Safety evaluation of a sinus surfactant in an explant-based cytotoxicity assay.

OBJECTIVES/HYPOTHESIS: Biofilms are associated with clinical relapse following surgery for chronic rhinosinusitis. Encased bacteria are protected from innate immunity and antimicrobial therapy. Surfactants can disperse the biofilm into its planktonic phenotype so that traditional treatments may be effective. The aim of this study was to assess a surfactant for its cytotoxicity profile. STUDY DESIGN: In vitro explant-based cytotoxicity study. METHODS: Sinonasal mucosa harvested from patients undergoing sinus surgery was tested using an air-liquid interface explant system. Surfactant at 1×, 2×, and 3× manufacturer's recommended concentrations were compared to control (saline) and Zinc Sulphate (ZnSO4 ), a known cytotoxic agent. Culture supernatant was analyzed for lactate dehydrogenase (LDH) as a marker of cellular toxicity. After 7 days, specimens were imaged using structured histopathology and scanning electron microscopy. RESULTS: Application of surfactant at 1× concentration did not elicit an elevation in LDH, whereas ZnSO4 caused a significant rise 1 day after application. Specimens tested with a 2× and 3× surfactant demonstrated LDH rises 4 days and 2 days after application, respectively. Mucosa tested with the 1× surfactant and control demonstrated intact cellular structures on histopathology and preserved cilial ultrastructure on SEM. In ZnSO4 -treated specimens, marked cellular degradation and ciliary denudation occurred. CONCLUSION: The surfactant does not appear to elicit cellular toxicity using an in vitro explant model at the manufacturer's recommended concentration. At higher concentrations, there may be dose-related toxicity that requires further investigation. In vivo testing is required to prove its efficacy in the treatment of recalcitrant chronic rhinosinusitis. LEVEL OF EVIDENCE: N/A.

An in vitro model of Pseudomonas aeruginosa biofilms on viable airway epithelial cell monolayers.

BACKGROUND: Chronic rhinosinusitis (CRS) that is refractory to medical or surgical intervention may involve a particularly resistant form of infection known as a bacterial biofilm. Bacterial biofilms are three-dimensional aggregates of bacteria that often are recalcitrant to antibiotics secondary to physical barrier characteristics. To date, all studies investigating biofilms in CRS have been descriptive in either human or animal tissue. To better understand the interactions of bacterial biofilms with respiratory epithelium, we describe an in vitro model of biofilm sinusitis by establishing mature biofilms on airway epithelial air-liquid interface cultures. METHODS: Airway epithelial cell cultures were grown on collagen-coated semipermeable support membranes as an air-liquid interface on tissue culture inserts. Confluent air-liquid interface cultures were inoculated with the biofilm-forming PAO-1 strain of Pseudomonas aeruginosa and compared with cultures inoculated with two mutant strains (sad-31 and sad-36) unable to form biofilms. Inoculated tissue transwells were incubated for 20 hours, allowing for biofilm growth. The semipermeable membranes were then harvested and imaged with confocal laser scanning microscopy and scanning electron microscopy. RESULTS: Microscopic analysis revealed the formation of biofilm-forming towers in the PAO-1 inoculated wells. The bacterial biofilms were supported by a viable airway epithelial cell surface monolayer. CONCLUSION: This study shows a reliable method for analysis of in vitro interactions of bacterial biofilms and airway epithelium. The experimental manipulation of this air-liquid interface model will help explore novel treatment approaches for bacterial biofilm-associated CRS.

Histological aspects of rhinosinusal polyps.

UNLABELLED: Contemporary cohort cross-sectional study. INTRODUCTION: Despite its importance for an accurate diagnosis, histology differences among nasal polyps and its clinical implications are rarely reported in the literature. The existing papers classify polyp samples without concern for prior treatments, which could influence the results attained. AIMS: carry out a morphological study, through light microscopy, of nasal polyps' structural alterations in the absence of any type of prior treatment and histologically classify it in relation to studies published in the literature. MATERIALS AND METHODS: We studied 89 patients with nasosinusal polyps without prior treatment. Polyp samples were collected by outpatient biopsy and analyzed through light microscopy after dyeing with hematoxylin-eosin. RESULTS: Samples were classified in the following way: Edematous or eosinophilic polyp 65 cases (73%); fibro-inflammatory polyp: 16 cases (18%); Polyp with Sero-mucinose gland hyperplasia: 06 cases (6.7%) and polyp with stroma atypia: 2 cases (2.3%). DISCUSSION: eosinophilic pattern prevailed in the patients with nasosinusal polyps of the population studied. This pattern is similar to the ones found in the major studies, which, however, do not mention prior treatment. CONCLUSION: after analyzing the polyps' histological characteristics, we noticed that the untreated polyps present a predominantly eosinophilic pattern.

