) family of transcription factors. This finding is relevant, not only due to the observation on a novel mechanism that control estrogen-induced transcription, but also because it may encourage further investigation for better defining specific genes with an ERE that do not respond to estrogen signaling in MCF-7 cells, a cell line widely employed as an in vitro model in breast cancer research.

Association of uteroglobin-related protein 1 with smoke inhalation injury severity. / Associação entre proteína 1 relacionada à uteroglobulina e gravidade da lesão por inalação de fumaça.

OBJECTIVE: To evaluate serum uteroglobin-related protein 1 expression early after smoke inhalation injuries and its association with the severity of inhalation injury in burned patients. METHODS: Smoke or chemical inhalation injury is associated with morbidity and mortality. The consequences of inhalation result from an inflammatory response. Uteroglobin-related protein 1 is an anti-inflammatory protein and may improve lung inflammation. We hypothesized that uteroglobin-related protein 1 levels could reflect disease severity and predict outcome in patients with inhalation injury. Sixteen patients diagnosed with acute respiratory distress syndrome secondary to smoke inhalation injury were prospectively included in the study. Plasma was collected upon intensive care unit admission and within 24 hours of the inhalation injury. Bronchoscopies were carried out in all patients to assess the severity of inhalation injury within 72 hours. Uteroglobin-related protein 1 plasma levels were determined in duplicate with enzyme-linked immunosorbent assay. RESULTS: The mean age was 23 ± 5 years, and the inhalation injury distribution was as follows: three of grade 1, four of grade 2, and nine of grade 3. The level of uteroglobin-related protein 1 was related to inhalation severity (grade 1: 0.389 ± 0.053 arbitrary units versus grade 2: 0.474 ± 0.0423 arbitrary units versus grade 3: 0.580 ± 0.094 arbitrary units; p = 0.007). CONCLUSION: Plasma levels of uteroglobin-related protein 1 are associated with the degree of lung inhalation injury.

OBJETIVO: Avaliar a expressão sérica da proteína 1 relacionada à uteroglobulina na fase inicial após lesões por inalação de fumaça e sua associação com a gravidade da lesão por inalação em pacientes queimados. MÉTODOS: A lesão por inalação de fumaça ou produtos químicos se associa com morbidade e mortalidade. As consequências da inalação resultam de uma resposta inflamatória. A proteína 1 relacionada à uteroglobulina é anti-inflamatória e pode melhorar a inflamação pulmonar. Nossa hipótese é que os níveis de proteína 1 relacionada à uteroglobulina podem refletir a gravidade da doença e predizer o desfecho em pacientes com lesão por inalação. Incluíram-se prospectivamente neste estudo 16 pacientes com diagnóstico de síndrome do desconforto respiratório agudo decorrente de lesão por inalação de fumaça. Em todos os pacientes, colheu-se amostra de plasma quando da admissão à unidade de terapia intensiva, para avaliar a gravidade da lesão por inalação dentro de 72 horas. Os níveis plasmáticos de proteína 1 relacionada à uteroglobulina foram determinados em duplicata por meio de ensaio de imunoabsorção ligado à enzima. RESULTADOS: A média de idade foi de 23 ± 5 anos, e a distribuição da lesão por inalação foi: três em grau 1, quatro em grau 2 e nove em grau 3. O nível de proteína 1 relacionada à uteroglobulina foi relacionado ao grau de severidade (grau 1: 0,389 ± 0,053 unidade arbitrária versus grau 2: 0,474 ± 0,0423 unidade arbitrária versus grau 3: 0,580 ± 0,094 unidade arbitrária; p = 0,007). CONCLUSÃO: Os níveis plasmáticos de proteína 1 relacionada à uteroglobulina se associam com o grau da lesão pulmonar por inalação.

