<p>Topical treatment of several inner ear diseases through the round window membrane (RWM) might be feasible in the near future. Bacteria toxins, ototoxic drugs and noise trauma seem to harm the inner ear by a common pathway which involves, excessive outflow of the afferent neurotransmitter glutamate and formation of nitric oxide (NO), which can severely damage cells/nerve endings and lead to cell death.</p><p>In this study we used 98 Sprague-Dawley rats and seven human temporal bones. Various substances were instilled into the middle ear of the rat, such as Pseudomonas Aeruginosa Exotoxin (PaExoA), gentamicin, NO-inhibitor N-Omega-Nitro-L-Arginine Methyl Ester (L-NAME), and glucocorticoids. The effects of the substances were studied by morphological analysis of RWM and the endolymphatic sac (ES) by light and electron microscopic. Hearing level was measured in the rats by ABR technique. The human temporal bones were studied immunomorphologically to search for glutamate.</p><p>In the human inner ear, glutamate receptors and glutamine synthetase, were identified. In the rat, we found, following PaExoA exposure, reversible and permanent hearing loss and morphological changes in the RWM. The ES showed increased numbers of macrophages and thickening of the epithelia. When L-NAME was used as an otoprotector from gentamicin ototoxicity a therapeutic effect in the high frequency area was found. Hydrocortisone (but not dexamethasone) exposure of the RWM resulted in membrane thickening, and adjacent to the membrane, inflammatory cells.</p><p>The importance of the RWM as a portal for toxic substances and topical treatment of inner ear diseases was highlighted in this study. The difficulties of applying drugs in the round window niche were exposed. The results of this study add important knowledge concerning certain mechanisms of inner ear injury and help us to understand possibilities and problems of local treatment of inner ear diseases in patients.</p>

Topical treatment of several inner ear diseases through the round window membrane (RWM) might be feasible in the near future. Bacteria toxins, ototoxic drugs and noise trauma seem to harm the inner ear by a common pathway which involves, excessive outflow of the afferent neurotransmitter glutamate and formation of nitric oxide (NO), which can severely damage cells/nerve endings and lead to cell death. In this study we used 98 Sprague-Dawley rats and seven human temporal bones. Various substances were instilled into the middle ear of the rat, such as Pseudomonas Aeruginosa Exotoxin (PaExoA), gentamicin, NO-inhibitor N-Omega-Nitro-L-Arginine Methyl Ester (L-NAME), and glucocorticoids. The effects of the substances were studied by morphological analysis of RWM and the endolymphatic sac (ES) by light and electron microscopic. Hearing level was measured in the rats by ABR technique. The human temporal bones were studied immunomorphologically to search for glutamate. In the human inner ear, glutamate receptors and glutamine synthetase, were identified. In the rat, we found, following PaExoA exposure, reversible and permanent hearing loss and morphological changes in the RWM. The ES showed increased numbers of macrophages and thickening of the epithelia. When L-NAME was used as an otoprotector from gentamicin ototoxicity a therapeutic effect in the high frequency area was found. Hydrocortisone (but not dexamethasone) exposure of the RWM resulted in membrane thickening, and adjacent to the membrane, inflammatory cells. The importance of the RWM as a portal for toxic substances and topical treatment of inner ear diseases was highlighted in this study. The difficulties of applying drugs in the round window niche were exposed. The results of this study add important knowledge concerning certain mechanisms of inner ear injury and help us to understand possibilities and problems of local treatment of inner ear diseases in patients.

Otitis media (OM), one of the commonest of childhood diseases, causes much suffering. OM exists in a variety of forms, two of which are acute otitis media (AOM) and otitis media with effusion (OME). The clinical courses of these conditions differ, AOM usually presenting with earache, fever and/or aural discharge, and the OME usually with hearing impairment. The tympanic membrane (TM) mirrors the events in the middle ear cavity, and pars flaccida (PF) is the initial site of inflammatory changes in the TM. PF is rich in mast cells (MCs), which by releasing various mediators, may trigger TM inflammation. The aims of the present studies were to investigate early inflammatory changes in the TM in rat models of OM; after mast cell degranulation, in response to AOM, and OME, after myringotomy in AOM and in normal ears. Furthermore, we developed a new rat AOM model, that excludes surgical trauma and resembles the natural route of infection in man. AOM and OME elicited the first inflammatory response in PF of the TM. The response to OME was discrete, but a slight increase in macrophages was found. During the first 48 hours of AOM, the inflammatory response was intense, following a bimodal pattern. This reaction is similar to that found after MC degranulation. In AOM, macrophages were the predominant cell in PF, while in pars tensa (PT), polymorphonuclear cells (mainly neutrophils) predominated. When myringotomy was performed in AOM ears, the healing time was shorter than that of myringotomy in normal ears. The highly inflamed lamina propria seemed to promote healing. During early AOM, as well as following myringotomy, fibrin extravasates into PF and PT. This fibrin deposition may be involved in regulating the inflammatory response. Repeated nasal challenge with the otitis media pathogen Streptococcus pneumoniae provoked AOM and concomitant TM stimulation reduced the number of AOM cases. This new rat AOM model has the advantage of avoiding trauma in the middle ear cavity, while eliciting an intense inflammatory response in the middle ear cavity (MEC).

