Nasal bioavailability of peptide T in rabbits: absorption enhancement by sodium glycocholate and glycofurol.

The aim of the present study was to investigate the absolute nasal bioavailability of Peptide T from aqueous formulations containing sodium glycocholate, an absorption enhancer with known effect on epithelial tight junctions, and/or glycofurol in a crossover study in rabbits. Additionally, the reversibility of the absorption enhancing effect of sodium glycocholate was studied by applying enhancer and peptide T with different time intervals and calculating Area Under the Curve of the peptide in plasma. It was shown that the bioavailability of Peptide T was significantly enhanced when glycofurol or sodium glycocholate was added to a nasal formulation. The nasal bioavailability of Peptide T in water (control formulation), 5% glycofurol, 5% glycofurol+1% sodium glycocholate and 1% sodium glycocholate was 5.9, 22, 29 and 59%, respectively. As indicated by the differences in t(max), C(max) and time-concentration profiles different patterns of Peptide T absorption were seen from the vehicles containing glycofurol and sodium glycocholate. In the reversibility study, the enhancing effect of sodium glycocholate on nasal absorption of Peptide T was found to be reversible within 4 h. It was concluded, that nasal absorption of Peptide T in rabbits was effectively enhanced by co-administration of sodium glycocholate, which also provided very fast absorption rates as well as a relatively short lasting effect of the absorption enhancing effect. Co-administration of glycofurol leads to enhanced and prolonged absorption of the peptide. Combining the two enhancers did not lead to increased peptide T absorption compared to 5% glycofurol alone.

Solubilization and stability of bumetanide in vehicles for intranasal administration, a pilot study.

The solubility of bumetanide in vehicles of various polarities, suitable for intranasal administration in acute situations, has been investigated. The solubility at 4 degrees C in glycofurol and polyethylene glycol 200 was high (167 and 143 mg/mL, respectively), decreasing exponentially with addition of phosphate buffer or coconut oil. Vehicles containing coconut oil and glycofurol did not seem to improve the solubility relative to mixtures between glycofurol and buffer. Adequate solubility (approximately 50 mg/mL) was achieved in vehicles containing about 80% cosolvent. The stability of bumetanide was studied at 5 degrees C and 57 degrees C. No degradation was observed at low temperature. At high temperature, bumetanide decomposes in nonaqueous vehicles with half-lifes ranging from 69 to 400 days, but sufficient stability may be obtained by adjustment of pH to 7.4. It may be concluded that it is possible to prepare a clinically relevant formulation for intranasal delivery of bumetanide.

Intranasal administration of different liquid formulations of bumetanide to rabbits.

The bioavailability of bumetanide in rabbits after intranasal administration of eight formulations intended for use in acute situations has been studied. The vehicles tested were combinations of phosphate buffer, pH 7.4, glycofurol 75. polyethylene glycol 200 and coconut oil. A mixture of 51% glycofurol in polyethylene glycol 200 was administered containing doses of 1 and 8 mg bumetanide respectively. For all other formulations the lower dose level only was studied. The tmax obtained ranged from 3 to 10 min. The vehicles resulting in the highest rate of absorption were 60% glycofurol in coconut oil and pure glycofurol. The observed bioavailability for the different formulations ranged from 16 to 37% for the time period 0-120 min. The bioavailability was also calculated omitting the initial peak seen after i.v. injection, which may be undesirable. Using this method bioavailabilities of 33-82, for the time interval 5-120 min was found. The study also demonstrated that the total amount of bumetanide absorbed increased proportionally to the dose administered. The rate of absorption of bumetanide from all formulations tested may be relevant for the treatment of acute oedematous states. The tmax obtained after intranasal administration was shorter than reported for other non-parenteral routes of administration.

The prognostic importance of volume-weighted mean nuclear volume, mitotic index, and other stereologically measured quantitative parameters in supraglottic laryngeal carcinoma.

