Tumores primitivos de pulmón en niños: imágenes y hallazgos / Primitive lung neoplasmas in children: imaging and findings

Los tumores primitivos pulmonares endobronquiales o parenquimatosos, son extremadamente raros en niños por lo que la experiencia individual en el diagnóstico, tratamiento y pronóstico es limitada. Nuestro trabajo muestra las formas de presentación, métodos de diagnóstico empleados y resultados en 21 pacientes que se atendieron en nuestro hospital entre los años 1991 a 2005. Los métodos de imágenes utilizados fueron: la radiología simple que permitió evaluar la sospecha de masa pulmonar, el US que demostró su naturaleza sólida, quística o mixta y la presencia o no de derrame pleural, mientras que la TC delimitó su localización extensión y metástasis, datos imprescindibles para el tratamiento quirúrgico adecuado. En los pacientes pedátricos que tienen lesión pulmonar ocupante de espacio, así como en los que debutan con neumotórax o hemotórax espontáneos debe tenerse en cuenta la posibilidad de tumor primitivo de pulmón, debiéndose profundizar los estudios mediante US, TC y endoscopia. Las lesiones congénitas quísticas tienen riesgo de malignización, por lo que su tratamiento debe ser quirúrgico.

The role of antioxidants in protection from ototoxic drugs.

A number of studies have shown that cisplatin and gentamicin ototoxic effects may result from free radical-mediated damage due to the reduction of antioxidant substances and an increased lipid peroxidation. The authors summarize the results obtained evaluating the auditory and vestibular functions and the inner ear hair cell morphology and survival after administration of antioxidant agents against cisplatin and gentamicin. In the first experiment, albino guinea pigs were treated with gentamicin (100 mg/kg per day, i.m.) alone or gentamicin (100 mg/kg per day, i.m.) plus alpha-tocopherol (100 mg/kg per day, i.m.) for 2 weeks. In a second experiment, albino guinea pigs were injected with cisplatin (2.5 mg/kg per day) or cisplatin (2.5 mg/kg per day) plus tiopronin (300 mg/kg) for 6 days. Electrocochleographic recordings were made from an implanted round window electrode. In all experiments compound action potentials (CAPs) were measured at 2-16 kHz. Changes in cochlear function were characterized as CAP threshold shifts. To evaluate vestibular function, the animals underwent sinusoidal oscillations in the dark about their vertical and longitudinal axes to evoke horizontal and vertical vestibulo-ocular reflexes (VOR). Frequency stimulation parameters ranged from 0.02 to 0.4 Hz and peak-to-peak amplitude was 20 degrees. Morphological changes were analysed by light microscopy and scanning electron microscopy. Both hearing loss and vestibular dysfunction induced by gentamicin were significantly attenuated by alpha-tocopherol. However, tiopronin co-therapy slowed the progression of hearing loss in cisplatin-treated animals and significantly attenuated the final threshold shifts. Cisplatin had little effect on the hair cells of cristae ampullares and maculae. Vestibular function was completely preserved in tiopronin co-treated animals. In conclusion, antioxidants such as alpha-tocopherol or tiopronin interfere with gentamicin and cisplatin damage and this suggests that they may be useful in preventing oto-vestibulotoxicity. Therefore, it is important to develop protective strategies that permit the avoidance of the toxic side effects of these drugs without interfering with their therapeutic effects.

The role of antioxidants in protection from ototoxic drugs.