Aspectos histológicos do pólipo rinossinusal / Histological aspects of rhinosinusal polyps

Estudo de coorte contemporânea com corte transversal. As diferenças histológicas dos pólipos nasais e a sua possível implicação clínica são escassas em literatura, apesar de sua importância para um diagnóstico preciso. Os trabalhos existentes classificam amostras de pólipos sem a preocupação quanto à influência de tratamentos prévios, o que influenciaria o resultado obtido. OBJETIVO: Estudar morfologicamente, através da microscopia ótica, as alterações estruturais do pólipo nasal na ausência de qualquer tratamento prévio e classificá-lo, histologicamente, correlacionando com os estudos de literatura. MATERIAL E MÉTODOS: Foram estudados 89 pacientes com polipose rinossinusal sem tratamento prévio. As amostras dos pólipos foram colhidas por biópsia ambulatorial e analisadas através de microscopia ótica após coloração com hematoxilina e eosina. RESULTADOS: As amostras foram classificadas da seguinte forma: pólipo Edematoso ou Eosinofílico: 65 casos (73 por cento); pólipo Fibroinflamatório: 16 casos (18 por cento); pólipo com Hiperplasia de Glândulas Seromucinosas: 06 casos (6,7 por cento) e pólipo com Atipia de Estroma: 2 casos (2,3 por cento). DISCUSSÃO: O padrão eosinofílico predominou nos pacientes com polipose rinossinusal na população estudada. Este padrão assemelha-se com os principais estudos que, no entanto não mencionam sobre tratamentos prévios. CONCLUSÃO: Após análise das características histológicas dos pólipos, observou-se que pólipos não tratados apresentam um padrão predominantemente eosinofílico.

Contemporary cohort cross-sectional study. Introduction: Despite its importance for an accurate diagnosis, histology differences among nasal polyps and its clinical implications are rarely reported in the literature. The existing papers classify polyp samples without concern for prior treatments, which could influence the results attained. AIMS: carry out a morphological study, through light microscopy, of nasal polyps' structural alterations in the absence of any type of prior treatment and histologically classify it in relation to studies published in the literature. MATERIALS AND METHODS: We studied 89 patients with nasosinusal polyps without prior treatment. Polyp samples were collected by outpatient biopsy and analyzed through light microscopy after dyeing with hematoxylin-eosin. RESULTS: Samples were classified in the following way: Edematous or eosinophilic polyp 65 cases (73 percent); fibro-inflammatory polyp: 16 cases (18 percent); Polyp with Sero-mucinose gland hyperplasia: 06 cases (6.7 percent) and polyp with stroma atypia: 2 cases (2.3 percent). DISCUSSION: eosinophilic pattern prevailed in the patients with nasosinusal polyps of the population studied. This pattern is similar to the ones found in the major studies, which, however, do not mention prior treatment. CONCLUSION: after analyzing the polyps' histological characteristics, we noticed that the untreated polyps present a predominantly eosinophilic pattern.

Rehabilitation of surgically traumatized paranasal sinus mucosa using retinoic acid.

BACKGROUND: Paranasal sinus mucosa that regenerates after surgical stripping is characterized by sparse, dysmorphic cilia. Although such dysfunctional mucosal changes are thought to be irreversible, the potential rehabilitation of deciliated mucosa by topical therapies has not been explored. This study evaluated the effect of topical retinoic acid (RA) in rehabilitating deciliated sinus mucosa that has regenerated after surgical stripping. METHODS: Twelve New Zealand rabbits underwent bilateral maxillary sinus mucosal stripping and were allowed to remucosalize for 10 days. In the first treatment arm (n = 6), 0.01% RA was applied to the regenerated mucosa of the left maxillary sinus and an aqueous control was placed contralaterally. In the second treatment arm, the regenerated mucosa was surgically restripped bilaterally before applying RA and control. After 14 additional days, mucosa was harvested and examined by scanning electron microscopy for ciliary density, orientation, and morphology. RESULTS: In the first treatment arm, no significant differences in ciliary density, orientation, or morphology were noted between RA-treated sinuses and controls. In the second arm, RA-treated sinuses showed improved ciliary density, morphology, and orientation compared with controls. CONCLUSION: RA does not appear to significantly enhance ciliogenesis when applied after mucosal regeneration has occurred. However, if deciliated mucosa is restripped and allowed to regenerate in the presence of RA, ciliary ultrastructural features may be enhanced. RA may have therapeutic value in rehabilitating deciliated sinus mucosa when applied before mucociliary differentiation of regenerating mucosa.