Associação entre proteína 1 relacionada à uteroglobulina e gravidade da lesão por inalação de fumaça / Association of uteroglobin-related protein 1 with smoke inhalation injury severity

RESUMO Objetivo: Avaliar a expressão sérica da proteína 1 relacionada à uteroglobulina na fase inicial após lesões por inalação de fumaça e sua associação com a gravidade da lesão por inalação em pacientes queimados. Métodos: A lesão por inalação de fumaça ou produtos químicos se associa com morbidade e mortalidade. As consequências da inalação resultam de uma resposta inflamatória. A proteína 1 relacionada à uteroglobulina é anti-inflamatória e pode melhorar a inflamação pulmonar. Nossa hipótese é que os níveis de proteína 1 relacionada à uteroglobulina podem refletir a gravidade da doença e predizer o desfecho em pacientes com lesão por inalação. Incluíram-se prospectivamente neste estudo 16 pacientes com diagnóstico de síndrome do desconforto respiratório agudo decorrente de lesão por inalação de fumaça. Em todos os pacientes, colheu-se amostra de plasma quando da admissão à unidade de terapia intensiva, para avaliar a gravidade da lesão por inalação dentro de 72 horas. Os níveis plasmáticos de proteína 1 relacionada à uteroglobulina foram determinados em duplicata por meio de ensaio de imunoabsorção ligado à enzima. Resultados: A média de idade foi de 23 ± 5 anos, e a distribuição da lesão por inalação foi: três em grau 1, quatro em grau 2 e nove em grau 3. O nível de proteína 1 relacionada à uteroglobulina foi relacionado ao grau de severidade (grau 1: 0,389 ± 0,053 unidade arbitrária versus grau 2: 0,474 ± 0,0423 unidade arbitrária versus grau 3: 0,580 ± 0,094 unidade arbitrária; p = 0,007). Conclusão: Os níveis plasmáticos de proteína 1 relacionada à uteroglobulina se associam com o grau da lesão pulmonar por inalação.

ABSTRACT Objective: To evaluate serum uteroglobin-related protein 1 expression early after smoke inhalation injuries and its association with the severity of inhalation injury in burned patients. Methods: Smoke or chemical inhalation injury is associated with morbidity and mortality. The consequences of inhalation result from an inflammatory response. Uteroglobin-related protein 1 is an anti-inflammatory protein and may improve lung inflammation. We hypothesized that uteroglobin-related protein 1 levels could reflect disease severity and predict outcome in patients with inhalation injury. Sixteen patients diagnosed with acute respiratory distress syndrome secondary to smoke inhalation injury were prospectively included in the study. Plasma was collected upon intensive care unit admission and within 24 hours of the inhalation injury. Bronchoscopies were carried out in all patients to assess the severity of inhalation injury within 72 hours. Uteroglobin-related protein 1 plasma levels were determined in duplicate with enzyme-linked immunosorbent assay. Results: The mean age was 23 ± 5 years, and the inhalation injury distribution was as follows: three of grade 1, four of grade 2, and nine of grade 3. The level of uteroglobin-related protein 1 was related to inhalation severity (grade 1: 0.389 ± 0.053 arbitrary units versus grade 2: 0.474 ± 0.0423 arbitrary units versus grade 3: 0.580 ± 0.094 arbitrary units; p = 0.007). Conclusion: Plasma levels of uteroglobin-related protein 1 are associated with the degree of lung inhalation injury.

The role of the non-ciliated bronchiolar cell in tolerance to inhaled vanadium of the bronchiolar epithelium.

The Non-Ciliated Bronchiolar Cell (NCBC) is responsible for the defense and maintenance of the bronchiolar epithelium. Several cellular defense mechanisms have been associated with an increase in the secretion of CC16 and changes in the phenotype of the cell; these mechanisms could be linked to tolerance to the damage due to exposure to inhaled Particulate Matter (PM) of the epithelium. These defense mechanisms have not been sufficiently explored. In this article, we studied the response of the NCBC to inhaled vanadium, an element which adheres to PM. This response was measured by the changes in the phenotype of the NCBC and the secretion of CC16 in a mouse model. Mice were exposed in two phases to different vanadium concentrations; 1.27 mg/m³ in the first phase and 2.56 mg/m³ in the second phase. Mice were sacrificed on the 2nd, 4th, 5th, 6th and 8th weeks. In the second phase, we observed the following: sloughing of the NCBC, hyperplasia and small inflammatory foci remained without changes and that the expression of CC16 was higher in this phase than in phase I. We also observed a change in the phenotype with a slow decrease in both phases. The increase in the secretion of CC16 and the phenotype reversion could be due to the anti-inflammatory activity of CC16. The changes observed in the second phase could be attributed to the tolerance to inhaled vanadium.