A number of studies have shown that cisplatin and gentamicin ototoxic effects may result from free radical-mediated damage due to the reduction of antioxidant substances and an increased lipid peroxidation. The authors summarize the results obtained evaluating the auditory and vestibular functions and the inner ear hair cell morphology and survival after administration of antioxidant agents against cisplatin and gentamicin. In the first experiment, albino guinea pigs were treated with gentamicin (100 mg/kg per day, i.m.) alone or gentamicin (100 mg/kg per day, i.m.) plus α-tocopherol (100 mg/kg per day, i.m.) for 2 weeks. In a second experiment, albino guinea pigs were injected with cisplatin (2.5 mg/kg per day) or cisplatin (2.5 mg/kg per day) plus tiopronin (300 mg/kg) for 6 days. Electrocochleographic recordings were made from an implanted round window electrode. In all experiments compound action potentials (CAPs) were measured at 2-16 kHz. Changes in cochlear function were characterized as CAP threshold shifts. To evaluate vestibular function, the animals underwent sinusoidal oscillations in the dark about their vertical and longitudinal axes to evoke horizontal and vertical vestibulo-ocular reflexes (VOR). Frequency stimulation parameters ranged from 0.02 to 0.4 Hz and peak-to-peak amplitude was 20°. Morphological changes were analysed by light microscopy and scanning electron microscopy. Both hearing loss and vestibular dysfunction induced by gentamicin were significantly attenuated by α-tocopherol. However, tiopronin co-therapy slowed the progression of hearing loss in cisplatin-treated animals and significantly attenuated the final threshold shifts. Cisplatin had little effect on the hair cells of cristae ampullares and maculae. Vestibular function was completely preserved in tiopronin co-treated animals. In conclusion, antioxidants such as α-tocopherol or tiopronin interfere with gentamicin and cisplatin damage and this suggests that they may be useful in preventing oto-vestibulotoxicity. Therefore, it is important to develop protective strategies that permit the avoidance of the toxic side effects of these drugs without interfering with their therapeutic effects.

Tumores primitivos de pulmón en pediatría / Primary pulmonary neoplasms in children

Los tumores primitivos pulmonares, endobronquiales y parenquimatosos, son extremadamente raros en niños, por lo que la experiencia individual en el diagnóstico, tratamiento y pronóstico es limitada. Nuestro trabajo muestra las formas de presentación, métodos de diagnóstico empleados y resultados en 17 pacientes que se atendieron en nuestro hospital entre los años 1991 y 2000. De los métodos de imágenes, la radiología permitió evaluar la sospecha de masa pulmonar, el US demostró su naturaleza sólida, quística o mixta y la presencia o no de derrame pleural, mientras que la TC delimitó su localización, extensión y metástasis, datos imprescindibles para el tratamiento quirúrgico adecuado. En los pacientes pediátricos que tienen lesión pulmonar ocupante de espacio, así como en los que debutan con neumotórax o hemotórax espontáneos, debe tenerse en cuenta la posibilidad de tumor primitivo de pulmón, debiéndose profundizar los estudios mediante US, TC y endoscopía. Las lesiones congénitas quísticas tienen riesgo de malignización, por lo que su tratamiento debe ser quirúrgico (AU)

Tumores primitivos de pulmón en pediatría / Primary pulmonary neoplasms in children

Los tumores primitivos pulmonares, endobronquiales y parenquimatosos, son extremadamente raros en niños, por lo que la experiencia individual en el diagnóstico, tratamiento y pronóstico es limitada. Nuestro trabajo muestra las formas de presentación, métodos de diagnóstico empleados y resultados en 17 pacientes que se atendieron en nuestro hospital entre los años 1991 y 2000. De los métodos de imágenes, la radiología permitió evaluar la sospecha de masa pulmonar, el US demostró su naturaleza sólida, quística o mixta y la presencia o no de derrame pleural, mientras que la TC delimitó su localización, extensión y metástasis, datos imprescindibles para el tratamiento quirúrgico adecuado. En los pacientes pediátricos que tienen lesión pulmonar ocupante de espacio, así como en los que debutan con neumotórax o hemotórax espontáneos, debe tenerse en cuenta la posibilidad de tumor primitivo de pulmón, debiéndose profundizar los estudios mediante US, TC y endoscopía. Las lesiones congénitas quísticas tienen riesgo de malignización, por lo que su tratamiento debe ser quirúrgico

Human papillomavirus infection and epidermal growth factor receptor expression in primary laryngeal squamous cell carcinoma.