A sheep model for the study of biofilms in rhinosinusitis.

BACKGROUND: Bacterial biofllms have been shown in chronic diseases such as cystic fibrosis, cholesteatoma, and otitis media with effusion. Recently, their detection on the mucosal tissue of sinusitis patients has implicated them in the pathogenesis of this condition. We present an animal model using sheep experimentally infected with Staphylococcus aureus to study the possible association between biofilm and sinusitis. METHODS: Twenty-four sheep underwent bilateral endoscopic sinus surgery to identify their frontal ostia. The frontal sinuses were treated in one of the following ways according to preoperative randomization: (1) ostium left patent, (2) ostium left patent and bacteria instilled, (3) ostium occluded, or (4) ostium occluded and bacteria instilled. The frontal mucosa was harvested at day 7 and examined for biofilm presence using confocal scanning laser microscopy (CSLM) as well as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). RESULTS: All three modalities showed different rates of biofilm detection. Three-dimensional structures that could be interpreted as biofilms were documented in 86% (n = 36) of the sinuses analyzed using SEM. These structures were seen in all four study groups. The detection rate using the other two modalities was much lower with CSLM, showing biofilms in 48% (n = 20) and TEM in only 29% (n = 12) of the sinuses analyzed. Unlike SEM, these two modalities only detected bacterial biofilms in sinuses randomized to bacterial instillation. CONCLUSION: The demonstration of bacterial biofilms in this animal model of sinusitis further supports the hypotheses that biofilms may play a role in the pathogenesis of this condition. There is an obvious discrepancy in the sensitivity and specificity of biofilm detection using the three modalities mentioned. CSLM appears to be the most objective technique. The inherent flaws, sampling error, and subjectivity involved in SEM and TEM make these less reliable in documenting biofilm existence.

A comparative study between ciliary count and the degree of opacity of paranasal sinus CT scans in chronic sinusitis pre and post FESS.

OBJECTIVE: To investigate the relationship between the extent of sinus disease in chronic sinusitis as detected radiologically by computed tomography (CT) scan and the population of cilia (ciliary area) both before and after functional endoscopic sinus surgery (FESS). In a simple way this is a trial to statistically prove that the CT scan could be a valid indicator and a mirror of the histological status of the sinus mucosa. DESIGN: Twenty adult patients were enrolled in this study. Radiological extension of the sinus disease was quantitated using the classification proposed by Kennedy in 1992 and the ciliary population was studied using scanning electron microscopy and image analysis softwares. RESULTS AND CONCLUSION: The more advanced the sinusitis, as evidenced by CT scans, the more the expected reduction in the ciliary area (CA) and in the ciliary count. But after FESS the degree of ciliary regeneration does not depend statistically on the radiological condition of the sinuses and the degree of opacity prior to intervention, i.e. a statistically valid inverse relationship exists between the radiological stage of sinusitis and the ciliary population pre-operatively but the same relation does not extend to the ciliary population post-operatively.

Presence of surfactant lamellar bodies in normal and diseased sinus mucosa.