Primary infection by Pneumocystis induces Notch-independent Clara cell mucin production in rat distal airways.

Clara cells are the main airway secretory cells able to regenerate epithelium in the distal airways through transdifferentiating into goblet cells, a process under negative regulation of the Notch pathway. Pneumocystis is a highly prevalent fungus in humans occurring between 2 and 5 months of age, a period when airways are still developing and respiratory morbidity typically increases. Pneumocystis induces mucus hyperproduction in immunocompetent host airways and whether it can stimulate Clara cells is unknown. Markers of Clara cell secretion and Notch1 activation were investigated in lungs of immunocompetent rats at 40, 60, and 80 days of age during Pneumocystis primary infection with and without Valproic acid (VPA), a Notch inducer. The proportion of rats expressing mucin increased in Pneumocystis-infected rats respect to controls at 60 and 80 days of age. Frequency of distal airways Clara cells was maintained while mRNA levels for the mucin-encoding genes Muc5B and Muc5ac in lung homogenates increased 1.9 and 3.9 times at 60 days of infection (P. = 0.1609 and P. = 0.0001, respectively) and protein levels of the Clara cell marker CC10 decreased in the Pneumocystis-infected rats at 60 and 80 days of age (P. = 0.0118 & P. = 0.0388). CC10 and Muc5b co-localized in distal airway epithelium of Pneumocystis-infected rats at day 60. Co-localization of Muc5b and Ki67 as marker of mitosis in distal airways was not observed suggesting that Muc5b production by Clara cells was independent of mitosis. Notch levels remained similar and no transnucleation of activated Notch associated to Pneumocystis infection was detected. Unexpectedly, mucus was greatly increased at day 80 in Pneumocystis-infected rats receiving VPA suggesting that a Notch-independent mechanism was triggered. Overall, data suggests a Clara to goblet cell transdifferentiation mechanism induced by Pneumocystis and independent of Notch.

Burnt sugarcane harvesting work: effects on pulmonary and systemic inflammatory markers.

Objective: To evaluate the effects of burnt sugarcane harvesting on the plasmatic and urinary concentrations of the club cell secretory protein (CC16) and inflammatory systemic biomarkers in a group of sugarcane cutters. Methods: Seventy-eight sugar cane workers were evaluated. The plasmatic and urinary concentrations of CC16, a pulmonary damage marker and inflammatory systemic biomarkers were collected at three time points: before, three months after and six months after the onset of the burnt sugarcane harvesting period. All evaluations were performed at ∼7 am, before the daily work shift. In the three-month evaluation, a post-work shift assessment (acute effect) was also performed. Results: The age of the workers was 37.9 ± 11.0 years. The PM2.5 concentrations were 27.0 (23.0-33.0) and 101.0 (31.0-139.5) µg/m3 in the pre harvest and harvest periods, respectively (p < .001). Burnt sugarcane harvesting was associated with a reduction, throughout the work during burnt sugarcane harvesting (subchronic effect), in plasmatic and urinary CC16 concentrations. Acutely, there was a decrease in plasmatic concentrations. There were acute and subchronic increases in inflammatory markers (neutrophils, monocytes) and muscle damage markers (CK and LDH) and a decrease in red blood cells. Conclusions: Harvesting of burnt sugarcane was associated with acute and subchronic reductions in the plasmatic and urinary concentrations of CC16 protein and changes in systemic inflammatory markers.

Clara Cell Protein Expression in Mechanically Ventilated Term and Preterm Infants with Respiratory Distress Syndrome and at Risk of Bronchopulmonary Dysplasia: A Pilot Study.

