Low lymphatic vessel density associates with chronic rhinosinusitis with nasal polyps.

OBJECTIVES: Chronic rhinosinusitis with and without nasal polyps (CRSwNP and CRSsNP) and antrochoanal polyps (ACP) are different upper airway inflammation phenotypes with different pathomechanisms. In order to understand the development of tissue edema, the present study aimed to evaluate lymphatic vessel density in CRSsNP, CRSwNP and ACP. MATERIALS AND METHODS: 120 retrospective nasal and maxillary sinus specimens were stained immunohistochemically with a von Willebrand factor polyclonal antibody recognizing vascular and lymphatic endothelium, and with a podoplanin monoclonal antibody recognizing lymphatic endothelium. Vessels were studied by microscopy in a blinded fashion, and the vessel density and the relative density of lymphatic vessels were calculated. Patient characteristic factors and follow-up data of in average 9 years were collected from patient records. RESULTS AND CONCLUSION: In the nasal cavity, the low absolute and relative density of vessels and of lymphatic vessels was associated with CRSwNP and ACP tissues compared to control inferior turbinate. This was observed also in the inflammatory hotspot area. In the maxillary sinus, lower absolute and relative density of lymphatic vessels associated with the CRSwNP phenotype. High lymphatic vessel density in polyp tissue associated with the need for revision CRS-surgery. As a conclusion, low density of lymphatic vessels distinguished patients with CRSwNP not only in the hotspot area of polyp tissue, but also in maxillary sinus mucosa. Yet, higher lymphatic vessel density seems to associate with polyp recurrence. Further studies are still needed to explore if formation of nasal polyps could be diminished by intranasal therapeutics affecting lymphangiogenesis.

Computed tomography findings after endoscopic sinus surgery with preserving or enlarging maxillary sinus ostium surgery.

Endoscopic sinus surgery (ESS) is the main surgical approach in the treatment of chronic rhinosinusitis (CRS) after failure of medical treatment. ESS is based on the theory that obstruction of the maxillary sinus ostium is mainly behind the pathogenesis of CRS. Controversy remains concerning the enlargement of the natural maxillary sinus ostium. The aim of this study was to compare computed tomography (CT) findings after preservation or enlargement of the maxillary sinus ostium. Thirty patients with non-polypous CRS underwent randomized endoscopic sinus surgery with uncinectomy on one side and additional middle meatal antrostomy on the other side. Lund-Mackay (LM) scores and the ostium diameters were analysed from CT scans taken preoperatively and nine months postoperatively, and were used for comparison of the two operative techniques. In addition, the correlation between CT findings and subjective outcomes was studied. Comparison of the preoperative and postoperative CT scans revealed that significant reduction of LM score was achieved on both sides, regardless of the type of procedure performed. The postoperative area of the ostium remained significantly larger on the antrostomy side compared to the uncinectomy side. A large maxillary sinus ostium size seems to associate with lower postoperative LM score, but does not seem to provide superior symptom relief.

Effect of endoscopic sinus surgery on antral mucociliary clearance.

Endoscopic sinus surgery (ESS) is the most used surgical approach in the treatment of chronic and recurrent maxillary rhinosinusitis. However, it still remains unclear how well surgery restores the mucociliary function in damaged maxillary sinus mucosa. There is also controversy whether to enlargen the natural ostium or not. We examined the mucociliary clearance (MCC) of maxillary sinuses in 27 patients with chronic and recurrent rhinosinusitis. On one side only an uncinectomy was done, on the contralateral side a middle meatal antrostomy was additionally performed. The mucociliary clearance (MCC) was measured in both sides preoperatively and 9 months after the operation. Measurements of the mucociliary clearance in maxillary sinuses were done using an isotope method. Preoperative mean residual activity on the uncinectomy side was 87.2 % and postoperative mean residual activity 94.1 %. On the middle meatal antrostomy side mean preoperative residual activity was 92.3 % and postoperative mean residual activity 88.4 %. Residual activity was considered as good (< or = 50 %) on the uncinectomy side in 2 sinuses (7.4 %) preoperatively and in 1 sinus (3.7 %) postoperatively. On the middle meatal antrostomy side residual activity was considered good in 1 sinus (3.7 %) preoperatively and in 4 sinuses (14.8 %) postoperatively. Mucociliary function remained poor even 9 months postoperatively. Surgery did not significantly improve the mucociliary function of maxillary sinus mucosa in chronic or recurrent rhinosinusitis. There was no statistical difference between operative techniques. In this study it seemed however, that uncinectomy combined with the enlargening of the natural ostium may restore maxillary sinus mucociliary clearance (MCC) better than uncinectomy alone.