PURPOSE: This study was designed to add new data about laryngeal carcinogenesis, a multistep process in which chemical and/or viral agents induce and promote successive alterations in growth factor-linked signal transmission pathways, genetic instability, and mutations in key genes involved in cell growth control. Epidemiological evidence suggests that human papillomavirus (HPV) infection may be associated with the development of laryngeal cancer. EXPERIMENTAL DESIGN: In this report, we have analyzed the prevalence of HPV infection and epidermal growth factor receptor (EGFR) expression in a series of 42 laryngeal squamous cell carcinomas by PCR with HPV consensus primers and by a radioligand receptor assay, respectively. RESULTS: HPV DNA was detected in 15 of 42 (35.7%) tumors, and it belonged almost exclusively to the highly oncogenic HPV-16, HPV-18, and HPV-33 genotypes. At analysis by Mann-Whitney nonparametric statistical test, EGFR level was found to be significantly higher in HPV-infected than in HPV-negative cases (T = 440; P = 0.002). EGFR overexpression (EGFR-positive status >6 fmol/mg protein, the arbitrary cutoff value chosen) was found in 20 of 42 (47.6%) tumors, and it was associated with HPV infection in a statistically significant extent (chi(2) = 4.686; P = 0.03). CONCLUSIONS: Viral oncoproteins have been shown to induce a perturbation of the cell response to signals for growth and differentiation; these findings confirm that enhanced EGFR expression and activation in laryngeal squamous cell carcinoma may occur also as a consequence of HPV infection and support the hypothesis of an involvement of HPV infection in laryngeal carcinogenesis.

Functional domains of the ClpA and ClpX molecular chaperones identified by limited proteolysis and deletion analysis.

Escherichia coli ClpA and ClpX are ATP-dependent protein unfoldases that each interact with the protease, ClpP, to promote specific protein degradation. We have used limited proteolysis and deletion analysis to probe the conformations of ClpA and ClpX and their interactions with ClpP and substrates. ATP gamma S binding stabilized ClpA and ClpX such that that cleavage by lysylendopeptidase C occurred at only two sites. Both proteins were cleaved within in a loop preceding an alpha-helix-rich C-terminal domain. Although the loop varies in size and composition in Clp ATPases, cleavage occurred within and around a conserved triad, IG(F/L). Binding of ClpP blocked this cleavage, and prior cleavage at this site rendered both ClpA and ClpX defective in binding and activating ClpP, suggesting that this site is involved in interactions with ClpP. ClpA was also cut at a site near the junction of the two ATPase domains, whereas the second cleavage site in ClpX lay between its N-terminal and ATPase domains. ClpP did not block cleavage at these other sites. The N-terminal domain of ClpX dissociated upon cleavage, and the remaining ClpXDeltaN remained as a hexamer, associated with ClpP, and expressed ATPase, chaperone, and proteolytic activity. A truncated mutant of ClpA lacking its N-terminal 153 amino acids also formed a hexamer, associated with ClpP, and expressed these activities. We propose that the N-terminal domains of ClpX and ClpA lie on the outside ring surface of the holoenzyme complexes where they contribute to substrate binding or perform a gating function affecting substrate access to other binding sites and that a loop on the opposite face of the ATPase rings stabilizes interactions with ClpP and is involved in promoting ClpP proteolytic activity.

Translocation pathway of protein substrates in ClpAP protease.