The aim of this pilot study was to determine Clara cell protein (CC16) concentration in bronchoalveolar lavages (BAL) fluid from full-term and preterm (<37 weeks' gestational age) neonates requiring respiratory support, having symptoms of neonatal respiratory distress syndrome, and at risk of bronchopulmonary dysplasia (BPD). We hypothesized that CC16 may be predictive of BPD diagnosis regardless of gestational age. BAL fluid CC16 was measured by ELISA at birth and at day 7 of life. Both groups that developed BPD showed significantly decreased BAL fluid CC16 levels compared to those infants that did not develop the disease. CC16 positively correlated with diagnosis of BPD and negatively with the severity of the disease. These results suggest that BAL fluid CC16 levels may have a diagnostic value at day 7 for BPD in both term and preterm infants. This study demonstrates the potential utility of BAL fluid CC16 levels as a biomarker for BPD in term infants.

Protective phenotypes of club cells and alveolar macrophages are favored as part of endotoxin-mediated prevention of asthma.

Atopic asthma is a chronic allergic disease that involves T-helper type 2 (Th2)-inflammation and airway remodeling. Bronchiolar club cells (CC) and alveolar macrophages (AM) are sentinel cells of airway barrier against inhaled injuries, where allergy induces mucous metaplasia of CC and the alternative activation of AM, which compromise host defense mechanisms and amplify Th2-inflammation. As there is evidence that high levels of environmental endotoxin modulates asthma, the goal of this study was to evaluate if the activation of local host defenses by Lipopolysaccharide (LPS) previous to allergy development can contribute to preserving CC and AM protective phenotypes. Endotoxin stimulus before allergen exposition reduced hallmarks of allergic inflammation including eosinophil influx, Interleukin-4 and airway hyperreactivity, while the T-helper type 1 related cytokines IL-12 and Interferon-γ were enhanced. This response was accompanied by the preservation of the normal CC phenotype and the anti-allergic proteins Club Cell Secretory Protein (CCSP) and Surfactant-D, thereby leading to lower levels of CC metaplasia and preventing the increase of the pro-Th2 cytokine Thymic stromal lymphopoietin. In addition, classically activated alveolar macrophages expressing nitric oxide were promoted over the alternatively activated ones that expressed arginase-1. We verified that LPS induced a long-term overexpression of CCSP and the innate immune markers Toll-like receptor 4, and Tumor Necrosis Factor-α, changes that were preserved in spite of the allergen challenge. These results demonstrate that LPS pre-exposition modifies the local bronchioalveolar microenvironment by inducing natural anti-allergic mechanisms while reducing local factors that drive Th2 type responses, thus modulating allergic inflammation.

Estrogen-regulated transcription of the uteroglobin gene from the brown hare (Lepus capensis).

To get further insights on the estrogen regulation of the uteroglobin (UG) gene, the 5'-flanking region of the UG gene from the brown hare (Lepus capensis) (Lc) was cloned and compared with those from two phylogenetically related species: the rabbit (Orictolagus cuniculus) (Oc) and the volcano rabbit (Romerolagus diazi) (Rd). The Lc-UG gene is very similar to those from rabbits (94%) and volcano rabbits (95%), and shares a number of genetic elements, including an estrogen response element (ERE). The estrogen-regulated transcription of a series of progressive 5'-deletion mutants of the Lc-UG gene, identified a functional ERE in the promoter region exhibiting the same orientation and relative position than that previously described in rabbits. The Lc-ERE is identical to the Oc-ERE, but different from both the Rd-ERE and the consensus ERE (c-ERE) by one nucleotide. We also detected important species-specific differences in the estrogen-regulated transcription of the UG gene. A luciferase reporter driven by 333 base pairs (bp) of the Lc-UG promoter elicited a higher response to estradiol than its related counterparts when expressed in estrogen-sensitive MCF-7 cells. Several ERE-like motifs which failed to act as functional EREs were also identified; one of them exhibited two mismatches in its palindromic sequence, a characteristic exhibited in many other natural occurring EREs, including the Rd-ERE.

Preoperative plasma club (clara) cell secretory protein levels are associated with primary graft dysfunction after lung transplantation.

