The involvement of clathrin-mediated endocytosis and two Sid-1-like transmembrane proteins in double-stranded RNA uptake in the Colorado potato beetle midgut.

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double-stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient-1 (Sid-1) transmembrane channel-mediated uptake and clathrin-mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi-of-RNAi experiments, we demonstrated the contribution of two different sid-1-like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H(+) ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid-1-like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well-devised reporter system in any future experiments on cellular dsRNA uptake.

Mast cell degranulation contributes to susceptibility to Leishmania major.

Leishmaniasis causes high morbidity and mortality in tropical and subtropical areas. Mast cells can be activated by Leishmania or Leishmania products in vitro and in vivo. Several innate immunity mediators, including some released by mast cells, play roles in the outcome of the disease. In this study, we examined whether pharmacological inactivation of mast cells before infection with L. major interferes with the progressive disease in BALB/c mice. The results show that, when mast cells are degranulated before challenge with L. major, susceptible mice become more resistant to infection, as measured by decrease of lesion size and lower parasite loads. Mast cell degranulation reduced IL-4 production. Moreover, mast cells degranulation enhanced mRNA expression for IFN-gamma, inducible nitric oxide, CCL2 and CCL5 in response to infection. Mast cell degranulation also decreased parasite loads in IL-4 KO animals, indicating that mediators other than IL-4 are involved in susceptibility in vivo. Taken together, our results disclose a role for mast cells in the induction of susceptibility to infection. This work contributes to a better understanding of the role of mast cells in Leishmania infection, and suggests a new field of study for strategies to contain the parasite, restricting its dissemination.

Chemotherapy and the future: microdialysis as a local administration technique.

Chemotherapy has an important role in cancer treatment. Although there have been developments and good results, chemotherapy still has many limitations mainly due to its toxicity and to the resistance mechanisms of tumour cells. Therefore, besides other important improvements in chemotherapy agents and indications, recent researches have focused on the development of locoregional administration techniques, with which therapeutic weapons can reach the tumour with a higher concentration and fewer side-effects. At present, local chemotherapy includes delivery systems or prodrug strategies, arterial infusions, intraperitoneal administration and aerosolised agents. We will describe a new local cancer chemotherapy method, using microdyalisis procedures, which may revolutionise the actual tumour management because of the higher effectiveness and the absence of side-effects. Finally, the applications and limitations of this technique will be considered.

Breast cancer patients with micrometastases in sentinel lymph nodes: differences considering additional metastatic lymph nodes.

AIMS: Characterization of breast cancer patients with micrometastases in sentinel lymph node (SLN) and establish differences between micrometastatic breast cancers with additional metastatic lymph nodes (LNS) versus no other lymph node invasion. METHODS: Analysis of 30 breast cancers, N1mi or pN0(i+), diagnosed and treated in our department from July 2000 to July 2008. RESULTS: Micrometastases in SLNs were found in 30 patients. Complete axillary dissection revealed other metastatic LNs in 24%. Concerning breast cancers with additional LN invasion versus no other LN invasion, tumors located in the superior-external quadrant were more frequent in the former group. Other characteristics as clinical presentation, histological subtype, focality, cytonuclear grade, hormone receptors and Her2 expression were not significantly different in either group. Regarding SLN invasion, the presence of at least two micrometastatic foci were significantly more relevant in patients with other metastatic LN invasion (p < 0.01). Micrometastases diagnosed only after immunohistochemistry (IHC) were exclusively found in patients without other LN invasion, reaching statistical significance (p < 0.05). CONCLUSIONS: Complete axillary dissection revealed additional LN invasion in 24% of patients with micrometastases in the SLN. Tumors with additional LN invasion were more frequently found in the superior external quadrant and SLNs harbored at least two micrometastatic foci. Micrometastases diagnosed exclusively by IHC techniques were more relevant in cases without additional lymph node invasion.

What is the diagnostic value of nipple discharge cytology and galactography in detecting duct pathology?

