Biomedical subjects

C Alvarez

Publications and source records attributed to C Alvarez.

At least 55 records · Page 3Linked to original sources

PubMed2001

Progesterone and 17 beta-estradiol acutely stimulate nitric oxide synthase activity in rat aorta and inhibit platelet aggregation.

The rapid non-genomic stimulatory action of progesterone (Pg) and estradiol (E2) on nitric oxide synthase (NOS) activity of endothelium intact aortic rings and its effect on platelet aggregation was investigated. First we measured the effect of the hormones on platelet aggregation when added to rat aortic strips (RAS) incubated in a PRP. RAS induced an antiaggregatory activity, which was enhanced by the presence of the hormones. The inhibitory action induced by the hormones was evoked in a dose dependent manner (10 pM-100 nM). These effects are specific for progesterone and 17-beta-estradiol, since either testosterone and 17-alpha-estradiol were devoid of activity. The hormones induced rapid responses, producing significant inhibition within 1 to 5 minutes of hormonal exposure. The addition of 10(-5) M L-NAME suppressed the antiaggregatory effect of 1 nM E2 or 10 nM Pg. Furthermore, we specifically quantified the NO generation by the 3H-citrulline technique. 10(-8) M E2 induced 2-fold increase of RAS citrulline production, while the increment induced by 10(-7) M Pg was 55% over control. Preincubation with 10(-5) M L-NAME completely suppressed the stimulatory action of 10(-9) M E2 or 10(-8) M Pg, confirming that the antiaggregatory factor released from the aortic tissue was NO. Preincubation with cycloheximide did not block the increment in NO induced by the hormones. In conclusion the present study provides for the first time evidence of acute, non-genomic effects of Pg on rat aorta NOS activity and platelet aggregation in coincidence with the results obtained with estradiol treatment.

Animals↗

PubMed2001

Human and monkey fetal brain development of the supramammillary-hippocampal projections: a system involved in the regulation of theta activity.

The supramammillary (SUM)-hippocampal pathway plays a central role in the regulation of theta rhythm frequency. We followed its prenatal development in eight Cynomolgus monkeys (Macaca fascicularis) from embryonic day E88 to postnatal day 12 (term 165 days) and in eight human fetuses from 17.5 to 40 gestational weeks, relying on neurochemical criteria established in the adult (Nitsch and Leranth [1993] Neuroscience 55:797-812). We found that 1) SUM afferents reached the dentate juxtagranular and CA2 pyramidal cell layers at midgestation in human fetuses, earlier than in monkeys (two-thirds of gestation [E109]). They co-expressed calretinin, substance P, and acetylcholinesterase but not gamma-aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD); 2) the presumed parent neurons in the monkey SUM expressed calretinin or both calretinin and substance P; 3) most of them were surrounded by GAD-containing terminals that might correspond to the septo-SUM feedback pathway (Leranth et al. [1999] Neuroscience 88:701); and 4) in addition, a large band of calretinin-labeled terminals that did not co-express substance P, GAD, or acetylcholinesterase was present in the deepest one-third of the dentate molecular layer in both the Cynomolgus monkey and human fetuses. It persisted in the adult monkey but not in adult human hippocampus; it remains questionable whether it originates in the SUM. In conclusion, the early ingrowth of the excitatory SUM-hippocampal system in human and non-human primates may contribute to the prenatal activity-dependent development of the hippocampal formation. The possibility and the functional importance of an in utero generation of hippocampal theta-like activity should also be considered.

Afferent Pathways↗

PubMed2001

Influence of type II 5' deiodinase on TSH content in diabetic rats.

The influence of hypothalamic and pituitary type II 5'deiodinase (5'D-II) activities and T3 content on pituitary TSH content was investigated in streptozotocin (STZ)-induced diabetic rats (D). The results show, first, that hypothalamic and pituitary 5'D-II activities were lower in neonatal D rats versus control (C) rats, and the normal developmental pattern was altered. Secondly, when D and C rats were thyroidectomized (Tx) at 25 days of age (D+Tx, C+Tx), pituitary and hypothalamic 5'D-II activities increased ten days later in both populations vs. intact rats, but the percentage of increase was smaller in D+Tx than in C+Tx. The hypothalamic T3 to T4 ratios were also decreased in D+Tx animals (0.38) as compared to C+Tx rats (1.64). The hypothalamic T3 content was reduced by 30% in D as compared to C rats and by 80% in D+Tx as compared to C+Tx rats, showing a defect in hypothalamic T4 deiodination. Pituitary TSH content increased after Tx in D+Tx, but not in C+Tx. These results in diabetic rats indicate that the hypothalamic and pituitary 5'D-II activity and hypothalamic T3 content are affected by diabetes and play a role in the regulation of pituitary TSH content.