Inherent recipient factors, including pretransplant diagnosis, obesity and elevated pulmonary pressures, are established primary graft dysfunction (PGD) risks. We evaluated the relationship between preoperative lung injury biomarkers and PGD to gain further mechanistic insight in recipients. We performed a prospective cohort study of recipients in the Lung Transplant Outcomes Group enrolled between 2002 and 2010. Our primary outcome was Grade 3 PGD on Day 2 or 3. We measured preoperative plasma levels of five biomarkers (CC-16, sRAGE, ICAM-1, IL-8 and Protein C) that were previously associated with PGD when measured at the postoperative time point. We used multivariable logistic regression to adjust for potential confounders. Of 714 subjects, 130 (18%) developed PGD. Median CC-16 levels were elevated in subjects with PGD (10.1 vs. 6.0, p<0.001). CC-16 was associated with PGD in nonidiopathic pulmonary fibrosis (non-IPF) subjects (OR for highest quartile of CC-16: 2.87, 95% CI: 1.37, 6.00, p=0.005) but not in subjects with IPF (OR 1.38, 95% CI: 0.43, 4.45, p=0.59). After adjustment, preoperative CC-16 levels remained associated with PGD (OR: 3.03, 95% CI: 1.26, 7.30, p=0.013) in non-IPF subjects. Our study suggests the importance of preexisting airway epithelial injury in PGD. Markers of airway epithelial injury may be helpful in pretransplant risk stratification in specific recipients.

Restoration of the normal Clara cell phenotype after chronic allergic inflammation.

Bronchiolar Clara cells play a critical role in lung homoeostasis. The main goal of this study was to evaluate the effects of chronic allergy on these cells and the efficacy of budesonide (BUD) and montelukast (MK) in restoring their typical phenotypes after ovalbumin-induced chronic allergy in mice. Chronic allergy induced extensive bronchiolar Alcian blue-periodic acid-Schiff (AB/PAS)-positive metaplasia. In addition, cells accumulated numerous big electron-lucent granules negative for Clara cell main secretory protein (CC16), and consequently, CC16 was significantly reduced in bronchoalveolar lavage. A concomitant reduction in SP-D and CYP2E1 content was observed. The phenotypic changes induced by allergy were pharmacologically reversed by both treatments; MK was more efficient than BUD in doing so. MK decreased AB/PAS reactivity to control levels whereas they remained persistently elevated after BUD. Moreover, most non-ciliated cells recovered their normal morphology after MK, whereas for BUD normal cells coexisted with 'transitional' cells that contained remnant mucous granules and stained strongly for CC16 and SP-D. Glucocorticoids were also less able to reduce inflammatory infiltration and maintained higher percentage of neutrophils, which may have contributed to prolonged mucin expression. These results show that chronic allergy-induced mucous metaplasia of Clara cells affects their defensive mechanisms. However, anti-inflammatory treatments were able to re-establish the normal phenotype of Clara cell, with MK being more efficient at restoring a normal profile than BUD. This study highlights the role of epithelial cells in lung injuries and their contribution to anti-inflammatory therapies.

Clara cell protein expression in human neonates during respiratory distress syndrome.

BACKGROUND/AIMS: Clara cell protein (cc-10) has been shown to negatively regulate inflammation, protect pulmonary surfactant from degradation in the lung, and administration of this recombinant protein improves the condition of infant respiratory distress syndrome (iRDS), a disease that occurred mainly in preterm infants. In view of the possibility that altered expression of cc-10 might regulate its protective function, we attempted to characterize this protein in infants with iRDS. METHODS: Using bronchotraqueal aspirates from human infants, we analyzed cc-10 in two-dimensional gel electrophoresis (2-DE) by combining immunoprecipitation, carbonyl groups and total protein immunoblotting. RESULTS: Seven forms of cc-10 were detected with western immunoblots in infants with iRDS while only four forms were present in neonates who needed mechanical ventilation for other reasons without any lung disease (control group). The overall levels of cc-10 in iRDS were lower and differences were seen in isoform pattern and distribution. CONCLUSION: Our demonstration that cc-10 is differentially expressed in infants with iRDS may point the way towards one possible mechanism that potentially involves modifications of the protein structure with its anti-inflammatory and surfactant protective function and could be detrimental for this airway disorder.