The present study aimed to evaluate the diagnostic value of nipple discharge (ND) cytology and galactography. Ninety-four patients submitted to duct excision, representing a total of 98 duct excisions, were retrospectively analyzed from January 1997 to May 2007. Histology of ducts excised revealed 35% duct ectasia (DE), 31% duct papilloma (DP), 20% potential malignant transforming lesions (PMTL), 6% breast cancer (BC), 1% adenoma and 6% normal breast tissue. Cytology had a sensibility and specificity in detecting duct pathology of, respectively, 40% and 61.3%, a positive predictive value (PPV) of 53.8% and a negative predictive value (NPV) of 47.5%. Concerning malignant and PMTL, cytology had a sensibility and specificity of, respectively, 46.2% and 62.3%, a PPV of 25% and a NPV of 82.5%. Breast cancer was never suggested by positive cytology. Galactography had a sensibility and specificity in detecting duct pathology of, respectively, 77.4% and 29.2%, a PPV of 58.5% and a NPV of 50%. For malignant and PMTL, galactography had a sensibility and specificity of, respectively, 80% and 26.7%, a PPV of 19.5% and a NPV of 85.7%. However, galactography never suggested a diagnosis of cancer. Cytology and galactography performed together in 49% showed a low Kappa Index (KI < 1), allowing us to conclude that cytology and galactography detect different pathologies. Cytology showed a poor performance in predicting histological diagnosis in face of ND. Galactography had a good sensibility in excluding malignant lesions and PMTL. Galactography was significantly more sensitive for duct pathology but cytology was more specific for malignant lesions and PMTL.

Soluble VCAM-1 and E-selectin in breast cancer: relationship with staging and with the detection of circulating cancer cells.

In breast cancer, the correct evaluation of cancer dissemination is essential to establish prognosis and treatment choices. This study analyses the relationship between circulating levels of soluble VCAM-1 and E-selectin and the presence of circulating cancer cells in breast cancer patients. Plasma levels of VCAM-1 and E-selectin were measured by enzyme-linked immunosorbent assay (ELISA). The presence of circulating cancer cells was diagnosed using a RT nested-PCR assay detecting the cancer specific transcript, epidermal growth factor receptor variant III (EGFRvIII) mRNA. Blood samples were collected from 64 patients divided in three groups: group A of 11 women selected for neoadjuvant chemotherapy; group B of 13 women with metastatic disease and group C, with 40 women having completed their treatment at least one year ago and with no evidence of relapse. The mutant transcript was detected in 45.5% of patients from group A, in 61.5% of patients from group B and in none of the group C patients. For both VCAM-1 and E-selectin, plasma levels increased with disease staging and with the presence of EGFRvIII mRNAin peripheral blood. The differences were statistically significant (p < 0.025) when group C was compared with all patients from group B, with patients from group B with EGFRvIII positive results or with all patients with EGFRvIII positive results. Increased plasma levels of VCAM-1 and E-selectin are associated with advanced stage of breast cancer and with the presence of circulating cancer cells. The combined analysis of these parameters may contribute to a more accurate evaluation of cancer dissemination.

Endometrial adenocarcinoma after endometrial ablation. A case report.

The authors present a case of endometrial adenocarcinoma after endometrial ablation, emphasizing the importance of close surveillance of these patients, patient selection and education. Even patients with none of the risk factors for endometrial cancer or contraindications to endometrial ablation should be checked carefully.

Primary breast lymphoma.

OBJECTIVES: Retrospective evaluation of the clinical behavior, treatment and prognosis in five cases of primary breast lymphoma. METHODS: From 1999 to 2003, five patients with primary breast lymphoma were diagnosed in our department. RESULTS: Primary breast lymphoma (PBL) was diagnosed in five patients, whose median age was 63.4 (41-79) years. In four out of five patients, a diagnosis of lymphoma was made after the evaluation of a palpable breast mass measuring 1.5 to 6 cm. All of them were classified as non-Hodgkin's B cell lymphomas and three of five cases were diffuse large cell lymphomas. All patients were submitted to chemotherapy; in only one patient was surgery performed. CONCLUSIONS: A relatively high rate of PBL was observed in our department compared with other oncology centers. Beyond its scarce appearance, PBL is very difficult to distinguish from primary breast carcinoma. Histology remains the major diagnostic tool.