Intracellular protein degradation, which must be tightly controlled to protect normal proteins, is carried out by ATP-dependent proteases. These multicomponent enzymes have chaperone-like ATPases that recognize and unfold protein substrates and deliver them to the proteinase components for digestion. In ClpAP, hexameric rings of the ClpA ATPase stack axially on either face of the ClpP proteinase, which consists of two apposed heptameric rings. We have used cryoelectron microscopy to characterize interactions of ClpAP with the model substrate, bacteriophage P1 protein, RepA. In complexes stabilized by ATPgammaS, which bind but do not process substrate, RepA dimers are seen at near-axial sites on the distal surface of ClpA. On ATP addition, RepA is translocated through approximately 150 A into the digestion chamber inside ClpP. Little change is observed in ClpAP, implying that translocation proceeds without major reorganization of the ClpA hexamer. When translocation is observed in complexes containing a ClpP mutant whose digestion chamber is already occupied by unprocessed propeptides, a small increase in density is observed within ClpP, and RepA-associated density is also seen at other axial sites. These sites appear to represent intermediate points on the translocation pathway, at which segments of unfolded RepA subunits transiently accumulate en route to the digestion chamber.

The RssB response regulator directly targets sigma(S) for degradation by ClpXP.

The sigma(S) subunit of Escherichia coli RNA polymerase regulates the expression of stationary phase and stress response genes. Control over sigma(S) activity is exercised in part by regulated degradation of sigma(S). In vivo, degradation requires the ClpXP protease together with RssB, a protein homologous to response regulator proteins. Using purified components, we reconstructed the degradation of sigma(S) in vitro and demonstrate a direct role for RssB in delivering sigma(S) to ClpXP. RssB greatly stimulates sigma(S) degradation by ClpXP. Acetyl phosphate, which phosphorylates RssB, is required. RssB participates in multiple rounds of sigma(S) degradation, demonstrating its catalytic role. RssB promotes sigma(S) degradation specifically; it does not affect degradation of other ClpXP substrates or other proteins not normally degraded by ClpXP. sigma(S) and RssB form a stable complex in the presence of acetyl phosphate, and together they form a ternary complex with ClpX that is stabilized by ATP[gamma-S]. Alone, neither sigma(S) nor RssB binds ClpX with high affinity. When ClpP is present, a larger sigma(S)--RssB--ClpXP complex forms. The complex degrades sigma(S) and releases RssB from ClpXP in an ATP-dependent reaction. Our results illuminate an important mechanism for regulated protein turnover in which a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific protease.

[Diagnostic value of defecography]. / Valor diagnóstico de la defecografía.

The aim of this study was to assess the importance of defecatography in the diagnosis of lower chronic constipation (4) or rectal type (12), principally in those patients on whom other diagnostic methods had not produced supportive data. Over a 64 month period, 65 patients who had consulted because of chronic constipation, were studied; they were suffering from low bowel symptoms like difficulty in the evacuation of the rectum. The average age was 48 and mostly female. All of them were asked to prepare the same mixture for the defecatory study, using the same type of contrast material and study technique. In most of the cases correlated functional elements were found, while very few cases resulting from just organic causes were found, and only one without functional or organic reason was found. Our results were as follows. 1) Insufficient laxity of the pubo-rectal beam related to forward or backward rectocele or lowering of the increased pelvic floor, a fact that was found in 19 patients (29.23%). 2) Inadequate laxity of the pubo rectal beam in 12 patients (18.46%). 3) Paradoxical contraction of the pubo rectal beam related to forward rectocele or lowering of the increased pelvic floor, in 11 patients (16.92%). 4) Lowering of the increased pelvic floor, related to forward or backward rectocele in 8 patients (12.30%). 5) Paradoxical contraction of the pubo rectal beam in 7 patients (10.76%). 6) Forward or backward rectocele in 3 patients (4.61%). 7) Lowering of the increased pelvic floor in 2 patients (3.07%). 8) Rectal intususception in 1 patient (1.53%). 9) Average study in 1 patient (1.53%). Therefore, the defecatography is a very useful method of study to appraise constipation with anorectoperineal symptoms, as it allows us to diagnose organic and functional problems in the area (6). Likewise, the importance of pre and post surgical tests, both therapeutic and reconstructive must be underlined.

