Tratamiento rehabilitador en la incontinencia fecal / Rehabilitation treatment in fecal incontinence

Introducción. Podríamos definir la incontinencia fecal como la pérdida involuntaria de heces sólidas y líquidas, siempre que esta pérdida supone un problema higiénico o social en la persona que lo padece. La prevalencia de esta patología no está clara puesto que supone una importante afectación de la calidad de vida de los pacientes que la padecen, y muchas veces origina un encubrimiento de este problema. Material y método. Realizamos un estudio prospectivo, en el mismo han participado 24 pacientes diagnosticados de incontinencia fecal, todos ellos realizaron un programa de tratamiento que incluyó: normas educativas, ejercicios de fortalecimiento del suelo pélvico, y técnicas de biorretroalimentación con electroestimulación. Resultados. El 79,2% eran mujeres, la edad media fue de 60,8 años, el 20,8% de los pacientes habían tenido una neoplasia intestinal. En la pruebas complementarias encontramos en la ecografía que en un 25% de los casos había una rotura de alguno de los esfínteres anales, en un 70,8% un adelgazamiento de los esfínteres, en la manometría inicial encontramos, que la media de la presión máxima basal en mmHg fue de 36,37 mmHg (D.E. 13,13), y la media en la presión máxima en la contracción voluntaria fue de 82,25mmHg (D.E. 21,45) puntuación media inicial obtenida en la escala de Wexner fue de 15,79, y tras el tratamiento 8,16. Obtenemos diferencias estadísticamente significativas en todos los ítems de la escala de Wexner. Conclusiones. El tratamiento conservador combinado mejora la puntuación obtenida en la escala de Wexner en la incontinencia fecal moderada-severa, además de presentar mínimos efectos secundarios. Las pruebas complementarias son de utilidad para evaluar la incontinencia fecal, pero la evaluación clínica es fundamental para determinar la gravedad de esta patología, y la afectación que produce en la vida del paciente (AU)

Introduction. We can define fecal incontinence as the involuntary loss of solid and liquid stools, whenever this loss poses a hygiene or social problem for the person suffering it. Prevalence of this condition is not clear since it means an important affection in the quality of life of the patient suffering it and therefore, they often hide this problem. Material and methods. We have performed a prospective study in which 24 patients diagnosed of fecal incontinence participated. All of them underwent a treatment program that included: education guidelines, pelvic floor strengthening exercises and biofeedback with electrical stimulation. Results. A total of 79.2% were women, with mean age of 60.8 years, and 20.8% of the patients had suffered an intestinal neoplasm. In the complementary tests, the ultrasonography showed that 25% of the cases had a rupture of the anal sphincters. In 70.8%, there was thinning of the sphincters. In the initial manometry, we found that the measurement of maximum baseline pressure in mmHg was 36.37 mmHg (SD 13.13), and the mean maximum pressure involuntary contraction was 82.25mmHg (SD 21.45), initial mean score obtained on the Wexner scale was 15.79 and after treatment, 8.16. We obtained statistically significant differences in all of the items on the Wexner scale. Conclusion. Combined conservative treatment improves the score obtained on the Wexner scale in moderate to severe fecal incontinence and also shows minimum side effects. Complementary tests are useful to evaluate fecal incontinence, however clinical evaluation is fundamental to determine the severity of this condition and how it affects the patient's life (AU)

PEA-15 mediates cytoplasmic sequestration of ERK MAP kinase.

The ERK 1/2 MAP kinase pathway controls cell growth and survival and modulates integrin function. Here, we report that PEA-15, a protein variably expressed in multiple cell types, blocks ERK-dependent transcription and proliferation by binding ERKs and preventing their localization in the nucleus. PEA-15 contains a nuclear export sequence required for its capacity to anchor ERK in the cytoplasm. Genetic deletion of PEA-15 results in increased ERK nuclear localization with consequent increased cFos transcription and cell proliferation. Thus, PEA-15 can redirect the biological outcome of MAP kinase signaling by regulating the subcellular localization of ERK MAP kinase.

DNA double helix destabilizing properties of cyclobisintercaland compounds and competition with a single strand binding protein.