Transdentinal cell photobiomodulation using different wavelengths.

OBJECTIVE: The aim of this study was to investigate the effects of transdentinal irradiation with different light-emitting diode (LED) parameters on odontoblast-like cells (MDPC-23). METHODS AND MATERIALS: Human dentin discs (0.2 mm thick) were obtained, and cells were seeded on their pulp surfaces with complete culture medium (Dulbecco modified Eagle medium). Discs were irradiated from the occlusal surfaces with LED at different wavelengths (450, 630, and 840 nm) and energy densities (0, 4, and 25 J/cm(2)). Cell viability (methyltetrazolium assay), alkaline phosphatase activity (ALP), total protein synthesis (TP), and cell morphology (scanning electron microscopy) were evaluated. Gene expression of collagen type I (Col-I) was analyzed by quantitative polymerase chain reaction (PCR). Data were analyzed by the Mann-Whitney test with a 5% significance level. RESULTS: Higher cell viability (21.8%) occurred when the cells were irradiated with 630 nm LED at 25 J/cm(2). Concerning TP, no statistically significant difference was observed between irradiated and control groups. A significant increase in ALP activity was observed for all tested LED parameters, except for 450 nm at 4 J/cm(2). Quantitative PCR showed a higher expression of Col-I by the cells subjected to infrared LED irradiation at 4 J/cm(2). More attached cells were observed on dentin discs subjected to irradiation at 25 J/cm(2) than at 4 J/cm(2). CONCLUSION: The infrared LED irradiation at an energy density of 4 J/cm(2) and red LED at an energy density of 25 J/cm(2) were the most effective parameters for transdentinal photobiomodulation of cultured odontoblast-like cells.

Modulation of coronary flow and cardiomyocyte size by sensory fibers.

Cardiac tissue is densely innervated by sensory neurons that are believed to play important modulatory roles in cardiac functions. In this study, pretreatment of neonate rats with capsaicin was performed. In adult rats, cardiomyocyte size and amount of fibrous tissue in left ventricles as well as in vitro coronary flow were evaluated. The chronotropic and inotropic responses to beta-adrenoceptor agonists (norepinephrine and isoproterenol), muscarinic agonists (carbachol and pilocarpine), and calcitonin gene-related peptide (CGRP) were also investigated with the use of the isolated right atria preparation. Capsaicin pretreatment significantly (P<0.05) reduced both basal coronary flow (18% reduction) and cardiomyocyte size (34% reduction) without affecting the amount of fibrous tissues in the left ventricles. The positive inotropic and chronotropic effects in response to norepinephrine in the isolated rat heart did not significantly differ between control and capsaicin-treated rats. Similarly, the positive chronotropic effects in response to norepinephrine, isoproterenol, and CGRP as well as the negative chronotropic responses to carbachol and pilocarpine in the isolated right atria were not affected by capsaicin pretreatment. Our data are consistent with the suggestion that reductions of both basal coronary flow and cardiomyocyte size seen in hearts from capsaicin-pretreated rats may be consequences of CGRP depletion. The cardiomyocyte size reduction produced by capsaicin treatment may be related to a modulatory role of CGRP as a growth factor.

Phase II study of carboplatin in patients with advanced or recurrent endometrial carcinoma. A trial of the EORTC Gynaecological Cancer Group.