Valor diagnóstico de la defecografía. / [Diagnostic value of defecography]

The aim of this study was to assess the importance of defecatography in the diagnosis of lower chronic constipation (4) or rectal type (12), principally in those patients on whom other diagnostic methods had not produced supportive data. Over a 64 month period, 65 patients who had consulted because of chronic constipation, were studied; they were suffering from low bowel symptoms like difficulty in the evacuation of the rectum. The average age was 48 and mostly female. All of them were asked to prepare the same mixture for the defecatory study, using the same type of contrast material and study technique. In most of the cases correlated functional elements were found, while very few cases resulting from just organic causes were found, and only one without functional or organic reason was found. Our results were as follows. 1) Insufficient laxity of the pubo-rectal beam related to forward or backward rectocele or lowering of the increased pelvic floor, a fact that was found in 19 patients (29.23

). 2) Inadequate laxity of the pubo rectal beam in 12 patients (18.46

). 3) Paradoxical contraction of the pubo rectal beam related to forward rectocele or lowering of the increased pelvic floor, in 11 patients (16.92

). 4) Lowering of the increased pelvic floor, related to forward or backward rectocele in 8 patients (12.30

). 5) Paradoxical contraction of the pubo rectal beam in 7 patients (10.76

). 6) Forward or backward rectocele in 3 patients (4.61

). 7) Lowering of the increased pelvic floor in 2 patients (3.07

). 8) Rectal intususception in 1 patient (1.53

). 9) Average study in 1 patient (1.53

). Therefore, the defecatography is a very useful method of study to appraise constipation with anorectoperineal symptoms, as it allows us to diagnose organic and functional problems in the area (6). Likewise, the importance of pre and post surgical tests, both therapeutic and reconstructive must be underlined.

Subunit-specific degradation of the UmuD/D' heterodimer by the ClpXP protease: the role of trans recognition in UmuD' stability.

The Escherichia coli UmuD' protein is a subunit of the recently described error-prone DNA polymerase, pol V. UmuD' is initially synthesized as an unstable and mutagenically inactive pro-protein, UmuD. Upon processing, UmuD' assumes a relatively stable conformation and becomes mutagenically active. While UmuD and UmuD' by themselves exist in vivo as homodimers, when together they preferentially interact to form heterodimers. Quite strikingly, it is in this context that UmuD' becomes susceptible to ClpXP-mediated proteolysis. Here we report a novel targeting mechanism designed for degrading the mutagenically active UmuD' subunit of the UmuD/D' heterodimer complex, while leaving the UmuD protein intact. Surprisingly, a signal that is essential and sufficient for targeting UmuD' for degradation was found to reside on UmuD not UmuD'. UmuD was also shown to be capable of channeling an excess of UmuD' to ClpXP for degradation, thereby providing a mechanism whereby cells can limit error-prone DNA replication.

Unfolding and internalization of proteins by the ATP-dependent proteases ClpXP and ClpAP.

ClpX and ClpA are molecular chaperones that interact with specific proteins and, together with ClpP, activate their ATP-dependent degradation. The chaperone activity is thought to convert proteins into an extended conformation that can access the sequestered active sites of ClpP. We now show that ClpX can catalyze unfolding of a green fluorescent protein fused to a ClpX recognition motif (GFP-SsrA). Unfolding of GFP-SsrA depends on ATP hydrolysis. GFP-SsrA unfolded either by ClpX or by treatment with denaturants binds to ClpX in the presence of adenosine 5'-O-(3-thiotriphosphate) and is released slowly (t(1/2) approximately 15 min). Unlike ClpA, ClpX cannot trap unfolded proteins in stable complexes unless they also have a high-affinity binding motif. Addition of ATP or ADP accelerates release (t(1/2) approximately 1 min), consistent with a model in which ATP hydrolysis induces a conformation of ClpX with low affinity for unfolded substrates. Proteolytically inactive complexes of ClpXP and ClpAP unfold GFP-SsrA and translocate the protein to ClpP, where it remains unfolded. Complexes of ClpXP with translocated substrate within the ClpP chamber retain the ability to unfold GFP-SsrA. Our results suggest a bipartite mode of interaction between ClpX and substrates. ClpX preferentially targets motifs exposed in specific proteins. As the protein is unfolded by ClpX, additional motifs are exposed that facilitate its retention and favor its translocation to ClpP for degradation.