BACKGROUND: Stereologically measured mean nuclear volume has been proven to have prognostic importance in several types of cancer, such as malignant melanoma and carcinomas of the breast, oral region, bladder, and uterine cervix. The main purpose of the current study was to investigate the possible prognostic importance of mean nuclear volume and mitotic index in carcinoma of the supraglottic larynx. METHODS: The study was performed with a stratified, random sample of 113 patients from a well-defined group of 386 patients with supraglottic laryngeal carcinoma treated with radiotherapy at the Finsen Institute in Copenhagen. Histologic sections from pretreatment biopsies were used to estimate the following parameters: mean nuclear volume (Vv(3)(0)), mitotic index (MI), number of nuclei per mm(2) (QA), mean nuclear profile area (Anuc), and the area fraction of nuclei in cancer tissue (AA). The geometric means of the parameters were used as cutoff points in a single factor and in a multivariate survival analysis with relapse free survival as the primary endpoint. RESULTS: The geometric means of the measured parameters were (Vv(3)(0)) = 480 micro(3), QA = 3630 nuclei/micro(2) cancer tissue, MI = 0.48 mitosis/100 nuclei, AA = 0.21, and Anuc = 57.9 micro(2). CONCLUSIONS: None of the stereologically estimated parameters proved to have prognostic importance, whereas tumor size, and lymph node status did. The method of adaptive, stratified, random sampling used in this study can save a great deal of work and is highly recommended by the authors.

High-performance liquid chromatographic analysis of Peptide T in rabbit plasma with on-line column enrichment.

The present paper describes the development of a simple and sensitive analytical method for quantification of Peptide T (PT) in rabbit plasma, using standard analytical equipment and on-line column enrichment, without prior extraction, clean-up or derivatization. The analytical procedure was found to be accurate, precise and linear. The accuracy was 100% (range 97-103%) and the mean precision was 8% (range 3-14%) for all (n=6) concentrations (0, 15, 50, 100 and 200 ng/ml). The total recovery was found to be approximately 80%, and it was found to be dependent upon the injection rate onto the extraction column. The correlation between added and found concentrations was 0.9982, and the limit of detection was estimated to be around 5 ng/ml. The method is therefore found to be suitable for bioavailability studies, involving Peptide T, in rabbits.

Bikunin and alpha 1-microglobulin in human zona pellucida and connective tissue.

The occurrence of bikunin and alpha 1-microglobulin was investigated in human ovary and Fallopian tubes. Bikunin and alpha 1-microglobulin are transcribed in the liver from a common gene. Bikunin immunoreactivity was detected in the zona pellucida. A positive reaction for bikunin was also observed in connective tissue of the oviduct. In addition, mast cells showed a more intense positive reaction than the surrounding connective tissue. Specific displaceable alpha 1-microglobulin immunoreactivity was revealed in the zona pellucida. The data suggest that bikunin and alpha 1-microglobulin are trapped in the zona pellucida.

[Validity of the diagnosis "essential hypertension" in the National Patient Registry]. / Validiteten af diagnosen essentiel hypertension i Landspatientregistret.

This study validated hypertension (ICD-8: 401.99) in The National Inpatient Register with reference to the use of the diagnosis in the Occupational Hospitalization Register. A university hospital and a regional hospital were chosen for the evaluation. A sample of case records with the discharge diagnosis essential hypertension and additional case records with other diagnoses were re-coded blindly and independently by two doctors. Cause of admission or admission diagnosis was recorded for essential hypertension cases. The agreement with The National Inpatient Register ranged from 60 to 40%. About half of the cases with the discharge diagnosis "essential hypertension" were admitted to hospital due to hypertension, about a quarter due to diagnoses within "other arteriosclerotic diseases" (ICD-8: 400, 402-440). The misclassification may lead to an underestimation of risks of hypertension in various occupations in the Occupational Hospitalization Register or bias the occupational risk pattern of essential hypertension to become more alike that of arteriosclerotic diseases.

Observer variability in the histologic assessment of oral premalignant lesions.

Histopathologic examination of oral leukoplakias has a major impact on the assessment of prognosis and treatment planning. We investigated the extent of agreement in grading epithelial dysplasia between pathologists with the same or different educational backgrounds. Two general pathologists and two oral pathologists were each given 100 sections of oral leukoplakia to grade from no dysplasia to carcinoma in-situ. The interobserver agreement rates were in the range of 49% to 69%. The calculated kappa values were in the range of 27% to 45%, showing poor to moderate agreement between the pathologists. When comparing the kappa values between the two pairs of pathologists with the same education, these values did not diverge from the general level of kappa values, indicating that the interobserver variability was due to individual differences rather than to educational background.

Are pathologists biased by clinical information?: A blinded cross-over study of the histopathological diagnosis of mesothelial tumours versus pulmonary adenocarcinoma.