Animals↗

PubMed2001

Effect of pH on the conformation, interaction with membranes and hemolytic activity of sticholysin II, a pore forming cytolysin from the sea anemone Stichodactyla helianthus.

Sticholysin II (St II) is a pore forming cytolysin obtained from the sea anemone Stichodactyla helianthus. Incubation of diluted St II solutions at different pHs (ranging from 2.0 to 12) slightly changes the secondary structure of the protein. These changes are particularly manifested at high pH. Similarly, the intrinsic fluorescence of the protein indicates a progressive opening of the protein structure when the pH increases from acidic (2.0) to basic (12). These modifications are only partially reversible and do not produce any significant increase in the small capacity of the protein to bind hydrophobic dyes (ANS or Prodan). Experiments carried out with model membranes show a reduced capacity of binding to egg phosphatidyl choline:sphingomyelin (1:1) liposomes both at low (2.3) and high (11.5) pH. Preincubation of the protein in the 2. 5-9.0 pH range does not modify its hemolytic activity, measured in human red blood cells at pH 7.4. On the other hand, preincubation at pH 11.5 drastically reduces the hemolytic activity of the toxin. This strong reduction takes place without measurable modification of the toxin ability to be adsorbed to the red blood cell surface. This indicates that preincubation at high pH irreversibly reduces the capacity of the toxin to form pores without a significant decrease in its binding capacity. The present results suggest that at pH > or = 10 St II experiences irreversible conformational changes that notably reduce its biological activity. This reduced biological activity is associated with a partial defolding of the protein, which seems to contradict what is expected in terms of a molten globule formalism.

Animals↗

PubMed2001

Primary structure of two cytolysin isoforms from Stichodactyla helianthus differing in their hemolytic activity.

Sticholysin I (St-I) and sticholysin II (St-II) are cytolysins purified from the sea anemone Stichodactyla helianthus with a high degree of sequence identity (93%) but clearly differenced in their hemolytic activity. In order to go further into the structural determinants for the different behavior of St-I and St-II, we report here the complete amino acid sequences and the consensus secondary structure prediction of both proteins. The complete determination of St-II primary structure confirms the partial revision of cytolysin III amino acid sequence. All nonconservative changes between St-I and St-II are located at the N-terminal. According to our prediction these changes could be located at the same face of an alpha-helix during pore formation events and could account for the observed differences in hemolytic activity between St-I and St-II.

Amino Acid Sequence↗

PubMed2001

Properties of St I and St II, two isotoxins isolated from Stichodactyla helianthus: a comparison.

Sticholysins I and II are two highly hemolytic polypeptides purified from the Caribbean Sea anemone Stichodactyla helianthus. Their high sequence homology (93%) indicates that they correspond to isoforms of the same hemolysin. The spectroscopic measurements show a close similarity in the secondary structure content, conformation and stability of both toxins. Exposure of the toxins to high pHs (>11), a free radical source (AAPH), urea or temperature produce permanent changes in the toxin that lead to a significant loss of HA. It is significant to note that this loss of hemolytic activity occurs when other indicators, probably with the only exception of near-UV CD spectra, barely detect changes in the protein structure. This emphasizes the sensitivity of the protein function to changes in the macromolecule conformation. The most noticeable difference between both toxins is the considerably higher activity of St II, both measured in terms of erythrocyte internal K(+) exit or hemolysis; which is related to enthalpic factors. This difference is not due to an incomplete association of St I to the membrane. We consider then that the different pore forming capacity of both toxins in erythrocytes can be explained in terms of the difference in charge of the N-terminal fragment, than can considerably reduce the St I insertion rate in the membrane probably due to the negatively charged outer leaflet of the red blood cell, without a significant reduction of its capacity to bind to the cell membrane. This electrostatic effect, together with a slightly more relaxed structure in St II, could explain the higher pore forming capacity of St II in the red blood cell membrane.