The DNA helix destabilizing activity of a series of cyclobisintercaland compounds (CBIs) has been evaluated by measuring their ability to displace a 32P-labelled oligonucleotide primer (17-mer) hybridized to the single stranded DNA of M13. This destabilizing activity appears to be strongly dependent on the cyclic structure (the linear acyclic references are inactive) and the size of the macrocycle; both features being known to determine the preferential binding of the compound to ssDNA. Interestingly, CBIs induced the dissociation of the duplex template in a concentration range (0.5-1 microM) close to that required for the destabilizing activity of single stranded DNA binding proteins (SSBs). Therefore competition experiments between CBIs and an SSB protein (Eco SSB) for binding to a single stranded oligonucleotide target (36-mer) have been performed through gel electrophoresis and nitrocellulose binding assays and strong inhibitory effects on the formation of the SSB:36-mer complex have been observed.

Involvement of intronic sequences in cell-specific expression of the peripherin gene.

Peripherin is an intermediate filament protein expressed in restricted populations of neurons. Our previous study of the chromatin structure of the mouse peripherin gene in cells that do or do not express peripherin suggested that the region located between -1,500 and +800 bp of the gene could be involved in its cell specificity. In the present work, we performed an in vitro functional analysis of the 5' flanking region of the mouse peripherin gene and observed that this region up to 9 kb contained both enhancer and inhibiting activities; however, it was insufficient to achieve a complete extinction of reporter gene expression in peripherin-negative cells. Furthermore, analysis of the first three introns with the 5' flanking sequences of the gene showed that intron I greatly increased specificity of the gene expression. Intron I also conferred the same properties to thymidine kinase heterologous promoter. DNase I footprinting experiments performed with intron I revealed at least two protected regions (Inl A and Inl B). Inl A encompasses an AP-2-like binding site that interacted with both neuroblast and fibroblast nuclear factors, as well as with the recombinant AP-2alpha protein. However, gel shift experiments suggested that the interacting nuclear factors are distinct from AP-2alpha itself and probably belong to the AP-2 family. Inl B perfectly matched the consensus binding site for Sp1 and specifically interacted with nuclear protein factors that showed the same binding properties as the Sp1 family members. Fine deletion analysis of intron I indicated that the Inl A element alone is responsible for its enhancing properties, whereas a region located between +789 and +832 gives to intron I its silencer activity.

Knock-out of the neural death effector domain protein PEA-15 demonstrates that its expression protects astrocytes from TNFalpha-induced apoptosis.

Apoptosis is a very general phenomenon, but only a few reports concern astrocytes. Indeed, astrocytes express receptors for tumor necrosis factor (TNF) alpha, a cytokine demonstrated on many cells and tissues to mediate apoptosis after recruitment of adaptor proteins containing a death effector domain (DED). PEA-15 is a DED-containing protein prominently expressed in the CNS and particularly abundant in astrocytes. This led us to investigate if PEA-15 expression could be involved in astrocytic protection against deleterious effects of TNF. In vitro assays evidence that PEA-15 may bind to DED-containing protein FADD and caspase-8 known to be apical adaptors of the TNF apoptotic signaling. After generation of PEA-15 null mutant mice, our results demonstrate that PEA-15 expression increases astrocyte survival after exposure to TNF.

Transcriptional activation of the mouse peripherin gene by leukemia inhibitory factor: involvement of STAT proteins.

Peripherin is a neuron-specific intermediate filament protein whose expression is activated in vitro by the neuropoietic cytokines leukemia inhibitory factor (LIF) and interleukin-6. We have studied the mechanisms of transcriptional activation of the peripherin gene by LIF. In particular, we have identified a 70-bp element [peripherin cytokine-responsive element (Pe-CyRE)] within the 5'-flanking sequences of the mouse peripherin gene (between -930 and -860) that enhances transcription in two neuroblastoma cell lines, NBFL and LA-N-2, in response to LIF treatment. We have also shown by DNA mobility shift assays that treatment of cells by LIF induces the binding of protein complexes composed of at least two members of the signal transducers and activators of transcription (STAT) factor family to a cis element (Pe-APRE2) within Pe-CyRE. Furthermore, the entire Pe-CyRE, as well as Pe-APRE2, conferred responsiveness onto a heterologous thymidine kinase promoter. However, the response amplitude of the heterologous promoter to LIF was lower than that observed with the 5'-flanking sequences of the peripherin promoter, suggesting that cooperative interactions with surrounding sequences of the peripherin gene are required for a full transcriptional activation.

