Sino-nasal cancer in Denmark 1982-1991--a nationwide survey.

Cancer of the nasal cavity and paranasal sinuses is a rare disease. The many different histologies and sites make the management of this disease a challenge. The current report from the Danish Society for Head and Neck Oncology comprises a joint analysis of five retrospective series covering the entire country, with 315 patients seen in the 10-year period from 1 January 1982 to 31 December 1991. Tumour sites were nasal cavity (n = 156), maxillary sinus (n = 139), ethmoid sinus (n = 14), sphenoid sinus (n = 5) and frontal sinus (one case). The most common histologies included squamous cell carcinoma (126 cases), adenocarcinoma (41 cases), malignant melanoma (38 cases) and malignant lymphoma (34 cases). A total of 284 patients (90%) received treatment with curative intent; most of these patients were treated with radiotherapy, either alone (120 patients) or in combination with surgery (111 patients). There was no significant difference between the five centres in disease specific survival and overall survival. The results showed that histology, localization and nodal involvement were significant prognostic factors for locoregional control and survival. Patients with squamous cell carcinoma had a significantly poorer prognosis compared with patients with adenocarcinoma. However, a Cox multivariate analysis revealed that this was likely the result of tumour localization, as most adenocarcinomas were in the nasal cavity. The experience from this data collection has inspired the Danish Society for Head and Neck Oncology to arrange common data registration of several other clinical head and neck series. In the future, the Society plans to expand this activity further.

New technology for regiospecific covalent coupling of polysaccharide antigens in ELISA for serological detection.

In this study we demonstrate a new UV irradiation technique for covalent coupling of bacterial polysaccharides derived from lipopolysaccharides to microtiter plates and the use of such plates in an enzyme linked immunosorbent assay (ELISA). Lipopolysaccharides were cleaved by mild acid hydrolysis into the lipid A part and the polysaccharide part. The polysaccharide was conjugated regiospecifically to a photochemically active compound, anthraquinone, resulting in a polysaccharide-anthraquinone conjugate. Anthraquinones forms active radicals when exposed to soft UV irradiation (350 nm) permitting the formation of stable covalent bonds to polymers e.g. microtiter plates. By this technique the polysaccharides are bound through the anthraquinone part of the polysaccharide-anthraquinone conjugates to the microtiter plates. This minimizes denaturation of O-antigen epitopes during binding to the microtiter plates and avoids cross-reactivity due to conserved domains in the lipid A. Furthermore, the covalent binding of the polysaccharide antigens are compatible with harsh assay conditions, such as extensive washing procedures and buffers with high salt concentrations with no risk of antigen leakage. Here we describe the use of this technique for the immobilization of lipopolysaccharide derived polysaccharides from Salmonella Typhimurium and Salmonella Choleraesuis lipopolysaccharides, representing the O-antigens 1, 4, 5, 6, 7, and 12. The functional polysaccharide surface gave similar ELISA results to plates coated passively with the corresponding unmodified lipopolysaccharide antigens. The plates were highly reproducible, showed very low inter- and intra-plate variation and were stable at room temperature for more than 8 months.

Photochemical immobilization of anthraquinone conjugated oligonucleotides and PCR amplicons on solid surfaces.

Ligand immobilization on solid surfaces is an essential step in fields such as diagnostics, bio sensor manufacturing, and new material sciences in general. In this paper a photochemical approach based on anthraquinone as the chromophore is presented. Photochemical procedures offer special advantages as they are able to generate highly reactive species in an orientation specific manner. As presented here, anthraquinone (AQ) mediated covalent DNA immobilization appears to be superior to currently known procedures. A synthetic procedure providing AQ-phosphoramidites is presented. These reagents facilitate AQ conjugation during routine DNA synthesis, thus enabling the AQ-oligonucleotides to be immobilized in a very convenient and efficient manner. AQ-conjugated PCR primers can be used directly in PCR. When the PCR is performed in solution, the amplicons can be immobilized after the PCR. Moreover, when the primers are immobilized prior to the PCR, a solid-phase PCR can be performed and the amplicons are thus produced directly on the solid support.

A quantitative enzyme-linked immunoassay for the detection of 2, 6-dichlorobenzamide (BAM), a degradation product of the herbicide dichlobenil.

2,6-Dichlorobenzamide (BAM) is the dominant degradation product in soil of the widely used herbicide dichlobenil. To detect BAM in water, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed. As an alternative to conventional coating of ELISA plates, the assay is based on direct covalent immobilisation. We achieved a surface which requires a short time for the immobilisation of ligand, is stable under dry storage, and which permits assays with a low CV. The performance of the assay was demonstrated by an inter-well CV that was generally less than 6%, a detection limit (DL(15)) of 0.02 microg/l and an IC(50) of 0.19 microg/l. Cross-reactivity was measured against nine analytes with structural homology to BAM. The highest degree of cross-reactivity (10.8%) was seen with 2,6-dichlorothiobenzamide (Chlorthiamid). Considering an EU-limit of 0.1 microg/l as the permissible maximum for the presence of pesticides in drinking water, this ELISA-procedure is suitable for large-scale screening of water samples suspected of being contaminated with BAM.