Cloning the uteroglobin gene promoter from the relic volcano rabbit (Romerolagus diazi) reveals an ancient estrogen-response element.

To gain further insight on the estrogen-dependent transcriptional regulation of the uteroglobin (UG) gene, we cloned the 5'-flanking region of the UG gene from the phylogenetically ancient volcano rabbit (Romerolagus diazi; Rd). The cloned region spans 812 base pairs (bp; -812/-1) and contains a noncanonical TATA box (TACA). The translation start site is 48 bp downstream from the putative transcription initiation site (AGA), and is preceded by a consensus Kozak box. Comparison of the Rd-UG gene with that previously isolated from rabbits (Oryctolagus cuniculus) showed 93% in sequence identity as well as a number of conserved cis-acting elements, including the estrogen-response element (ERE; -265/-251), which differs from the consensus by two nucleotides. In MCF-7 cells, 17ß-estradiol (E(2)) induced transcription of a luciferase reporter driven by the Rd-UG promoter in a similar manner as in an equivalent rabbit UG reporter; the Rd-UG promoter was 30% more responsive to E(2) than the rabbit promoter. Mutagenesis studies on the Rd-ERE confirmed this cis-element as a target of E(2) as two luciferase mutant reporters of the Rd-promoter, one with the rabbit and the other with the consensus ERE, were more responsive to the hormone than the wild-type reporter. Gel shift and super-shift assays showed that estrogen receptor-α indeed binds to the imperfect palindromic sequence of the Rd-ERE.

Effect of vitamin supplementation on lung injury and running performance in a hot, humid, and ozone-polluted environment.

In this study, the effect of vitamin C and E supplementation on lung injury and performance of runners were analyzed. Using a randomized, double-blinded, crossover design, nine runners participated in two experimental trials: a 2-week Vitamin trial (vitamin C = 500 mg/day + vitamin E = 100 IU/day) and a 2-week Placebo trial. At the end of each supplementation period the runners performed an 8-km time-trial run in a hot (31°C), humid (70% rh), and ozone-polluted (0.10 ppm O(3)) environmental chamber. Nasal lavage and blood samples were collected pre-, post-, and 6-h post-exercise to assess antioxidant status and CC16 as lung injury marker. Higher plasma (pre- and post-exercise) and nasal lavage (post-exercise) antioxidant concentration were found for the Vitamin trial. Nevertheless, this did not result in performance differences (Vitamin trial: 31:05 min; Placebo trial: 31:54 min; P = 0.075) even though significant positive correlations were found between antioxidant concentration and improvement in time to complete the run. CC16 was higher post-exercise in the Placebo trial (P < 0.01) in both plasma and nasal lavage. These findings suggest that antioxidant supplementation might help to decrease the lung injury response of runners when exercising in adverse conditions, but has little effect on performance.

Neutrophil predominance in induced sputum from asthmatic patients. Therapeutic implications and role of clara cell 16-KD protein.

UNLABELLED: Eosinophil is considered to be a main protagonist in asthma; however, often discordances between clinical manifestations and response to treatment are observed. We aimed to determine the occurrence of neutrophil predominance in asthma and to identify its characteristics on the basis of clinical-functional features, induced sputum cellular pattern and soluble molecules, to guide the appropriated anti-inflammatory therapy. A total of 41 patients were included in randomized groups: 21-40 year-old, with stable mild-to-severe asthma, steroid-naïve and non-smokers. An induced sputum sample was obtained under basal conditions, a second one after treatment with budesonide (400 ug b.i.d.) or montelukast (10 mg/d) for six weeks, and a final one after a 4-week washout period. By cytospin we evaluated eosinophil (EP) or neutrophil predominance (NP), and in supernatant we determined LTE4, and CC16. Peak expiratory flow variability (PEFV) was measured. A total of 23/41 patients corresponded to EP and 18/41 patients to NP. The PEFV was higher in EP than in NP. LTE4 was higher with NP than with EP. No difference was found for CC16. Montelukast reduced the predominant cell in both subsets, whereas budesonide only reduced eosinophils in EP. Budesonide and montelukast reduced PEFV in EP but not in NP. Considering the total treated-samples in each subset, CC16 level increased significantly in EP. IN CONCLUSION: a NP subset of asthmatic patients was identified. These patients show a lower bronchial lability; the leukotriene pathway is involved which responds to anti-leukotriene treatment. This phenotype shows a poor recovery of CC16 level after treatment.