Selective photocleavage of single-stranded nucleic acids by cyclobisintercaland molecules.

Irradiation of mixtures of a single-stranded circular plasmid and of a double-stranded supercoiled DNA in presence of the cyclobisintercaland compounds 2 or 3 shows that these reagents effect the selective photocleavage of the single-stranded entity. Furthermore, 2 also cleaves tRNAasp preferentially at single-stranded domains.

Guanidinium-cholesterol cationic lipids: efficient vectors for the transfection of eukaryotic cells.

Two cationic lipids, bis-guanidinium-spermidine-cholesterol (BGSC) and bis-guanidinium-trencholesterol (BGTC)-cholesterol derivatives bearing two guanidinium groups-have been synthesized and tested as artificial vectors for gene transfer. They combine the membrane compatible features of the cholesterol subunit and the favorable structural and high pKa features of the guanidinium functions for binding DNA via its phosphate groups. Reagent BGTC is very efficient for transfection into a variety of mammalian cell lines when used as a micellar solution. In addition, both BGTC and BGSC present also a high transfection activity when formulated as liposomes with the neutral phospholipid dioleoylphosphatidyl ethanolamine. These results reveal the usefulness of cholesterol derivatives bearing guanidinium groups for gene transfer.

Three different states of the chromatin structure of the mouse peripherin gene.

The chromatin structure of the mouse peripherin gene domain was analyzed in peripherin-positive and -negative cell lines. At least nine DNase I hypersensitive sites (HSS) are present within the 20-kb peripherin domain in the mouse neuroblastoma cell lines which express peripherin. Three of them are situated in intron I and intron III, the others being distributed within the 5' flanking region up to -5.5 kb. The presence of these sites was also investigated in the peripherin chromatin domain of peripherin-negative cell lines. Two other types of HSS distribution were observed along the peripherin gene according to the category of cell considered: constantly peripherin-negative cells, or negative cells arising from transiently peripherin-expressing precursors. From comparison of HSS patterns in these cell lines with those of neuroblastoma cells, it can be predicted that HSS located in the region -1500/+800 bp participate in cell-specific expression of the mouse peripherin gene.

Evolution of acetylcholinesterase transcripts and molecular forms during development in the central nervous system of the quail.

We studied the expression of acetylcholinesterase (AChE) in the nervous system (cerebellum, optic lobes and neuroretina) of the quail at different stages of development, from embryonic day 10 (E10) to the adult. Analyzing AChE mRNAs and AChE molecular forms, we observed variations in the following: (a) production of multiple mRNA species (4.5 kb, 5.3 kb, and 6 kb); (b) translation and/or stability of the AChE protein; (c) production of active and inactive AChE molecules; (d) production of amphiphilic and nonamphiphilic AChE forms; and (e) proportions of tetrameric G4, dimeric G2, and monomeric G1 forms. The large transcripts present distinct temporal patterns and disappear in the adult, which possesses only the 4.5-kb mRNA; these changes are unlikely to be related to those observed for the AChE protein, because all transcripts seem to encode the same catalytic subunit (type T). In addition, the levels of mRNA and AChE are not correlated in the three regions, especially at the adult stage. The proportion of inactive AChE was found to be markedly higher at the hatching period (E16) than at earlier stages (E10 and E13) or in the adult. The G4 form is predominant already at E10, and in the adult its proportion reaches 80% of the activity in the cerebellum and optic lobes, and 65-70% in the neuroretina. This form is largely nonamphiphilic in embryonic tissues, but it becomes progressively more amphiphilic with development. Thus, the different processing and maturation steps appear to be regulated in an independent manner and potentially correspond to physiologically adaptative mechanisms.

The tyrosine hydroxylase gene is expressed in endoderm and pancreas of early quail embryos.