In a blinded cross-over design, we studied whether three pathologists were biased by clinical information when making histopathological diagnoses of adenocarcinoma of the lung and benign and malignant mesothelial tumours. Furthermore, the interobserver variation of these diagnoses was assessed. Forty-one cases of adenocarcinoma of the lung and mesothelial tumours were assessed by three pathologists in four rounds. In the first two rounds, slides stained by H&E and clinical information were available. Slides and information were matched so that a specific slide in one round was given clinical information suggesting adenocarcinoma and in the other round, the clinical information suggested mesothelial tumour. In the third and fourth rounds, a panel of immunohistochemical stains was added. The clinical information was matched in the same way as in the first and second rounds. Bias by clinical information was observed when the diagnoses were made on slides stained by H&E, while no bias could be demonstrated when immunohistochemical reactions were included. The reproducibility also improved significantly when these slides were available.

Differentiation of adenocarcinoma of the lung and malignant mesothelioma: predictive value and reproducibility of immunoreactive antibodies.

A panel of antibodies against keratins, epithelial membrane antigen (EMA), epithelial antigen (Ber-EP4), carcinoembryonic antigen (CEA), tumour-associated glycoprotein (B72.3), vimentin and LeuM1 was applied to sections of adenocarcinoma of the lung and malignant mesothelioma in a randomized design. The proportion of stained tumour cells within each section was estimated independently in five categories by three pathologists (no positive tumour cells, 1-10%, 11-33%, 34-66% and more than 67% positive tumour cells). The kappa values representing the chance corrected interobserver agreement for the different antibodies in such a five group assessment were between 0.38 and 0.72. In two group assessment the kappa values were between 0.53 and 0.94. Nosological sensitivity and nosological specificity were calculated for all antibodies, and diagnostic sensitivity and diagnostic specificity (predictive values) were calculated for the Ber-EP4, CEA, B72.3, LeuM1 and vimentin. The difference between nosological sensitivity and nosologic specificity and the clinically relevant predictive values of positive and negative tests were demonstrated. In respect of the reproducibility and the diagnostic power defined by the predictive values, we demonstrated that a panel of antibodies, including CEA, Ber-EP4 and B72.3 and, to a lesser degree, LeuM1 and vimentin is applicable for the histopathological distinction between adenocarcinoma of the lung and malignant mesotheliomas. Before introduction of new diagnostic tests, including new antibodies, the prevalence of the tested tumours should be estimated. Nosological sensitivity and nosological specificity should be converted to predictive values.

Human protein HC (alpha 1-microglobulin) and inter-alpha-trypsin inhibitor in connective tissue.

The presence of protein HC (alpha 1-microglobulin) and inter-alpha-trypsin inhibitor was investigated in different human tissues. Inter-alpha-trypsin inhibitor is a complex protein composed of bikunin and two heavy polypeptide chains. Protein HC and bikunin are transcribed from a common gene. Inter-alpha-trypsin inhibitor immunoreactivity was detected in mast cells. The positive reaction could be blocked by antisera absorption with bikunin, indicating that mast cells contain only bikunin. Protein HC immunoreactivity was revealed on elastic fibres in connective tissue of skin, colon and lung, and on the internal elastic lamina of blood vessels. In the testis, the basement membrane of the seminiferous tubules reacted positively with protein HC antibodies.

[Continuous monitoring of pulse and blood pressure among telephone operators]. / Kontinuerlig puls- og blodtryksmåling hos telefonister i arbejde.

The purpose of the study was to evaluate a) whether the monitoring device was usable during ordinary working conditions, b) whether job-strain and self-reported stress influenced the blood pressure and pulse and c) whether there was a correlation between job-strain and self-reported stress. Twenty-six female telephone-operators were monitored during a working day. Job-strain was defined as the number of calls to the telephone operator. Eighty-nine percent of the monitoring measurements were usable. No difference was found between the blood pressure and pulse during rest and during work. A significant negative correlation was found between the number of calls and the blood pressure, and a significant positive correlation between the number of calls and self-reported stress. The device can give information about variations in blood-pressure and pulse during physically quiet work. The results show surprisingly that the quantitative workload is in itself not necessarily a stress factor at work. Perhaps waiting time and unpredictability are of greater importance.

Carcinoid tumor of the middle ear.