Neutrophil predominance in induced sputum from asthmatic patients: Therapeutic implications and role of clara cell 16-KD protein / Predominio de neutrófilos en esputo inducido de asmáticos: Implicancias terapéuticas y rol de la proteína 16-kD de la célula clara

Eosinophil is considered to be a main protagonist in asthma; however, often discordances between clinical manifestations and response to treatment are observed. We aimed to determine the occurrence of neutrophil predominance in asthma and to identify its characteristics on the basis of clinical-functional features, induced sputum cellular pattern and soluble molecules, to guide the appropriated anti-inflammatory therapy. A total of 41 patients were included in randomized groups: 21-40 year-old, with stable mild-to-severe asthma, steroid-naïve and non-smokers. An induced sputum sample was obtained under basal conditions, a second one after treatment with budesonide (400 µg b.i.d.) or montelukast (10 mg/d) for six weeks, and a final one after a 4-week washout period. By cytospin we evaluated eosinophil (EP) or neutrophil predominance (NP), and in supernatant we determined LTE4, and CC16. Peak expiratory flow variability (PEFV) was measured. A total of 23/41 patients corresponded to EP and 18/41 patients to NP. The PEFV was higher in EP than in NP. LTE4 was higher with NP than with EP. No difference was found for CC16. Montelukast reduced the predominant cell in both subsets, whereas budesonide only reduced eosinophils in EP. Budesonide and montelukast reduced PEFV in EP but not in NP. Considering the total treated-samples in each subset, CC16 level increased significantly in EP. In conclusion: a NP subset of asthmatic patients was identified. These patients show a lower bronchial lability; the leukotriene pathway is involved which responds to anti-leukotriene treatment. This phenotype shows a poor recovery of CC16 level after treatment.

El eosinófilo es considerado la célula protagonista principal en el asma; sin embargo, a menudo se observan discordancias entre las manifestaciones clínicas y la respuesta de los pacientes al tratamiento. Nos propusimos determinar la ocurrencia de predominio de neutrófilos en el asma e identificar las características clínico-funcionales, el patrón celular y las moléculas solubles del esputo inducido, para guiar el tratamiento apropiado anti-inflamatorio. Se incluyeron 41 pacientes: 21 a 40 años de edad, con asma estable leve a grave, no tratados con esteroides tópicos ni sistémicos y no fumadores. Se obtuvo una muestra de esputo inducido en condiciones basales, una segunda muestra después del tratamiento al azar con budesonida (400 µg dos veces al día) o el montelukast (10 mg/d) durante seis semanas, y una final después de un período de lavado de 4 semanas. En el frotis por citocentrifugado se evaluó el predominio de eosinófilos (EP) o neutrófilos (NP), y en el sobrenadante se determinó LTE4, y CC16. Se midió la variabilidad del flujo espiratorio máximo (PEFV). Un total de 23/41 pacientes correspondieron al EP y 18/41 pacientes con NP. El PEFV fue mayor en el EP que en NP. LTE4 fue mayor en NP que en EP. No se encontraron diferencias de los niveles de CC16 en ambos grupos. Montelukast redujo la célula predominante en ambos subgrupos, mientras que budesonida sólo redujo los eosinófilos en EP. Tanto budesonida como montelukast redujeron PEFV en EP, pero no en NP. El nivel de CC16 aumentó significativamente en el EP luego del tratamiento antiinflamatorio. En conclusión: se identificó un subgrupo de asmáticos NP que presentan una menor labilidad bronquial, la vía de los leucotrienos parece estar involucrada y responde al tratamiento anti-leucotrienos. Este fenotipo muestra una escasa recuperación del nivel de CC16 posterior al tratamiento.