Amidines↗

PubMed2001

Different role of insulin in GLUT-1 and -4 regulation in heart and skeletal muscle during perinatal hypothyroidism.

Two groups of hypothyroid rats were used; one group was given 2-mercapto-1-methylimidazole (MMI) treatment in the drinking water of the mothers and was killed at 2 and 4 days of life, and the other group was given similar MMI treatment and then was thyroidectomized at 5 days of life and killed at 8 or 20 days. Serum insulin, growth hormone (GH), and insulin-like growth factor I (IGF-I) were decreased in MMI-treated rats but increased in MMI-treated plus thyroidectomized rats. No significant reduction of thyroid hormones was observed in 2-day-old MMI rats. Protein and mRNA expression of GLUT-1 increased, and those of GLUT-4 decreased, in the heart in all populations independent of changes in insulin, GH, and IGF-I levels. However, GLUT-4 protein and mRNA expression in quadriceps and gastrocnemius skeletal muscles decreased at 4 days and increased at 8 and 20 days of life in parallel with insulin, GH, and IGF-I levels. GLUT-1 in the skeletal muscles seemed regulated posttranscriptionally and presented a decrease of mRNA expression in all stages studied. A differential sensitivity to insulin regulation of GLUT-1 and GLUT-4 glucose transporters seems to be one of the causes for the tissue-specific regulation of these glucose transporters in heart and skeletal muscles during the perinatal period.

Animals↗

PubMed2001

Glucose uptake and glucose transporter proteins in skeletal muscle from undernourished rats.

Undernutrition in rats impairs secretion of insulin but maintains glucose normotolerance, because muscle tissue presents an increased insulin-induced glucose uptake. We studied glucose transporters in gastrocnemius muscles from food-restricted and control anesthetized rats under basal and euglycemic hyperinsulinemic conditions. Muscle membranes were prepared by subcellular fractionation in sucrose gradients. Insulin-induced glucose uptake, estimated by a 2-deoxyglucose technique, was increased 4- and 12-fold in control and food-restricted rats, respectively. Muscle insulin receptor was increased, but phosphotyrosine-associated phosphatidylinositol 3-kinase activity stimulated by insulin was lower in undernourished rats, whereas insulin receptor substrate-1 content remained unaltered. The main glucose transporter in the muscle, GLUT-4, was severely reduced albeit more efficiently translocated in response to insulin in food-deprived rats. GLUT-1, GLUT-3, and GLUT-5, minor isoforms in skeletal muscle, were found increased in food-deprived rats. The rise in these minor glucose carriers, as well as the improvement in GLUT-4 recruitment, is probably insufficient to account for the insulin-induced increase in the uptake of glucose in undernourished rats, thereby suggesting possible changes in other steps required for glucose metabolism.

Animals↗

PubMed2001

Effects of long-term treatment with antipsychotics on serum leptin levels.

BACKGROUND: Abnormal regulation of the adipocyte-derived hormone leptin could play a role in body weight gain induced by antipsychotics. AIMS: To study the effects of long-term antipsychotic treatment on leptin levels in patients with schizophrenia. METHOD: Serum leptin levels were determined in 59 out-patients with chronic schizophrenia and in the same number of healthy subjects controlled by gender, age and body mass index. RESULTS: Leptin levels did not differ between patients and controls. Leptin levels in patients with schizophrenia correlated with weight gain, even after controlling for current weight, but did not show any association with clinical variables. Antipsychotic class tended to exert different effects over leptin levels (among atypicals, olanzapine induced a greater increase). CONCLUSIONS: Elevation of leptin levels induced by chronic antipsychotic treatment can be attributed to weight gain, but other mechanisms could be involved.

Adult↗

PubMed2001

Regulation of IGF-I and -II by insulin in primary cultures of fetal rat hepatocytes.