Expression and purification of polyhistidine-tagged firefly luciferase in insect cells--a potential alternative for process scale-up.

The coleopteran firefly, Photinus pyralis, luciferase was produced in lepidopteran Trichoplusia ni insect cells using a baculovirus expression vector. The recombinant protein was equipped with a polyhistidine affinity tag at the carboxyl terminus and purified by immobilized metal-ion affinity chromatography in combination with an expanded bed adsorption system. This approach enabled an efficient, one-step purification protocol of a genetically modified luciferase with properties similar to those of the authentic counterpart. According to light emission measurements, the final yield of highly purified protein was 23 mg l(-1) of cell culture. In addition, no specific interaction of interfering substances, such as, ATP, adenylate kinase, nucleoside diphosphokinase, as well as, creatine kinase of the final preparation were identified. Together, the results presented here clearly show that the baculovirus expression system in combination with immobilized metal-ion affinity chromatography is a potential strategy for process scale-up of polyhistidine tagged insect luciferase.

Long-term follow-up of the clinical relevance of short outer dynein arms in human nasal cilia.

Clinical significance of short outer dynein arms was examined in a long-term follow-up study of 76 patients with various respiratory symptoms. Clinical evaluations, nasal mucociliary transport rate (NMTR) measurement and transmission electron microscopy were performed. Follow-up examinations took place 5-11 years later. In the initial examination four patients and on follow-up seven patients were found to have short outer dynein arms in their nasal mucosal biopsies. Short dynein arms were associated with a slow NMTR, poorly coordinated ciliary beat direction, and clinical symptoms of perennial rhinitis and recurrent sinusitis. Short dynein arms were found to be a significant histologic finding and probably represent a variant of primary ciliary dyskinesia. In some cases short outer dynein arms may be a reversible finding with improving clinical symptoms. It is obvious that at least part of dynein defects are acquired.

Overexpression of the Aspergillus niger pH 2.5 acid phosphatase gene in a heterologous host Trichoderma reesei.

An Aspergillus gene coding for a pH 2.5 acid phosphatase enzyme was successfully overexpressed in Trichoderma reesei under the strong main cellobiohydrolase I (cbh 1) promoter. The best transformants produced up to 240 times more of the acid phosphatase than the Aspergillus strain from which the phosphatase gene was originally isolated. The recombinant enzyme was effectively secreted into the culture medium both by its own and the cbh 1 secretion signal. The heterologous pH 2.5 acid phosphatase enzyme produced by the Trichoderma transformants was seen as four protein bands of about 55-66 kD resulting from variable glycosylation in Trichoderma. The activity of the recombinant enzyme was not affected. Enzyme preparations rich in both cellulose and phytate hydrolysing enzymes are of interest in the animal feed industry.

Clinical relevance of tubulus anomalies and compound cilia.

Transmission electron microscopy (TEM) from nasal mucosa and nasal mucociliary transport rate (MTR) with a radio-isotopic method were examined in 144 patients with various respiratory symptoms. Examinations were not performed during acute infections. In cases of increased amount of tubulus anomalies: nasal MTR was not significantly slower than in other patients, the disorientation of ciliary beat direction was significantly larger, there was no connection with increased amount of compound cilia and there was no correlation to any specific symptom or respiratory disease. In cases of increased amount of compound cilia: nasal MTR did not differ from other patients and the disorientation of ciliary beat direction did not differ from other patients. In a follow-up study of 76 patients 5-11 years (mean 9.3 years) later the amount of tubulus anomalies and compound cilia had changed in most patients. In patients with less symptoms at the follow-up than primarily, there was a non-significant tendency to have less tubulus anomalies, too, but no change in the amount of compound cilia. The amount of tubulus anomalies and compound cilia is not stable. Their number can either increase or decrease during a follow-up. Tubulus anomalies are non-specific and probably secondary changes of minor clinical importance and compound cilia would seem to be quite irrelevant findings in the ultrastructural evaluation of respiratory cilia.

The correlation of mucociliary transport and scanning electron microscopy of nasal mucosa.

The ciliary ultrastructure studied by scanning electron microscopy (SEM), nasal mucociliary transport rate (MTR) measured by a radioisotopic method and their correlation with each other were studied in 56 patients with recurrent or chronic respiratory infections. Patients were divided into three groups according to MTR. The number of pathological SEM findings increased remarkably in groups with moderate or poor MTR. Loss of ciliated cells and microvilli were seen in 86% of specimens with poor MTR, in 71% with moderate MTR, and in 50% with good MTR. There was ciliary disorientation in 45% of cases with poor MTR compared with 25% in cases with good MTR. Epithelial metaplasia and the number of short cilia increased in groups with poor or moderate MTR.