Evaluation of a novel enzyme-linked immunosorbent assay for detection of antibodies against Salmonella, employing a stable coating of lipopolysaccharide-derived antigens covalently attached to polystyrene microwells.

Polysaccharides derived from Salmonella typhimurium lipopolysaccharide (LPS) representing the O-antigen factors 1, 4, 5, and 12 and the O-antigen factors 6 and 7 from Salmonella choleraesuis LPS were derivatized with the photoreactive compound anthraquinone and subsequently covalently coupled to microtiter polystyrene plates by ultraviolet irradiation. Both polysaccharide antigens could be coupled simultaneously to the same microtiter plate. The coated surface was used in indirect ELISA for the determination of serum antibodies from pigs infected with bacteria of the two Salmonella groups and from uninfected pigs. This ELISA proved itself by having a good long-term durability and a high degree of reproducibility, including low day-to-day variations and low interplate variations. Furthermore, the ELISA showed good specificity and sensitivity when data were compared with the optical density levels of a panel of pig sera as determined by a conventional ELISA on the basis of passive coating of the two Salmonella LPS antigens (the mix-ELISA). The covalent anthraquinone mix-ELISA shows promise as a stable and durable alternative to the existing conventional ELISA for serological surveillance of Salmonella infections in pigs.

Detection of the factor V Leiden mutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids.

BACKGROUND: Individuals carrying the factor V Leiden mutation have been shown to have an increased risk of developing venous thromboembolism. Our aim was to develop an ELISA-like assay to detect the mutation in PCR-amplified genomic DNA using novel, high-affinity DNA analogs, termed locked nucleic acids (LNAs). METHODS: LNA octamer probes complementary to the factor V wild-type or mutated sequence were covalently attached to individual wells of a microtiter plate. Biotinylated factor V amplicons were added, and hybridization to the immobilized LNA probes was scored colorimetrically using a horseradish peroxidase-anti-biotin Fab conjugate and tetramethylbenzidine substrate. RESULTS: In a prospective study of 53 patients, the assay reproducibly scored both factor V homozygotes and heterozygotes with excellent sensitivity and specificity. All results were in complete agreement with the results obtained with the conventional PCR-restriction fragment length polymorphism technique. CONCLUSIONS: The simplicity of the assay and its procedural relatedness to the widely used ELISA format should make it useful for routine factor V testing in the clinical laboratory.

Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family.

Mucin-type O-glycosylation is initiated by a large family of UDP-GalNAc: polypeptide N -acetyl-galactosaminyltransferases (GalNAc-transferases). Individual GalNAc-transferases appear to have different functions and Northern analysis indicates that they are differently expressed in different organs. This suggests that O-glycosylation may vary with the repertoire of GalNAc-transferases expressed in a given cell. In order to study the repertoire of GalNAc-transferases in situ in tissues and changes in tumors, we have generated a panel of monoclonal antibodies (MAbs) with well defined specificity for human GalNAc-T1, -T2, and -T3. Application of this panel of novel antibodies revealed that GalNAc- transferases are differentially expressed in different cell lines, in spermatozoa, and in oral mucosa and carcinomas. For example, GalNAc-T1 and -T2 but not -T3 were highly expressed in WI38 cells, and GalNAc-T3 but not GalNAc-T1 or -T2 was expressed in spermatozoa. The expression patterns in normal oral mucosa were found to vary with cell differentiation, and for GalNAc-T2 and -T3 this was reflected in oral squamous cell carcinomas. The expression pattern of GalNAc-T1 was on the other hand changed in tumors to either total loss or expression in cytological poorly differentiated tumor cells, where the normal undifferentiated cells lacked expression. These results demonstrate that the repertoire of GalNAc-transferases is different in different cell types and vary with cellular differentiation, and malignant transformation. The implication of this is not yet fully understood, but it suggests that specific changes in sites of O-glycosylation of proteins may occur as a result of changes in the repertoire of GalNAc-transferases.

Interaction between haemoglobin and synthetic peptides of the N-terminal cytoplasmic fragment of trout band 3 (AE1) protein.

Two acidic peptides corresponding to the first 10 and 20 amino acid residues of the N-terminal, cytoplasmic fragment of rainbow trout band 3 (AE1) protein were synthesised in order to study their interaction with trout and human haemoglobin (Hb). The peptides did not influence the oxygen affinity of the main anodic trout Hb component (Hb IV) when tested at surplus peptide concentration ([peptide]/[Hb4]=16), at high and low ionic strength and at pH values ranging from 6.5 to 7.6. With human Hb, however, the 20-mer peptide markedly decreased the oxygen affinity and increased the Bohr effect. These data suggest that the trout band 3 peptide binds preferentially to the deoxy (T) conformation of human Hb, probably at the organic phosphate binding site in the central cavity between the beta-chains, which is known to be the binding site for the acidic N terminus of human band 3. In trout Hb IV, the presence of negatively charged Asp at position NA2 of the beta-chains (in contrast to positive or neutral residues in mammalian Hb) may weaken any interaction with the highly negatively charged peptides.