The aim of this study was to investigate the efficacy and toxicity of carboplatin given as monotherapy in endometrial adenocarcinoma. Cisplatin is one of the most active drugs in gynaecological cancer types, but at the cost of an associated high toxicity. In this high-risk population of endometrial cancer patients, it is necessary to have chemotherapy regimens with a low toxicity. Patients eligible for this study were those with histologically-confirmed endometrial adenocarcinoma with evidence of recurrent and/or metastatic disease. Carboplatin was administered every 4 weeks as a first- (dose: 400 mg/m(2)) or second- (dose: 300 mg/m(2)) line chemotherapy. Of the 64 patients who entered the trial, 60 were eligible, 53 patients were evaluable for toxicity and 47 for efficacy. A total of 169 cycles of carboplatin was given with a median of 2 cycles per patient (range 1-11 cycles) to a median cumulative dose of 798 mg/m(2) (range 290-3879 mg/m(2)). No grade 4 toxicity or toxic deaths occurred. White Blood Cell (WBC) toxicity grade 3 was noted five times, mainly in the radiotherapy pre-treated patients. Grade 3 non-haematological toxicity consisted mainly of nausea and vomiting (21%). There was a total of eight responses (3 Complete Responses (CR) and 5 Partial Responses (PR) with an overall response rate (ORR) of 13% (95% Confidence Interval (CI) 6-25). No responses occurred in patients treated with prior chemotherapy. In evaluable patients, the ORR in all patients (n=47) and in those receiving first-line chemotherapy (n=33) were, 17% (95% CI 8-31) and 24% (95% CI 11-42), respectively. After a median follow-up of 379 days, the median duration of response was 488 days (range 141-5303 days) with two very long responses in patients with a CR. Carboplatin has a low toxicity and is active in chemotherapy-naive advanced endometrial carcinoma patients. These results lead us to propose its use in association in first-line chemotherapy in recurrent or advanced endometrial carcinoma patients. The choice of the initial dose can be determined according to whether the patients have received prior radiotherapy treatment.

HIV type 1 tat gene heteroduplex mobility assay as a tool to establish epidemiologic relationships among HIV type 1-infected individuals.

Molecular biology techniques are increasingly used to study the molecular epidemiology of infectious diseases. Most of these methods are expensive and labor-intensive. The human immunodeficiency virus (HIV) has substantial genomic variation, such that HIVs from different individuals are genetically diverse, although mutation rates differ for distinct regions of the genome. Most studies of HIV linkage and molecular evolution have focused on env or gag regions. We show that heteroduplex mobility analysis of the first exon of the HIV tat gene provides a simple, rapid, inexpensive, and reliable discriminatory tool for the molecular differentiation of shared versus distinct HIV-1 quasispecies when epidemiologic relations need to be defined. tat, as a relatively conserved region, appears to be a better region than the more variable env region to establish HIV-1 epidemiological linkages.

Lymphocytes in patients with ovarian cancer.

Studies to determine the total number of circulating lymphocytes and their morphologic transformation in the presence of phytohemagglutinin and pokeweed mitogen were carried out in 156 women: 81 who currently had an ovarian cancer or who had had it in the past; 54 normal women; and 21 patients with malignant diseases. The latter two groups served as controls. The total number of circulating lymphocytes is an important cosideration, as there is a significant decrease of lymphocytes in patients with ovarian cancer, a factor with great bearing on prognosis. The lymphoblastic transformation test in the presence of phytohemagglutinin and pokeweed mitogen showed not only significant changes in patients with ovarian cancer but also a correlation with survival at 6 months.

Development of cardiomyocyte hypotrophy in rats under prolonged treatment with a low dose of a nitric oxide synthesis inhibitor.

Chronic administration of the nitric oxide (NO) synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats causes hypertension and morphological abnormalities in the heart, consisting mainly of ventricular hypertrophy and foci of necrosis and fibrosis. Since these phenomena have usually been described with high (or moderate) doses of L-NAME, this study was undertaken to evaluate the effects of a low dose of L-NAME on arterial blood pressure, heart weight index, left ventricular weight index, amount of ventricular fibrosis, and cardiomyocyte size. Male Wistar rats received L-NAME (7.5 mg/kg per day) in the drinking water for 2, 4, and 6 months, whereas control animals received tap water alone. At this dose, L-NAME caused 90% inhibition (P<0.001) of brain NO synthase (NOS) activity. The chronic L-NAME treatment caused an approximately 15% reduction in body weight of the animals, and no death was observed. The tail-cuff pressure was markedly (P<0.01) elevated in L-NAME-treated rats. A significant (P<0.05) reduction in both heart weight index (13-20% decrease) and left ventricular weight index (20-34% decrease) at 2, 4, and 6 months of treatment was observed in L-NAME-treated rats. The cardiomyocyte size in subendocardial, subepicardial, and midmyocardial regions of the left ventricles was time-dependently reduced, irrespective of the region studied, as measured at 2 (11% decrease), 4 (28% decrease, P<0.05), and 6 (45% decrease, P<0.05) months of chronic L-NAME treatment. The amount of fibrous tissue was unaltered at 2 and 4 months, but a small (but significant) increase in the amount of fibrous tissue was detected at 6 months (7.1+/-0.2 %, P<0.05) compared to that of control animals (5.9+/-0.2%). Our results show that chronic treatment of rats with a low dose of L-NAME for prolonged periods (up to 6 months) causes arterial hypertension accompanied by significant reductions in heart weight, left ventricular weight indexes, and cardiomyocyte size.