BACKGROUND: Pulmonary surfactant originates from phospholipid lamellar bodies secreted from the type II epithelial cell of the alveolus. In the lower airway, surfactant optimizes surface tension and oxygen exchange, decreases mucus viscosity and aids in mechanical elimination of inhaled pathogens. In addition to the lung, lamellar bodies have been identified in many other cell types throughout the human body. However, no prior studies have identified lamellar bodies in human sinus mucosa. OBJECTIVES: We performed ultrastructural studies to assess whether lamellar bodies are present in the human sinus in a variety of diseased and normal epithelium. METHODS: We biopsied sinus mucosa from 5 subjects, 1 each with allergic fungal sinusitis, eosinophilic mucin rhinosinusitis, cystic fibrosis, frontal sinus mucocele, and cerebrospinal fluid leak (healthy control). Mouse lung served as a positive control. Specimens were prepared using ferrocyanide-reduced osmium tetroxide and thiocarbohydrazide for fixation (R-OTO method) to avoid extraction of phospholipids during dehydration and were viewed with transmission electron microscopy. RESULTS: We identified lamellar bodies in the sinus mucosa of all patients. Additionally, preservation of mouse lung lamellar bodies confirms that the R-OTO method is a valid technique to preserve these structures. CONCLUSIONS: We describe a simpler, faster technique for identification of cellular phospholipid components than those used previously. Definitive identification of these lamellar bodies within ciliated pseudostratified epithelium of the upper airway indicates that surfactant may have a role in sinus function and pathophysiology.

[Pathological and ultrastructural observation on the epithelia of nasal cavity and nasal sinuses in chronic sinusitis and nasal polyps after endoscopic sinus surgery].

OBJECTIVE: To research the restoration characteristics of epithelia in nasal cavity and nasal sinuses with chronic sinusitis and nasal polyps after endoscopic sinus surgery,and to provide basis for improving the treatment after operation. METHOD: Twenty patients with chronic sinusitis and nasal polyps after endoscopic sinus surgery were observed. The "Vesicle tissues" situated ostiomeatal complex (OMC) were observed with light and transmission electron microscopy after operation of 7-14 days. RESULT: (1) The "Vesicle tissues" were off white or buff, some contained liquid, and some were parenchyma under the endoscopy. (2) The epithelia of "Vesicle tissues" were pseudostratified ciliated columnar epithelium (16/20), and some were stratified squamous epithelium (2/20), some were squamous metaplasia in pseudostratified ciliated columnar epithelium (2/20). Lamina propria were edema. Inflammation cells infiltrated epithelial lining and subepithelial lining. We found mucous gland, mixed gland and glandular duct under subepithelial lining,and small vascular ectasia under light microscopy. (3) Transmission electron microscope: epithelia were pseudostratified ciliated columnar epithelium, cilia were in good order, microvilli located between cilia. The cross section of majority cilia were 9 + 2 form microtubule, but in some cilia we could not find microtubule and could only find one central microtubule. Golgi complex were up growth, and endoplasmic reticulum expand, and multi-mitochondrion were coagulation degeneration in epithelial cells. Some mitochondrion were 1-2 degree swelling and the lysosome increased. Inflammation cells infiltrated epithelial lining. Majority were neutrophil and lymphocyte, but we also found eosinophil and plasmacyte. CONCLUSION: "Vesicle tissues" is the manifestation of active epithelia regeneration in nasal cavity and nasal sinuses after endoscopic sinus surgery, it's the earlier period of imbalance regeneration of the epithelization in postoperative cavity. "Vesicle tissues" is similar to the nasal polyp histopathology, and it is phenomena of limited pathological rebuilding in earlier period. Cleaning "Vesicle tissues" can effectively control the nasal polyps relapse and impel the postoperative cavity to epithelization.

Estudo da ultra-estrutura da face medial da bolha etmoidal em pacientes com rinossinusite crônica / Ultrastructural study of medial face of ethmoidal bulla in patients with chronic rhinosinusitis

As alterações ultra-estruturais da mucosa dos seios paranasais representam um papel importante na fisiopatologia da rinossinusite crônica. Com o objetivo de conhecer estas alterações, foi estudada a ultra-estrutura da mucosa da bolha etmoidal, As pesquisas foram feitas em 21 pacientes, submetidos à análise histopatológica e da ultra-estrutura, com microscópio eletrônico de varredura. Observou-se epitélio respiratório, cilíndrico ciliado e pseudo estratificado. Foi freqüente o achado de regiões com espaçamentos evidentes entre as células epiteliais. A rinossinusite crônica provavelmente provoca a decomposição da camada celular por falta de coesão entre seus elementos e metaplasia do epitélio de revestimentob / The ultrastructural changes in paranasal sinus mucosa play an important role in the pathophysiology of chronic rhinosinusitis. To improve knowledge about these alterations, the ultrastructure of medial wall of ethmoidal bulla was studied. Portions of the ethmoid bulla wall of twenty-one patients were submitted to histopathological examination with scanning electron microscope. Although the metaplasia was difficult to detect in the light microscopy, its presence was confirmed by electronic microscopy. Basal cells hyperplasia was observed in the majority of the cases. Seromucous glands and hyperplasic acinus were found in all cases. Chronic rhinosinusitis probably leads to a decomposition of cellular layer, due to a loss of cohesion between its elements and the covering squamous metaplasia...