Carcinoid tumor of the middle ear is a very rare benign neoplasm, which may be mistaken for a malignant tumor. We present two new cases together with a review of 28 previously reported cases. Nearly all patients had progressive hearing loss, most often of the conductive type. About half of the patients complained of tinnitus and fullness of the ear. The typical otoscopic picture was erythema or lateral bulging of the tympanic membrane, which was intact in most patients. The tumor was most often localized to the middle ear with varying degree of extensions into neighboring areas. It often encapsulated the ossicles, which sometimes were eroded. Systemic symptoms were only reported in two cases. The tumor is clinically benign and total excision of the tumor and affected ossicles is an adequate treatment. The correct diagnosis, which should be considered in case of any adenomatous tumor of the middle ear, requires immunohistochemical and ultrastructural procedures.

Epstein-Barr virus and Hodgkin's disease: a comparative immunological, in situ hybridization, and polymerase chain reaction study.

During recent years numerous studies have demonstrated the presence of Epstein-Barr virus (EBV) in tissues affected by Hodgkin's disease (HD). The percentage of cases with evidence of EBV infection has varied among the different studies, a positive result being highly dependent on the sensitivity of the method employed. In this study three different methods of detecting EBV in 48 cases of 'classical' HD (33 cases of nodular sclerosis and 15 cases of mixed cellularity) were compared: Immunohistochemistry (IH) for detection of latent membrane protein-1 (LMP-1), in situ hybridization (ISH) for detection of Epstein-Barr virus early RNAs (EBER 1 and 2), and polymerase chain reaction (PCR) for detection of a reiterated 110 base-pair EBV genomic sequence of the BamHI region. In 14 cases (29%) Hodgkin's (H) and Reed-Sternberg (RS) cells were positive for LMP-1 using IH, and in 21 cases (44%) positive signals were seen in H-RS cells with EBER 1 and 2 probes using ISH. A few EBER-positive non-malignant lymphocytes were seen in 17 cases. Thirty-two cases (71%) were EBV-positive by PCR. It is concluded that the PCR technique is the most sensitive method for detecting EBV in HD. However, this method cannot provide information about the cellular localization of EBV. ISH with EBER 1 and 2 probes is superior to immunohistochemical detection of LMP-1 with regard to sensitivity. The advantage that the latter two methods have over the PCR techniques is that it is possible to analyse whether the EBV infection occurs in the H-RS cells or in the admixed non-malignant cell population. Furthermore, this study supports the observation that EBV is associated with a considerable number of HD cases.

The histopathological diagnosis of malignant mesothelioma v. pulmonary adenocarcinoma: reproducibility of the histopathological diagnosis.

In a randomized design we examined the interobserver variation in the histopathological diagnosis of adenocarcinoma of the lung and malignant mesothelioma. In three rounds, three pathologists assessed slides from 42 tumours originally diagnosed as adenocarcinomas, malignant mesotheliomas or benign lesions in the pleura. In the first round the assessments were made on haematoxylin and eosin (H & E) stained sections; in the second, on H & E sections plus sections stained with histochemical mucin stains; and in the final round, the diagnoses were made on H & E sections and sections stained with a panel of antibodies against various antigens (cytokeratin, EMA, CEA, Ber-EP4, B72.3, Leu-M1, vimentin and S-100 protein) said to be of value in the differential diagnosis. The overall interobserver agreements for the three rounds were 0.659, 0.802 and 0.817; the kappa values were 0.461, 0.681 and 0.690. It is concluded that differentiation between adenocarcinoma of the lung and malignant mesothelioma should be made on sections stained with H & E and mucin and/or immunohistochemical staining reactions, including antibodies against B72.3, Ber-EP4 and CEA.

Human papillomavirus and Epstein-Barr virus in the etiology of testicular germ cell tumours.

Epidemiological features suggest that the risk of testicular cancer may be related to exposure to unknown infectious agents, including viruses. Therefore a series of twenty specimens of testicular germ cell tumours, including preinvasive carcinoma in-situ, were tested for the presence of DNA sequences of two viruses with known transforming abilities, human papillomavirus (HPV) and Epstein-Barr virus (EBV). The polymerase chain reaction (PCR) technique was used. In none of the 19 successfully amplified samples were DNA sequences of HPV type 16 or type 18 detected. In six cases a faint trace of EBV DNA was revealed in one of two experiments. These samples were examined by immunohistochemical staining with specific antibodies raised against the EBV protein products and in-situ hybridization with specific molecular probes, and were confirmed to be negative. The study indicates that a significant direct involvement of HPV and EBV in human testicular germ cell carcinogenesis is unlikely. However, a putative growth-stimulating role of EBV-transformed lymphocytes, which are frequently present in the stromal tissues of testicular tumours, cannot be excluded.