Secretion of the Hormoconis resinae glucoamylase P enzyme from Trichoderma reesei directed by the natural and the cbh1 gene secretion signal.

Secretion of the Hormoconis resinae glucoamylase P (GAMP) enzyme from Trichoderma reesei using either the natural N-terminal extension of the premature glucoamylase P or the cellobiohydrolase I (CBHI) signal peptide was examined. The expression conditions for the heterologous glucoamylase P (gamP) gene in T. reesei were standardized by targeting one copy of a plasmid fragment, containing the gamP gene, to the cbh1 locus of the host. The results showed that the transient N-terminal extension of the premature GAMP acts as an efficient secretion signal in T. reesei and leads to a higher yield of extracellular glucoamylase activity than does the signal peptide of CBHI.

Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei.

A cDNA encoding for the glucoamylase P enzyme (GAMP) of the fungus Hormoconis resinae was introduced into the cellulolytic filamentous fungus Trichoderma reesei under the control of the promoter of the major cellulase gene (cbh1) of Trichoderma. The transforming vector plasmid used was found to be integrated into the genome of T. reesei at various locations and in multiple copies. The size of the GAMP secreted by Trichoderma varied because of different glycosylation patterns. The best transformant strains secreted about 700 mg/l of active GAMP, which is 20-fold more than obtained with H. resinae.

The pH of secretions in sinusitis and otitis media.

Impaired mucociliary function is a common finding in patients with recurrent respiratory infections, sinusitis and otitis media. One main component of mucociliary transport, ciliary beating activity, is sensitive to environmental hydrogen ion concentration (pH). The pH of secretion in 103 specimens from acute or chronic sinusitis and in 98 specimens from acute or secretory otitis media were measured. There were no differences between acute and chronic sinusitis (mean of pH 7.5 +/- 0.5 and 7.4 +/- 0.6) or between mucous and purulent secretions. In acute otitis media, secretion was significantly more acidic than in secretory otitis media (mean of pH 7.7 +/- 0.5 versus 8.2 +/- 0.4). The pH was less than 7.0 in 16 (15.5%) patients with chronic sinusitis and in two (8%) patients with acute otitis. The study shows that pH of sinusal or tympanal secretion is, in the majority of patients, within limits that do not cause ciliostasis.

Glucoamylase P gene of Hormoconis resinae: molecular cloning, sequencing and introduction into Trichoderma reesei.

The glucoamylase P gene of the fungus Hormoconis resinae has been cloned and sequenced from a genomic library. The gene consists of a 2153-bp protein coding region including three introns. The usual number of introns in cloned fungal glucoamylase genes has been four and in some cases five. Two of the glucoamylase P gene introns contain a sequence resembling the consensus sequence found near the 3' splice site in the introns of the fungus Trichoderma reesei cellobiohydrolase 1 (cbh1) gene. The H. resinae glucoamylase P gene, under its own promoter, was introduced into T. reesei, but no expression could be detected.

Interactive morphometry: the influence of magnification.

The influence of magnification on light microscopic morphometry was studied. Morphometry was performed on 34 hepatocytes by using a digitizer plate with micrographs of the nuclei, and with digitized image of the nuclei on a separate monitor. Intraobserver variation was smallest at objective magnifications of 40 and 100 X (CV of form factor, maximum diameter and nuclear perimeter under 1%, of nuclear area under 2%, when measured from the monitor). The results show that more reproducible measurements are possible with higher magnifications. The authors recommend objective magnifications of 40 and 100 X for morphometric measurements on nuclei. The results also suggest that morphometric grading should apply an intermediate third grade in dichotomous grading scales.

Sampling in diagnostic morphometry: the influence of variation sources.

The variation sources relevant to a diagnostic morphometric study were analysed. The influence of each source was estimated in two experiments, performed in systems applying computer assisted interactive morphometry. In the first experiment one observer measured the areas of a large number of nuclei in a section from a grade II transitional cell carcinoma of the bladder. In the second experiment two groups of researchers, from Ancona and Kuopio, measured one field from five different samples of transitional cell tumours (including the case of grade II carcinoma). It turned out that pure interobserver variation was responsible for about a half of the total variation present in the diagnostic system. When the variation characteristics of the diagnostic system had been determined, the number of nuclei that had to be measured to reach a defined level of accuracy could be estimated. Such an estimate was also dependent on the predefined expectancy probability of reaching a correct estimate. The study showed that group morphometry (statistical, investigative morphometry) and diagnostic morphometry must be understood as two different approaches in histopathology. By applying group morphometry, good research results can be gathered with cruder measurements than in diagnostic morphometry. Because investigations in group morphometry are more standardized than in diagnostic morphometry, a larger number of structures has to be measured in diagnostic histopathology for the same level of accuracy.