[Therapeutic effect of roxithromycin on chronic sinusitis with nasal -- polyps clinical, computed tomography, and electron microscopy analysis].

To evaluate the therapeutic effect of Roxithromycin (RXM), we studied 56 chronic sinusitis patients with nasal polyps using computed tomography (CT) and electron microscopy in addition to conventional clinical assessment. The paranasal sinus of subjects was observed clinically before and after daily administration of RXM at 300 mg for 3 months all underwent allergy testing for possible complications of allergic rhinitis based on subjective symptoms and objective findings. Improvement after RXM treatment was seen in 50.3% based on subjective symptoms and 59.1% based on objective findings. Overall improvement was seen in 53.6%. In 41 cases (73.2%) of all patients with chronic sinusitis and complications of allergic rhinitis, no significant difference was seen between patients with and without complications (53.7% in those with complications and 53.3% in those without). In CT analysis the paranasal sinus in 51.8% of all posttreated patients showed obvious improvement. In electron microscopy in chronic sinusitis patients with complications of allergic rhinitis, pretreated ethmoidal sinus tissues showed high mucous epithelial cell apoptosis in addition to common histological lesions, while posttreatment patients showed only eosinophil apoptosis in the interstitium and no apoptotic epithelial cells. We divided ethmoidal sinus lesions in patients without complications into 3 types and evaluated them as follows: In type 1, pretreated ethmoidal sinus tissues showed plasma cell infiltration and posttreatment cell apoptosis. In type 2, pretreated tissues showed lymphocyte and plasma cell infiltration and posttreated showed only some lymphocytes and no plasma cells. In type 3, proliferation of fibroblasts, most of which showed apoptosis, was seen in addition to apoptotic epithelial cells before treatment, while after treatment, these lesions remained with some apoptotic bodies phagocytosed by macrophages. In type 3 patients relapsed after surgery. Our findings indicate that RXM treatment had a significant therapeutic effect on chronic sinusitis with nasal polyps with and without complications of allergic rhinitis. We clarify the morphological mechanism of therapeutic effect of RXM on each type of ethmoidal sinus lesion divided by light and electron microscopy.

Evaluation of ultrastructural changes in paranasal sinus mucosa after functional endoscopic sinus surgery in patients with chronic rhinosinusitis.

The purpose of this study was to compare the preoperative and postoperative ultrastructural changes of paranasal sinus mucosa in patients treated with functional endoscopic sinus surgery for chronic rhinosinusitis. Twelve patients undergoing functional endoscopic sinus surgery for the treatment of chronic rhinosinusitis were involved. The ethmoid sinus mucosa was sampled during the operation and approximately 6 months after the operation. The ciliated epithelium of sinus mucosa was evaluated with transmission electron microscopy. The samples were taken at the Otolaryngology Department of Istanbul University School of Medicine. Electron microscopic study was performed at the Histology and Embryology Department of the same University. Preoperatively, ciliated epithelial cells of the sinus mucosa of the patients showed degenerated ultrastructure with decreased number of cilia, cytoplasmic protrusions, cisternal dilatations of endoplasmic reticulum, and mitochondrial swellings. Remnants of degenerated cells and cellular separations at cell junctions were evident in the diseased epithelium. Goblet cells were frequent along the epithelial lining. Postoperatively, normal architecture and ultrastructure of the ciliated epithelium was restored. These observations showed that unlike other surgical operations, paranasal sinus mucosa can regenerate and the ciliated epithelium can return to normal after functional endoscopic sinus surgergy.

[Ultrastructure of nasal and paranasal mucosa in patients with chronic polypous rhinosinusitis]. / Ul'trastruktura slizistoi obolochki nosa i okolonosovykh pazukh u bol'nykh khronicheskim polipoznym rinosinusitom.