Effect of Ca2+ channel blockers on arterial hypertension and heart ischaemic lesions induced by chronic blockade of nitric oxide in the rat.

The effects of the Ca2+ channel blockers diltiazem, nifedipine and amlodipine were investigated on both arterial hypertension and myocardial changes induced by chronic blockade of nitric oxide synthesis. Control male Wistar rats received Nomega-nitro-L-arginine methyl ester (L-NAME; 20 mg rat(-1) day(-1)) in the drinking water for 8 weeks; blood pressure and body weight were monitored weekly. The Ca2+ channel blockers were given concomitantly to L-NAME, as follows: diltiazem (13.5 mg rat(-1) day(-1)) and amlodipine (6.25 mg rat(-1) day(-1)) were administered in the drinking water whereas nifedipine (6.25 mg rat(-1) day(-1)) was given in the chow. Nomega-nitro-L-arginine methyl ester induced a time-dependent increase in blood pressure which was significantly attenuated by diltiazem (154+/-1.6 vs. 139+/-1.6 mm Hg, p < 0.05), nifedipine (166+/-2.7 vs. 150+/-2.1 mm Hg, p < 0.05) and amlodipine (208+/-5.8 vs. 158+/-1.8 mm Hg, p < 0.05) at the last week of the treatment. Rats treated with the L-NAME also developed myocardial ischaemia, as indicated by the increased percentage of fibrous tissue found in the left ventricles of these animals (10.9+/-0.1%, p < 0.01) when compared to control ones (6.3+/-0.1%). Neither diltiazem (14.9+/-1.2%) nor nifedipine (11.1+/-1.5%) prevented this effect whereas amlodipine (6.9+/-1.1%, p < 0.01) virtually abolished the increase in fibrous tissue induced by L-NAME. The plasma concentration of the Ca2+ channel blockers was measured by liquid chromatography coupled to mass spectrometry at two different time points (morning and afternoon). Only amlodipine treatment was able to maintain constant levels (186+/-46 ng ml(-1) in the morning and 110+/-19 ng ml(-1) in the evening) compared to nifedipine (3003+/-578 ng ml(-1) in the morning and 436+/-100 ng ml(-1) in the evening) and diltiazem (77+/-51 ng ml(-1) in the morning and not detectable in the evening). In conclusion, our results indicate that amlodipine (but not diltiazem and nifedipine) can efficiently control myocardial ischaemia in nitric oxide deficient rats, probably due to its intrinsically long half-life.

Evaluation of the gynaecological oncology situation in the European Community countries.

At the end of 1991 an enquiry was sent to 40 doctors recognized as experts in Gynaecological Oncology in the E.C. countries to establish the situation of this sub or superspecialty and 32 (80%) answers were received. Inside the E.C. countries only the United Kingdom recognized the subspecialty of Gynaecological Oncology. Concerning the other 11 countries, except Denmark, their experts (invited to answer the questionnaire) consider it is necessary to individualize Gynaecological Oncology as a sub or a superspecialty. Before a training period of 24 or 36 months in Gynaecological Oncology, it is necessary to have the specialty of Obstetrics/Gynaecology. The training period must include 50% or 60% in diagnosis and surgical treatment, 10% in radiotherapy (only theoretical), 10% or 20% in medical oncology (theoretical and practical), 5% or 10% in oncological pathology, basic sciences and general aspects of cancer. Breast diseases, including breast cancer, must be a part of the training in Gynaecological Oncology and at the end of this period an evaluation test must be made. Lastly, the health authorities, of each country, are the best able to recognize the individualization of Gynaecological Oncology.