Conjugation to preadsorbed preactivated proteins and efficient generation of anti peptide antibodies.

A solid phase conjugation method is described based on the preadsorption of proteins to aluminium hydroxide adjuvant followed by activation of the adsorbed carrier proteins with iodoacetic acid N-hydroxysuccinimidester or other conjugation reagents. Cysteine-containing peptides were coupled to the iodoacetic acid-activated carrier-adjuvant particles through their SH groups. No dialysis is required since the reaction product is isolated at each step of the procedure by a simple centrifugation and can easily be extensively washed between individual manipulations. The method generates peptide-carrier-adjuvant particles with sterically defined presentation of the peptides at the surface of the particles. When used for immunization of mice and rabbits the conjugates elicited high-titered specific anti-peptide sera, which reacted well with the parent protein in ELISA. The strongest reactions were with the denatured form of the parent protein. On immunoblots antisera to the N- and C-terminus of calreticulin recognized the same M, 52,000 protein.

Cancer of the nasal cavity and paranasal sinuses. Prognosis and outcome of treatment.

A retrospective study of 121 patients, 77 men and 44 women, with sino-nasal cancer, admitted to the National University Hospital, Rigshospitalet, during the period 1983 1993, is presented. The median follow-up time was 21 months, (range 3 124). Forty-six percent of the tumors originated from the nasal cavity, 29% from the maxillary sinuses and 5% from the ethmoid sinuses. In 18% of the cases, the site of origin was not clear due to advanced local growth. Sixty-five patients received primary radiation therapy with curative intention of whom 5 underwent secondary surgery. Forty-nine patients underwent primary surgery, 38 of them received postoperative radiation therapy. The overall 5-year survival rate in this material was 35% and the disease-specific 5-year survival was 45%. Patients with well-differentiated squamous cell carcinomas had a significantly higher 5-year survival rate than patients with poorly differentiated carcinomas and patients with regional metastases had a significantly poorer 5-year survival than patients without. The 5-year local control was 48% (41/121). Six of 9 patients with regional metastases at admission were controlled locally, whereas 16 patients developed regional metastases after primary treatment.

[Peritonsillar abscess treated with puncture and aspiration--a prospective 3-year follow-up]. / Peritonsillaer absces behandlet med punktur og aspiration--en prospektiv treårsopgørelse.

Forty-one selected patients were treated with needle aspiration for peritonsillar abscess in our department in 1988. Two patients were treated with quinsy tonsillectomy secondary to needle aspiration, because of failure of treatment. The follow-up group consisted of 39 patients and the median follow-up time was 38 months (range 36-44 months). There were three recurrences of the abscess, and one patient had recurrent tonsillitis subsequent to discharge. The rate of recurrent tonsillitis/abscess among patients younger than 30 and 40 years was respectively 21% (4/19) and 14% (4/28). The rate in the corresponding older age groups was 0%. The overall recurrence rate was 10% (4/39). Needle aspiration is suggested as part of a selected strategy of treatment.

Multiple column peptide synthesis, Part 2 (1, 2).

A manually operated apparatus for parallel multiple column soli-phase peptide synthesis is described. It employs Fmoc-amino acid-O-Dhbt or -Pfp esters in the continuous flow version of the polyamide method on small packed columns of kieselguhr supported resin in a reaction block of Teflon. The solvents and deprotecting reagents are dispensed from two washers in a parallel fashion and reagent consumption is low. Activated and protected amino acids are transferred from a dispenser tray as solutions, eight at a time. The use of the method is demonstrated by the synthesis of overlapping peptides from a protein structure and of analogous protease substrates. The products have been characterized by HPLC, FAB mass spectroscopy and amino acid analysis.

C-terminal amidation of calcitonin by carboxypeptidase Y catalyzed transpeptidation with a photocleavable nucleophile.

The C-terminal amidation of calcitonin represents an important technological problem. A method using a serine carboxypeptidase-catalyzed transpeptidation reaction in combination with photochemical cleavage to give the warranted peptide amide is described. The overall yield is higher than 95%.

[Peritonsillar abscess treated with puncture and aspiration]. / Peritonsillaer absces behandlet med punktur og aspiration.

Out of 48 patients treated for peritonsillar abscess in our department in 1988, 45 were treated with needle aspiration. In 43 of these the aspiration was positive. More than one aspiration was only required in seven patients, no patient was needle aspirated more than twice. Five patients were treated with abscess tonsillectomy secondary to needle aspiration. In two cases the indication may have been failure of treatment. No complications were recorded in relation to aspiration. Followup time was seven months (median). There were two cases of recurrence of the abscess and five patients subsequently had one episode of tonsillitis after discharge. Needle aspiration is suggested as part of a selected strategy of treatment for peritonsillar abscess.