The initial expression of the gene encoding tyrosine hydroxylase (TH) was studied in the trunk of quail embryos by in situ hybridization. We detected the presence of quail TH mRNA on embryonic day 3.5 (E3.5) in the sympathetic ganglia and aortic plexus, both neural crest derived structures. In contrast, the TH gene was expressed much earlier in the endodermal layer of E2 embryos, i.e. from the 8-somite stage onwards. TH mRNA was found also in the pancreatic bud, an endoderm-derived structure. The TH protein and catecholamines were subsequently looked for in these structures. TH immunoreactivity was found in cells of E2 explanted endoderm, but no catecholamine histofluorescence was observed before or after a few days in culture. TH-positive cells were also detected in cultures of pancreatic rudiments, explanted from E3 to E6 quail embryos. We suggest that the TH-positive cells of the endoderm are the progenitors of the catecholaminergic cells of the pancreas and of the enterochromaffin cells of the gut. The hypothesis that the TH-positive cells of the endoderm are involved in the expression of the catecholaminergic phenotype by neural crest cells is discussed.

The neuropeptide VIP regulates the expression of the tyrosine hydroxylase gene in cultured avian sympathetic neurons.

The neuropeptide vasoactive intestinal polypeptide (VIP) increases the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, in cultured chicken sympathetic neurons. We report here that VIP acts by increasing TH mRNA levels in these cells. Induction of TH mRNA is transient and reaches maximal values 6-8 h after the addition of the peptide to the cultures. TH mRNA levels return to control values after 1-2 days. The quail cDNA probe detects a single mRNA species of approximately 9 kb in RNA extracted both from embryonic chicken sympathetic neurons and adult quail adrenal medulla.

Regulation of the quail tyrosine hydroxylase gene in neural crest cells by cAMP and beta-adrenergic ligands.

Expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine (CA) synthesis, was studied in quail neural crest (NC) cultures to investigate the role of environmental factors in the development of the adrenergic phenotype. First, the activity of the quail TH gene promoter was analyzed in immortalized quail NC cells by transient transfection assay. We found that chick embryo extract (CEE), which is known to trigger adrenergic differentiation of NC cells in vitro, strongly enhanced reporter gene transcription. A sequence of only 77 nucleotides, including a cAMP-responsive element, was sufficient to elicit this response. Implication of cAMP in the CEE effect was further supported by the fact that CEE produced a stimulation of adenylate cyclase activity that was sensitive to beta-adrenoreceptor antagonists. Moreover, stimulation of TH gene expression by CEE could be reduced by beta-adrenergic antagonists or CA-depleted extract. We have subsequently investigated the possibility that TH expression during differentiation of nontransformed NC cells could also be influenced by CA via the cAMP pathway. In dissociated cultures of 3-day-old quail sclerotomes and of 2-day-old quail trunk NC, differentiation of TH+ cells was induced in the presence of forskolin, an activator of adenylate cyclase, and modulated by beta-adrenoreceptor ligands. In particular, the adrenergic-promoting effect of 10% CEE on trunk NC cultures was inhibited by beta-adrenoreceptor antagonists or by eliminating CA from the extract. In conclusion, these data suggest that CA (through activation of beta-adrenergic receptors and cAMP production) can stimulate the initial expression of TH in cultured NC cells and enhance TH transcription in immortalized NC cells. This supports the notion that exogenous CA, which is present in the early embryo, may promote the differentiation of sympathoadrenal precursors in vivo by stimulating TH gene activity.

The quail tyrosine hydroxylase gene promoter contains an active cyclic AMP-responsive element.

A 1.5-kb genomic DNA fragment situated upstream from the quail tyrosine hydroxylase (TH) gene transcription site was isolated. This upstream region starts 15 bp from the translation initiation site. It contains two canonical TATA boxes, at positions -37 and -297, three putative glucocorticoid-responsive elements, at positions -1487, -1329, and -1268, and one putative cyclic AMP (cAMP)-responsive element (CRE) at position -53, as well as a putative negative regulatory element consensus sequence at position -735. The consensus POU-Oct site is partly conserved. Comparison of the 5' flanking sequences of quail and mammalian (bovine, human, and rat) TH genes revealed a strong sequence conservation within the 230 nucleotides upstream of the TATA box, with a distinct conservation of the CRE region. Constructs in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was linked to promoter stretches of increasing lengths were transfected into three cell lines, two of them originating from quail and rat neural crest and the third derived from mouse fibroblasts. Reporter gene expression was specifically high in the quail and rat neural crest-derived cells compared to the fibroblast cell line. The physiological activity of this putative quail CRE was analyzed further in transfected neural crest cells of quail origin. Both cAMP analogues and agents that enhance intracellular cAMP increased CAT activity. The physiological relevance of this finding is sustained by the presence, in quail nuclear extracts, of a protein(s) that binds CRE consensus sequences.