During perinatal development, insulin and nutrients, rather than GH, regulate the IGF system. A selective primary culture of fetal rat hepatocytes has been established in our laboratory to elucidate the molecular mechanism of action of the above regulatory factors on IGF-I and -II gene expression during the late fetal period of the rat. In this model we have previously reported a regulatory role for glucose on IGF-I and -II synthesis and secretion. In the same experimental model, we now report that doses of insulin (0.1-5 microM) within the physiological range in rat fetuses during the last stages of gestation evoke an increase of IGF-I and -II mRNA abundance. Insulin regulated in a parallel manner IGF peptide secretion, and an excellent correlation was observed between IGF-I and -II mRNA and IGF-I and -II peptide levels in the conditioned media in response to the hormone. Finally, the insulin-induced rise in IGF-I and -II mRNA was not mediated by stimulation of gene transcription but by increased transcript stability. The results support the hypothesis that insulin plays a major role in IGF regulation at immature stages of development.

Animals↗

PubMed2001

Effect of thyroxine administration on the IGF/IGF binding protein system in neonatal and adult thyroidectomized rats.

The effects of different doses of thyroxine (T(4)) delivered by injection or s.c. pellet implantation on alterations of the IGF/IGF binding protein (IGFBP) system were studied in neonatal and adult thyroidectomized (Tx) rats. Body weight, blood glucose, plasma insulin, TSH and GH and pituitary GH content, as well as serum IGF-I, IGF-II, IGFBP-1, -2 and -3 and their liver mRNA expression were assayed. Pellet implantation with the smaller dose of T(4) (1.5 microg/100 g body weight (b.w.) per day) in Tx neonatal rats decreased serum IGF-I, -II and the 30 kDa complex of IGFBPs (IGFBP-1 and -2), and increased serum IGFBP-3. Only the larger dose of T(4) (3 microg/100 g b.w. per day) recovered liver mRNA expression of IGF-I and ensured euthyroid status as shown by the normalized levels of plasma TSH. The rapid increase of body weight and serum GH after T(4) administration indicated a high sensitivity to T(4) during the neonatal period. Serum and liver mRNA expression of IGFs and plasma insulin and GH recovered in adult Tx rats after pellet implantation of 1.75 microg/100 g b.w. per day throughout 10 days. The continuous replacement of T(4) by pellet seems to be the most suitable method for thyroid rehabilitation. A very good correlation was found between insulin and IGF-II in Tx neonates treated with T(4) but not between insulin and IGF-I in Tx adults. IGFBP-2 seems to be up-regulated by T(4) deprivation in neonatal and adult rats. Finally, a good correlation as well as a partial correlation were found between IGFs and thyroid hormones in both neonatal and adult Tx populations, suggesting a direct effect in vivo of T(4) on the hepatic secretion of IGFs, as previously suggested in vitro.

Analysis of Variance↗

PubMed2001

[High prevalence of subclinical thyroidal disease among individuals attended in health control].

BACKGROUND: There is no information about the prevalence of thyroidal diseases in the general Chilean population. AIM: To assess the prevalence of thyroidal diseases in individuals attended in occupational health examinations. SUBJECTS AND METHODS: Four hundred seventy two individuals were examined between 1998 and 1999. In all, serum levels of thyroid hormones, TSH and anti thyroidal antibodies (anti microsomal, anti thyroid peroxidase and anti thyroglobulin) were measured. RESULTS: Forty four subjects were excluded from the study due to an incomplete medical record and 18 due to a personal history of thyroidal disease. Abnormal serum levels of thyroid hormones or TSH were detected in 28 subjects (6.8%). Four (1%) had hypothyroidism, 23 a subclinical hypothyroidism (5.6%) and one (0.2%) had hyperthyroidism. In 87 subjects (21.2%) at least one of the antibodies was positive. Positive anti thyroid antibodies were found in 14 of 28 subjects (50%) with abnormal thyroid hormone levels, compared with 73 of 382 subjects (19.1%) with normal thyroid hormone levels. Thyroid dysfunction was twice as frequent in women than in men. CONCLUSIONS: In this sample, a 6.8% prevalence of abnormal thyroid function tests was detected.

Adolescent↗

PubMed2001

[Computer analysis of respiratory sounds in bronchial obstruction evaluation in small children that do not collaborate with spirometry tests].