Electron microscopic study of operative samples of nasal and paranasal mucosa from patients with chronic polypous rhinosinusitis has discovered changes in the mucosa typical for inflammation and ultrastructural changes. Epithelial layer underwent destruction. Adequate function of the remaining ciliary cells is doubtful even in minimal changes of their ciliary apparatus. The absence of the specialized contacts between the epithelyocytes indicates dysfunction of the selective ionic permeability. There was overgrowth of the reticular fibers of the basal membrane. Plasma cells exhibited marked dilation of the cysterns of the rough endoplasmic reticulum probably because of active accumulation of immunoglobulins or their precursors.

[Comparative studies of ciliary epithelial morphological and radiologic findings in the osteomeatal complex].

In this study, we evaluated quantitatively preoperative coronal CT scan in 30 nasal cavities of 22 patients with chronic sinusitis who underwent functional endoscopic sinus surgery. The ciliated area of the osteomeatal complex (OMC) from these patients was quantitatively observed by scanning electron microscope and image analysis. Our results showed a positive correlation between ciliary epithelial pathological changes in OMC and disease degree of this area in CT scan.

[Primary nasal sinus malignant melanomas]. / Melanomas malignos primarios nasosinusales.

Primary malignant melanoma of the mucosa of the nasal and paranasal sinuses is rare, difficult to treat, and usually has a poor prognosis. Seven cases, six women and one man (mean age 76.5 years), were treated at the Hospital 12 de Octubre (Madrid, Spain) during the period 1982-1994. The presenting symptoms, diagnostic evaluation, morphology, therapeutic modalities, histological evaluation, and clinical course were reviewed. Three patients were treated surgically and three received palliative irradiation. All had local recurrence or distant metasfases within 15 months after concluding treatment. The seventh patient refused treatment.

Nebulization of S-carboxymethylcysteine does not adversely affect the mucociliary system in the paranasal sinus and trachea of the healthy rabbit.

Chronic sinusitis is a persistent inflammatory impairment of the paranasal sinus. Disturbance of the mucociliary function in the paranasal sinus is the most common finding in chronic sinusitis. S-carboxymethylcysteine (S-CMC) has been shown to directly enhance the ciliary activity of the chronic sinusitis mucosa. Direct contact of the disturbed cilia with S-CMC may recover the reduced beating activity of cilia in chronic sinusitis and the mucosal pathology of the disease can thus be improved. Before S-CMC as medicine for nebulization in the treatment of chronic sinusitis can be clinically applied, however, it should be experimentally established whether nebulization of S-CMC has any adverse effects on the mucociliary system of the respiratory mucosa. The present study was designed to experimentally examine the safety of nebulization of S-CMC especially with regard to the respiratory mucosa. Rabbits were treated with nebulization of three different concentrations of S-CMC solution for 20 min a day for 14 successive days, and their mucosal pathology of the sinus and trachea was examined and compared with that of healthy animals. Nebulization of concentrations of 0.5-10% of S-CMC solution did not affect the ciliary activity in the sinus and tracheal mucosa, nor did this treatment induce pathological changes such as epithelial injury and inflammatory cell accumulation. It is therefore concluded that concentrations of 0.5-10% S-CMC solution are quite safe for the use of nebulization in the treatment of chronic sinusitis.

Ciliary dyskinesia in the nose and paranasal sinuses.

Primary ciliary dyskinesia is a rare autosomal recessive disorder of which 50% with situs inversus Kartagener's syndrome. Secondary ciliary dyskinesia is a frequent observation, mostly in association with or after respiratory tract infections. Diagnosis and differential diagnosis are mostly based on the typical clinical picture, the absence of mucociliary clearance and ciliary activity and the electron microscopical demonstration of ultrastructural abnormalities. However, these investigations are not always conclusive. Functional and ultrastructural ciliary evaluation after ciliogenesis in tissue culture is essential and crucial.

[Hemangiosarcoma of the nasal cavity]. / Hemangiosarcoma de fosas nasales.

Hemangiosarcoma is a rarely diagnosed in the nasal cavity and there are few reports in the ENT literature. The most interesting aspect of our 82-year-old male's case was that he presented all the findings characteristic of angiosarcoma, but the final post-mortem diagnosis was unexpected because of the poor information available until that moment.