Diagnostic value of ultrasound and color Doppler in identifying axillary lymph node metastases in patients with breast cancer.

PURPOSE: The aim of this study was to evaluate the diagnostic ability of ultrasound and color Doppler in axillary lymph node metastases of patients with breast cancer. MATERIAL AND METHODS: A prospective study including 55 patients with primitive, invasive, node negative breast cancer who underwent preoperative axillary ultrasound and color Doppler. Doppler and morphologic ultrasound criteria were applied to the identification of axillary lymph node metastases. RESULTS: The imagery study of all 55 patients identified a total of 141 nodes; 44 were considered to be positive according to established criteria. The histological examination of the axillary dissection revealed a total of 989 nodes; 77 out of 989 presented metastases; all invaded nodes belonged to 21 patients. The previous imagiologic study was positive for axillary lymph node metastases in 15 out of these 21 patients. A sensitivity of 71.4%, a specificity of 71.4%, a negative predictive value of 80.6% and a positive predictive value of 60.0% were achieved. CONCLUSION: The imagery study of the axillary region through ultrasound and color Doppler might be useful in assessing axillary lymph node metastases in patients with breast cancer.

Endometrioid adenocarcinoma arising in endometriosis foci six years after estrogen replacement therapy: a case report.

We present a case of a 53-year-old woman who developed an endometrioid adenocarcinoma six years after total abdominal hysterectomy (TAH) and bilateral salpingo-oophorectomy (BSO), who was on estrogenic-only hormone replacement therapy (HRT).

Carcinoma in situ and early breast carcinoma survey of the Portuguese Senology Society on treatment in Portugal and its evolution between 1985 and 2000.

By means of a questionnaire sent to Portuguese hospitals which diagnose and treat most female patients with breast cancer, it was intended to assess the situation regarding the treatment of carcinoma in situ and early breast cancer (T1 or T2, N0 or N1), as well as their evolution between 1985 and 2000. The hospital participation rate was 65% and a sample of 865 patients was collected, distributed by the years 1985, 1990, 1995 and 2000. It was observed that, in terms of surgery, there was an increase in conservative surgery, which was over 40% in 2000, as well as an increase in the average of excised axillary lymph nodes. Progress in the surgical approach was similar both in cancer centres and in large and university hospitals, when compared with the other surveyed hospitals. Also, no differences between these two hospital groups in disease-free survival and overall survival were found. Postoperative radiotherapy was employed in more than 90% of the patients submitted to conservative surgery and adjuvant chemotherapy was used in 39% of all the patients, while tamoxifen as adjuvant treatment was used in 58% of the patients.

Carcinoma in situ and early breast carcinoma. Survey of the Portuguese Senology Society on the diagnostic tools used in Portugal and their evolution between 1985 and 2000.

By means of a questionnaire, sent to the Portuguese hospitals which diagnose and treat most female patients with breast cancer, it was intended to assess the situation regarding the diagnosis of carcinoma in situ and early breast cancer (T1 or T2, N0 or N1), as well as their evolution between 1985 and 2000. The hospital participation rate was 65% and a sample of 865 patients was collected, distributed in the years 1985, 1990, 1995 and 2000. It was found that the presentation form of breast cancer in 1985 was of palpable tumour in 87% of the cases, whereas in 2000 this situation only corresponded to 54% of the patients, being most of the remaining patients diagnosed by imaging without palpable tumour. In 94% of the patients, the first diagnostic investigation was mammography, associated or not to echography, and the second most frequent investigation was fine-needle aspiration biopsy. The time evolution of the tumour size showed an increasingly earlier diagnosis. Invasive tumours not more than 1 cm represented 13.2% in 1985 and 20.3% in 2000. On the other hand, breast cancers more than 2 cm and not more than 5 cm decreased from 67.2% in 1985 to 40% in 2000. When oncology centres and some large university hospitals (Group A) were compared to the other hospitals (Group B), there were no significant differences between the diagnostic methods, although the sequence of diagnostic methods was different in the hospitals in Group A versus those in Group B. It was observed that in more differentiated hospitals the diagnosis was achieved increasingly earlier along the studied periods, and this situation did not occur in the other hospitals.