Application of morphometry in tumor pathology.

The application of morphometry in tumor pathology is discussed, e.g., its use in studying the biology of tumors, in creating tumor classification(s), in creating methods for the identification of a tumor in the diagnostic context, and in characterizing diagnostic histopathology in absolute terms. In traditional subjective diagnostic histopathology, reproducibility can be defined satisfactorily, but the definition of accuracy is ambiguous; in morphometric histopathology, a satisfactory definition is found for both concepts but it may be difficult to separate them in practice. Morphometric histopathology can study parameters measured from sections or parameters derived from the primary measurements through calculations. In the histopathology of tumors, the following parameters have turned out to be specially valuable: densitometric measurements of nuclei, nuclear area, perimeter and form factors, nucleolar parameters, the number of mitotic cells per area, the cellularity, the volume fraction of the epithelium, and parameters associated with the fraction of tumor tissue in the sample. The standard deviation or other moments of the distribution of these measurements can be more relevant than the mean values of the results. This indicates that more attention should be given to sampling rules, which are important in defining the efficiency of the methods. For rational application of morphometric methods, it is very important to make a distinction between group morphometry and diagnostic morphometry. The latter engenders numerous sources of variation (variation in section thickness, variation in tissue processing, variation in the techniques of measurement, interobserver variation, interlaboratory variation, variation due to subjective interpretation, etc.), which are usually better controlled in group morphometry. The influence on morphometric parameters of variation in section thickness and tissue shrinkage during processing are discussed.

Observer variation in interactive computerized morphometry.

The validity of a test system in morphometric histopathology depends on the variation sources involved. Biological variation is one of the variation sources, but is also the object to be studied with morphometry. We studied the variation sources in interactive computerized morphometry in two test systems involving two different commercially available image analyzers. The measurements were made on nuclei in a microscopic field of 5 transitional cell tumors (papilloma and WHO grade I-III carcinomas) of the urinary bladder. It turned out that different observers selected a variable number of nuclei for measurements, the coefficient of variation (CV) in the number of nuclei being 11-11.5%. Mean CV in nuclear perimeter measurements was 4.4-4.8%, and in nuclear area measurements 8.2-8.6%. The variation in measurements by 1 observer was smaller, the CV values being 2.8% for the number of nuclei, 1.2% for nuclear perimeter, and 2.4% for nuclear area. The results showed that interobserver variation can be considerable in these systems. It is suggested that special sampling rules should be tested with the idea of finding the relevant approach with the smallest interobserver variation.

Androgen induction of ornithine decarboxylase mRNA in mouse kidney as studied by complementary DNA.

To investigate the mechanisms by which androgens regulate ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) in mouse kidney, a cDNA clone encoding OrnDCase mRNA was prepared. Purification of OrnDCase mRNA from kidneys of androgen-treated mice was accomplished by immunoadsorption of renal polysomes to a protein A-Sepharose column and enrichment for poly(A)-containing RNA by oligo(dT)-cellulose. Double-stranded cDNA synthesized from this mRNA was inserted into the Pst I site of plasmid pBR322 by using oligo(dG . dC)-tailing and was propagated in Escherichia coli. Plasmids containing cDNA sequences coding for OrnDCase were identified by differential colony hybridization, by radioimmunological detection of OrnDCase-like antigens in bacterial cultures, and by cell-free translation of hybrid-selected mRNA followed by immunoprecipitation with monospecific OrnDCase antiserum. A restriction endonuclease fragment of the selected plasmid DNA (pODC54) was labeled by nick-translation and used to study changes in OrnDCase mRNA concentration. After a single dose of testosterone, renal OrnDCase mRNA concentration increased as soon as 6 hr and peaked 24 hr after steroid injection, as measured by RNA blot hybridization. Continuous androgen treatment for 4 days resulted in a 10- to 20-fold increase in OrnDCase mRNA concentration in normal animals, but no induction of this mRNA was detected in mice that have an inherent defect of the androgen receptor (testicular feminization). These results indicate that androgens regulate OrnD-Case synthesis in mouse kidney, at least in part, by increasing OrnDCase mRNA accumulation.