Mammaglobin and maspin transcripts in blood may reflect disease progression and the effect of therapy in breast cancer.

Detection of residual tumor cells in the circulation can provide prognostic as well as therapeutic information and help in identifying patients at high risk for developing metastases. Maspin and mammaglobin are two molecules that are specifically associated with breast cancer. We looked for mammaglobin and maspin transcripts in the peripheral blood of patients with breast cancer and evaluated their utility as a marker of the response to therapy. Maspin and mammaglobin transcripts were analyzed in 85 breast-cancer patients by nested RT-PCR, prior to and after treatment. Before therapy, 10 patients were found positive for mammaglobin and 20 patients were positive for maspin. In four patients, both transcripts were detected. Immediately following treatment, only one patient was still positive for mammaglobin while maspin transcripts persisted in three patients. Disease progression was observed mainly in patients in whom maspin transcripts were not detectable. Molecular detection of circulating tumor cells during therapy based on analysis for mammaglobin and maspin transcripts is an easy and practical method that can be applied to follow-up patients. We suggest that detection of mammaglobin mRNA is useful to determine the effect of therapy while maspin transcripts may indicate more aggressive disease.

Sentinel lymph node molecular pathology in breast carcinoma.

OBJECTIVES: The prognosis of breast cancer patients depends on primary tumor resection and axillary lymph nodes examination. The purpose of this study was to analyze by molecular biology techniques the presence of mammaglobin A and B messenger RNA in breast sentinel lymph node (SLN) by reverse-transcription polymerase chain reaction (RT-PCR). METHODS: Sentinel lymph nodes from 50 patients with a diagnosis of breast cancer were prospectively studied between June 2004 and August 2006. Lymph nodes were all examined every 2 mm by intraoperative cytology. Hematoxylin-eosin (HE), immunohistochemistry (IHC) with cytokeratin (clone AE1-AE3, DAKO, dilution 1:100), and molecular biology techniques were used in all cases. RESULTS: Deferred study with routine techniques showed subcapsular metastasis in 3/50 cases. Out of 50 cases, 5 were detected with IHC, and 2 of them were negative for HE. Multiplex RT-PCR allowed the detection of 18/50 positive SLN, which included the 5 above-mentioned cases. The other SLN studied (32/50) showed no metastases with the methods herein implemented. CONCLUSIONS: The epidemiologic impact of incomplete SLN study has been observed, as the HE technique fails to identify all SLN with micrometastases. In our opinion, SLN should be studied with IHC and molecular biology techniques. The multiplex RT-PCR technique for A and B mammaglobin proves to be specific and sensitive. This study will serve to formulate hypotheses. Further research, including a larger population and a longer-term follow-up period, will be required to confirm these hypotheses. Should our findings be confirmed in the future, molecular biology determinations could modify patients' staging and treatment.

Increased expression of uteroglobin associated with tubal inflammation and ectopic pregnancy.

OBJECTIVE: Evaluation of uteroglobin (UG) expression in the fallopian tube in different tubal diseases. DESIGN: The UG was screened and quantified in samples of fallopian tubes from patients with salpingitis, hydrosalpinx, and ectopic pregnancy by exposing the UG with immunohistochemical techniques. SETTING: University hospital and electron microscopy center. PATIENT(S): Women with pelvic inflammatory disease (PID) and complicated tubal ectopic pregnancy consulting for medical care. INTERVENTION(S): Salpingectomy. MAIN OUTCOME MEASURE(S): Tubal tissues were collected and examined using regular pathologic techniques. The UG immunoreactivity in the tubal epithelium was also assessed. RESULT(S): Fallopian tube epithelium displayed an increased UG expression in patients with PID and complicated tubal pregnancy compared with control patients. CONCLUSION(S): Uteroglobin is present in the human fallopian tube as a secretory protein and appears to be involved in immunosuppressive responses in the fallopian tube.