BACKGROUND: There are changes in inspiratory breath sound intensity in patients with airway obstruction. Airway narrowing may change sound spectral characteristics. AIM: To define the characteristics of lung sounds at standardized air flow during methacholine challenge and to compare acoustic changes with transcutaneous oxygen tension (PtcO2) during induced airway narrowing. PATIENTS AND METHODS: Forty asthmatic children (20 male) aged 5.2 +/- 1 years and 40 normal children (18 male), aged 5.6 +/- 1 years were studied. All patients were free of respiratory tract infections one month before the study. A methacholine challenge from 0.06 to 8 mg/ml was performed; the test was ended when a fall in PtcO2 of > 20% from baseline was observed or if the final concentration was reached. Subjects breathed through a pneumotachograph aiming at flows of 0.4 to 0.6 l/s. Respiratory sounds were recorded using contact sensors at the suprasternal notch and at the posterior right lower lobe. From average spectra, power at low (100-200 Hz = P1) and high frequencies (400-2000 = P2) was calculated. Frequencies below which 50% (F50) and 99% (SEF90) of the spectral power between 100 and 2000 Hz was contained, were also calculated. RESULTS: In asthmatics, the metacholine concentration at which a 20% fall in PtcO2 was observed, was lower than in normal children (p < 0.05). There was an increase in P1 (p < 0.01) and a reduction in P2 (p < 0.01) during inspiration, in subjects that experienced a 20% reduction in PtcO2. Also, there was an increase in F50 and SEF99 during inspiration in lung sounds, but not over the trachea. CONCLUSIONS: Lung sounds analysis can be useful for the assessment of airway reactivity in asthmatic children.

Asthma↗

PubMed2001

[Pleural effusion and complicated empyema in children. Evolution and prognostic factors].

BACKGROUND: Complicated pleural empyema has a torpid and longer clinical evolution, requiring in some patients surgical management. The predictive factors for surgical treatment are not well known. AIM: To search for clinical, laboratory or radiological predictors for the requirement of surgical treatment in pediatric patients with empyema. PATIENTS AND METHODS: A retrospective review of the charts of 108 patients hospitalized for pneumonia plus pleural effusion at the Pediatric Service of the Catholic University Hospital between January 1985 and July 2000. RESULTS: Eighty one patients had complete radiological evaluation and pleural fluid biochemical analysis. Forty nine (60%) fulfilled the criteria for empyema and 32 (40%) for an exudate. Thirteen patients with empyema required surgery and 36 were treated medically. The mean age was 3 years (range 9 months-6 years) for the surgically treated and 4 years (range 12 months-14 years) for the non-surgical group. The male/female ratio was 5:1 in the surgical group and 1:1 for the non-surgical group. Pleural fluid cultures were positive in 21 of 79 patients. Streptococcus pneumonia was the most frequently isolated agent. No significant differences were found between groups for the average days of fever prior to the diagnosis or total days of fever, days of hospital stay, pleural fluid pH (6.8 and 7.0 respectively) and glucose (21 and 31 mg/dl respectively). No differences were either observed for pleural fluid risk factors (pH < 7 and glucose < 20 mgdl), the presence of extensive pleural effusions, pleural loculations or bands on pleural ultrasonography and positive Gram stain or cultures in the pleural fluid. Surgical patients required oxygen for more days than medical patients (7.7 and 5.1 days; p = 0.037). CONCLUSIONS: This study failed to find predictive clinical, radiological or pleural fluid parameters, for the requirement of surgical treatment of empyema.

Adolescent↗

PubMed2000

The p115-interactive proteins GM130 and giantin participate in endoplasmic reticulum-Golgi traffic.

The transport factor p115 is essential for endoplasmic reticulum (ER) to Golgi traffic. P115 interacts with two Golgi proteins, GM130 and giantin, suggesting that they might also participate in ER-Golgi traffic. Here, we show that peptides containing the GM130 or the giantin p115 binding domain and anti-GM130 and anti-giantin antibodies inhibit transport of vesicular stomatitis virus (VSV)-G protein to a mannosidase II-containing Golgi compartment. To determine whether p115, GM130, and giantin act together or sequentially during transport, we compared kinetics of traffic inhibition. Anti-p115, anti-GM130, and anti-giantin antibodies inhibited transport at temporally distinct steps, with the p115-requiring step before the GM130-requiring stage, and both preceding the giantin-requiring stage. Examination of the distribution of the arrested VSV-G protein showed that anti-p115 antibodies inhibited transport at the level of vesicular-tubular clusters, whereas anti-GM130 and anti-giantin antibodies inhibited after the VSV-G protein moved to the Golgi complex. Our results provide the first evidence that GM130 and giantin are required for the delivery of a cargo protein to the mannosidase II-containing Golgi compartment. These data are most consistent with a model where transport from the ER to the cis/medial-Golgi compartments requires the action of p115, GM130, and giantin in a sequential rather than coordinate mechanism.