Human papillomavirus types 11 and 16 detected in nasopharyngeal carcinomas by the polymerase chain reaction.

Most nasopharyngeal carcinomas (NPCs) are of the nonkeratinizing or undifferentiated types, which are consistently associated with Epstein-Barr virus (EBV). The smaller group of highly differentiated, keratinizing NPCs seems to be only infrequently associated with EBV. In order to examine whether these rare tumors were related to another oncogenic virus, the authors used the polymerase chain reaction to examine paraffin-embedded sections of 15 keratinizing NPCs for human papillomavirus (HPV) types 6, 11, 16, and 18 genomic sequences. HPV DNA was found in 4 tumors (1 HPV-11-positive, and 3 HPV-16-positive tumors). None of 23 undifferentiated or nonkeratinizing NPCs harbored HPV DNA. The putatively oncogenic HPV type 16 may thus be involved in the carcinogenesis of some EBV-negative keratinizing squamous cell nasopharyngeal carcinomas.

Nasopharyngeal carcinoma: histopathological types and association with Epstein-Barr Virus.

The polymerase chain reaction was used to examine paraffin-embedded tissues of 37 nasopharyngeal carcinomas (NPC) for Epstein-Barr virus (EBV) genomic sequences. EBV DNA was found in 2/14 keratinising squamous cell (WHO 1) carcinomas and in all of 23 non-keratinising and undifferentiated (WHO 2 and 3) NPC. The study confirms the infrequent association of keratinising NPC and EBV, in contrast with the 100% association of the less differentiated NPCs and the virus. The results may indicate a different carcinogenesis for the WHO 1 NPC subtype.

Neurofibrosarcoma of the intrathoracic vagus nerve. Case report.

A rare case of neurofibrosuarcoma of the intrathoracic vagus nerve in a man with von Recklinghausen's neurofibromatosis is reported. Magnetic resonance imaging accurately demonstrated the tumour and its relations to surgically important structures. Resection, in accordance with general recommendations for such tumours, was successfully performed.

Detection of immunoglobulin light chain mRNA in nodular sclerosing Hodgkin's disease by in situ hybridization with biotinylated oligonucleotide probes compared with immunohistochemical staining with poly- and monoclonal antibodies.

In order to elucidate the origin of the Hodgkin's and Reed-Sternberg cells, the expression of immunoglobulin kappa- and lambda light chain mRNA in 23 cases of nodular sclerosing and two cases of mixed cellularity Hodgkin's disease was examined by in situ hybridization using biotinylated oligonucleotide probes and compared with immunohistochemical staining with mono- and polyclonal antibodies against immunoglobulin kappa- and lambda light chains. No hybridization signals were seen in Hodgkin's or Reed-Sternberg cells in any of the cases. Polyclonal staining with polyclonal anti-immunoglobulin light chain antibodies was seen in Hodgkin's and Reed-Sternberg cells in 12 cases of nodular sclerosis and in two cases of mixed cellularity and with monoclonal antibodies in three cases of nodular sclerosis, but in no cases of mixed cellularity. In all cases, there was polyclonal labelling of plasma cells with both the oligonucleotide probes and the antibodies. In five cases, the Hodgkin's and Reed-Sternberg cells were also stained with one of the B-cell antibodies L26, MB2 or LN1. Lack of mRNA signals in Hodgkin's and Reed-Sternberg cells might indicate that these cells in Hodgkin's disease of the nodular sclerosis subtype are either not B-cell derived or they are early B-cells (precursor B-cells) not yet able to produce immunoglobulin light chain mRNA, at least not at a level detectable by in situ hybridization. Immunohistochemical staining of Hodgkin's and Reed-Sternberg cells, however, with antibodies against immunoglobulin kappa and lambda light chains may be explained by cellular uptake of the light chains, but the difference in reactivity between poly- and monoclonal antibodies cannot be explained at present.