Expression of the gene encoding tyrosine hydroxylase in a subpopulation of quail dorsal root ganglion cells cultured in the presence of insulin or chick embryo extract.

Avian sensory ganglia contain a population of normally latent autonomic precursors with catecholaminergic potentialities. The present study examines the expression of the tyrosine hydroxylase (TH) gene in quail dorsal root ganglia (DRG) by both in situ hybridization and polymerase chain reaction (PCR) techniques. In situ hybridization using quail TH cDNA as a probe demonstrated the presence in DRG cell cultures of TH mRNA in a subpopulation of cells that never express the adrenergic phenotype in vivo. Expression of the TH gene in autonomic precursor cells of DRG in culture is totally dependent on the presence either of insulin or chick embryo extract. The numbers of catecholaminergic cells expressing TH mRNA and TH immunoreactivity evolve in a closely similar manner during the culture period. Using two primers, specific for highly conserved 5' regions of TH cDNA, it was possible to detect the same band of DNA amplified by PCR in total RNA from DRG cultures grown in the presence of insulin, sympathetic ganglia and adrenal gland. No amplified DNA was detected in uncultured DRG cells. These data further indicate that, under the influence either of insulin or a still unknown factor contained in the CEE, the TH gene is induced in a subpopulation of DRG cells.

myc products induce the expression of catecholaminergic traits in quail neural crest-derived cells.

The avian myelocytomatosis virus strain MC29 v-myc oncogene transforms a wide panel of avian cells in vitro and either blocks or maintains differentiation, depending on the cell type. In the present work, we have investigated the effect of this oncogene on the differentiation of early embryonic cells, neural crest cells, grown in vitro. We report that the MC29 v-myc gene product induces a strong cellular proliferation of 2-day quail neural crest with the appearance of catecholaminergic traits. Other v-myc as well as the c-myc gene products also trigger this phenotype. Retroviruses carrying some other oncogenes do not elicit this phenotypic expression, although they activate cell multiplication. Thus, our results indicate that myc gene products induce (directly or indirectly) a differentiated phenotype in a subpopulation of neural crest cells.

A monoclonal antibody directed against quail tyrosine hydroxylase: description and use in immunocytochemical studies on differentiating neural crest cells.

Catecholamine (CA) synthesis is one of the phenotypic traits expressed by some neural crest-derived cells in vivo and in vitro. In the present study, we have evidenced, in quail embryos, the expression of the first enzyme of CA metabolism, tyrosine hydroxylase (TOH), using a monoclonal antibody raised against the quail enzyme. This antibody also recognizes TOH from chick and pleurodele, but not from several mammalian species (rat, human). We have also investigated the extent to which TOH-positive cells, differentiated in neural crest cultures, express structural neuronal markers and display vasoactive intestinal polypeptide (VIP) and substance P (SP) immunoreactivity. Double-immunolabeling experiments show that, in vitro, half of the population of TOH-positive cells exhibits tetanus toxin binding sites but none of them are recognized by a neurofilament antibody. On the other hand, some TOH-positive cells contain VIP or SP. These observations suggest that under our culture conditions autonomic neural crest precursors differentiate only into immature sympathoblasts, but are able to synthesize peptides in addition to CA.

Cloning of quail tyrosine hydroxylase: amino acid homology with other hydroxylases discloses functional domains.

A cDNA clone containing the entire coding region of quail tyrosine hydroxylase (TH) has been isolated and analyzed. Comparison with rat and human THs and phenylalanine hydroxylases reveals several highly conserved domains. Two of them, shared by all these hydroxylases, are localized in the central and C-terminal parts of the molecules, and most probably include the active site. Two others are found only in the TH molecules. One contains putative sites of phosphorylation and is implicated in the posttranslational regulation of the enzyme. The second highly preserved domain, consisting of a stretch of 21 amino acids, is presumably associated with an important feature of the enzyme that remains to be identified.