Antibodies↗

PubMed2000

Higher levels of serum copper in schizophrenic patients treated with depot neuroleptics.

The findings of previous research on the status of trace elements in patients with schizophrenia have been controversial. We studied 62 outpatients with a DSM-IV diagnosis of schizophrenia, and compared them with sex- and age-matched healthy controls. Serum copper levels were significantly higher in schizophrenic patients (mean 117.4 microg/dl; S.D. 23.4) than in healthy controls (105.6+/-27.9). Those patients on treatment with depot neuroleptics had higher copper levels. Zinc levels did not differ between patients and healthy controls. Altered levels of trace elements in schizophrenic patients may be a consequence of antipsychotic treatment.

Adult↗

PubMed2000

Prevalence of genotypic resistance to nucleoside analogues and protease inhibitors in Spain. The ERASE-2 Study Group.

OBJECTIVE: To examine the prevalence of resistance mutations to nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) in a representative HIV-1 population in Spain. METHODS: A cross-sectional study was conducted including 601 HIV-infected patients who attended 20 Spanish hospitals in June 1998. Drug resistant mutations were examined using hybridization line probe assays (LiPA). The 6 bp insert at position 69 and the codon 75 mutant were examined by sequencing analysis in specimens lacking reactivity to 69/70 and 74 bands on LiPA, respectively. RESULTS: Primary resistance to NRTI was recognized in nine out of 52 (17%) naive individuals, whereas primary resistance to PI was found in seven out of 126 (6%) PI-naïve patients. The codons most frequently involved in NRTI resistance were at positions 70 (66%), 184 (44%), 215 (33%), and 41 (11%), whereas the most common PI resistance mutation was at codon 82 (6/7 subjects). In pre-treated patients, the overall prevalence of resistant genotypes was 72.9% for NRTI and 27.2% for PI. The most frequent NRTI mutations occurred at codons 184 (38.5%), 215 (30.1%), and 41 (22.5%), whereas the most frequent PI mutations in pre-treated subjects were found at positions 82 (15.8%) and 84 (11.4%). Overall, patients who began triple combinations as initial therapy showed a lower number of key resistance mutations than those who began highly active antiretroviral therapy (HAART) after being exposed to NRTI for a period of time (mean number of mutations, 0.1 versus 1.8, P< 0.05). Codon 75 mutant was found in three out of 387 patients (0.7%), whereas no insertions at codon 69 were recognized. CONCLUSION: The prevalence of primary genotypic resistance to NRTI and PI in Spain was 17% and 6%, respectively. Zidovudine, lamivudine, indinavir and ritonavir were the drugs most frequently affected. These data support the use of resistance testing prior to the introduction of first-line antiretroviral therapies in Spain. Among pre-treated subjects, drug resistance genotypes were less prevalent in those who began HAART as initial therapy.

Adult↗

PubMed2000

Complicated enterocutaneous fistulas: failure of octreotide to improve healing.

Octreotide has been promoted as a potential aid during closure of enterocutaneous fistulas (ECFs) on the basis of clinical and experimental evidence that it can reduce gastrointestinal secretions. We retrospectively reviewed the records of patients admitted with ECF to our tertiary referral center to ascertain whether use of octreotide affected fistula duration, length of hospitalization, closure rate, and morbidity. Of 60 patients diagnosed and treated for ECF over a 4-year period, 13 underwent a therapeutic trial with octreotide. Thirteen patients from the group who did not receive octreotide were matched by cause, location, and output of the fistula, age, and primary diagnosis to the treatment group. Octreotide was administered in therapeutic dosage for a mean course of 57 +/- 29 days, resulting in a substantial acute decrease (84.7 +/- 4.8%) in fistula output. Prolonged therapy nevertheless failed to affect the outcome parameters studied, particularly fistula duration, spontaneous closure rate, and length of hospitalization. There was a significantly higher incidence of septic and thrombotic complications associated with octreotide use. In this patient population with complicated ECFs, use of octreotide showed no benefit and was associated with increased morbidity.

Cutaneous Fistula↗