Protein binding and unfolding by the chaperone ClpA and degradation by the protease ClpAP.

ClpA, a bacterial member of the Clp/Hsp100 chaperone family, is an ATP-dependent molecular chaperone and the regulatory component of the ATP-dependent ClpAP protease. To study the mechanism of binding and unfolding of proteins by ClpA and translocation to ClpP, we used as a model substrate a fusion protein that joined the ClpA recognition signal from RepA to green fluorescent protein (GFP). ClpAP degrades the fusion protein in vivo and in vitro. The substrate binds specifically to ClpA in a reaction requiring ATP binding but not hydrolysis. Binding alone is not sufficient to destabilize the native structure of the GFP portion of the fusion protein. Upon ATP hydrolysis the GFP fusion protein is unfolded, and the unfolded intermediate can be sequestered by ClpA if a nonhydrolyzable analog is added to displace ATP. ATP is required for release. We found that although ClpA is unable to recognize native proteins lacking recognition signals, including GFP and rhodanese, it interacts with those same proteins when they are unfolded. Unfolded GFP is held in a nonnative conformation while associated with ClpA and its release requires ATP hydrolysis. Degradation of unfolded untagged proteins by ClpAP requires ATP even though the initial ATP-dependent unfolding reaction is bypassed. These results suggest that there are two ATP-requiring steps: an initial protein unfolding step followed by translocation of the unfolded protein to ClpP or in some cases release from the complex.

A multicenter trial of specific local nasal immunotherapy.

OBJECTIVE: To assess the efficacy and safety of specific local nasal immunotherapy (LNIT) in powder form in patients with allergic rhinitis, using subjective and objective parameters. STUDY DESIGN: A double-blind randomized multicenter trial of 102 patients with allergic rhinitis who were treated with specific LNIT for 8 consecutive months. METHODS: After identifying allergens with the skin prick test and sensitization threshold dose with the specific nasal provocation test, 102 patients were selected, of whom 55 were allergic to mites and 47 were allergic to Graminaceae or Parietaria pollen. The specific treatments were self-administered using an insufflator in two phases (phase 1: increasing doses; phase: 2, maintenance dose). Patients were evaluated before and after 32 weeks of treatment by subjective analysis of their self-reported symptoms and by objective analysis of nasal provocation test, nasal resistance by anterior rhinomanometry, and mucociliary clearance time. RESULTS: Clinical efficacy of LNIT for allergy to mites and pollens was confirmed by the differences in the symptoms score between the active group and the placebo group. The nasal provocation test results confirmed that this difference was statistically significant. The rhinomanometric analysis gave positive results for the treated group mainly in LNIT for mites. No differences in mucociliary clearance time were found. CONCLUSIONS: Specific LNIT is effective for allergic rhinitis and appears to offer considerable advantages over other hyposensitization methods. It can be done at home, patient compliance is good, and the treatment is safe.

Visualization of substrate binding and translocation by the ATP-dependent protease, ClpXP.

Binding and internalization of a protein substrate by E. coli ClpXP was investigated by electron microscopy. In sideviews of ATP gamma S-stabilized ClpXP complexes, a narrow axial channel was visible in ClpX, surrounded by protrusions on its distal surface. When substrate lambda O protein was added, extra density attached to this surface. Upon addition of ATP, this density disappeared as lambda O was degraded. When ATP was added to proteolytically inactive ClpXP-lambda O complexes, the extra density transferred to the center of ClpP and remained inside ClpP after separation from ClpX. We propose that substrates of ATP-dependent proteases bind to specific sites on the distal surface of the ATPase, and are subsequently unfolded and translocated into the internal chamber of the protease.