<p>Antibody-based targeting techniques play an increasingly important role in cancer research. By targeting a structure that is abundant in tumour cells, but rare in healthy tissues, an antibody can mediate the delivery of radioactivity specifically to tumour cells in the body. This idea is particularly appealing for head and neck squamous cell carcinoma (HNSCC), as the advanced stages have a large fraction of spread disease that is difficult to treat with procedures available today. </p><p>In this thesis, we have investigated possible radioimmunotargeting structures for HNSCC, and found that CD44v6 is a suitable target for antibody-based radiotherapy and diagnostics in this patient group. We have identified radiohalogens as attractive nuclides for such use, and have investigated the possibility of radiohalogenating the anti CD44v6 chimeric monoclonal antibody (cMAb) U36. Several feasible labelling methods were identified, using both direct and indirect labelling. The cMAb U36 was then successfully labelled with ²¹¹At and ¹³¹I, and preclinically evaluated for therapeutic use. Results proved the astatinated conjugate to be most efficient in this context, demonstrating a specific and dose-dependent cytotoxicity. The cMAb U36 was then evaluated for diagnostic use in thyroid anaplastic carcinoma, using ¹²⁴I as the diagnostic nuclide. Results in tumour-bearing mice were promising, with all of the tumours identified in micro-PET studies.</p><p>These results demonstrate how antibody-based radionuclide targeting can provide more sensitive and specific methods for identifying and treating head and neck cancer, and hopefully help improve long-term survival rates for this patient group in the future.</p>

<p>Monogenic disorders offer a possibility for studies of genetic disturbances in hearing impairment—a knowledge which could be essential for development of future treatment options. In this thesis, the underlying genetic disturbances in neurofibromatosis 2 (NF2) and familial Meniere’s disease (FMD) were evaluated, and familial X-linked hearing impairment was described from a clinical point of view. </p><p>In paper I, constitutional DNA from 116 individuals with NF2 of variable severity was studied using the array-CGH method focusing on a 7.6-Mb area surrounding the NF2 gene on chromosome 22q. Deletions were found in 20.7% of samples. In mild NF2, the deletions were small, but variable sizes of deletions were found in cases that were moderately or severely affected. Disease phenotype could not be predicted from the size of the deletions.</p><p>In papers II and III, a single five-generation family with autosomal dominant FMD was described. Anticipation concerning age of onset was observed. Genome scan revealed five candidate gene regions with a LOD score of > 1. Two additional families with autosomal dominant MD were analyzed for linkage to these five regions. A cumulative Zmax of 3.46 was obtained for a single 463-kb region on chromosome 12p12.3, containing only one known gene: PIK3C2G. This encodes a protein with a proposed role in hair cell regeneration in mammalian ears. No mutations were found in protein-coding sequences or exon-intron borders. In two of the three families, a shared haplotype, suggested common ancestry, was found to extend over 1.7 Mb, which could be a genomic region of importance for FMD.</p><p>In paper IV, a family in which five males displayed progressive low- and mid-frequency hearing impairment from the first or second decade was described. Female carriers were affected by a high-frequency hearing impairment from the fourth decade. The family could represent a novel X-linked dominant audiophenotype.</p>

<p>The diagnosis and treatment of patients with advanced tumors in the head and neck is an interesting challenge where there is a need for new approaches in diagnostics and adjuvant treatment. Differences in antigen expression between tumors and normal tissues provide a means for application of antibody-based targeting techniques. By targeting a structure that is abundant on tumor cells and limited on normal cells, radioactivity can be delivered.</p><p>The use of positron emission tomography (PET) in patients with head and neck tumors is evaluated in this thesis. PET using the tracer fluorodeoxyglucose (FDG) is found to play an important diagnostic role and often has a direct clinical impact on planned surgery or other treatment. Possible targeting structures are also investigated in this thesis, and it is concluded that the EGFR and CD44v6 stand out as possible antigens for targeting approaches of squamous cell carcinomas in the head and neck (HNSCC). A radioimmunoassay for quantification of EGFR and CD44v6 is validated and concluded to be a valuable complement to immunohistochemistry for the analysis of tumors and for the planning of radioimmunotherapy. Finally, promising results of radioimmunotherapy in tumor bearing mice with the monoclonal antibody U36 labeled with the alpha emitter astatine-211 are presented.</p><p>These results demonstrate how differences between tumors and normal tissues can be used to improve diagnostic outcomes and indicate that radioimmunotherapy can be a future adjuvant therapy or treatment of residual disease in HNSCC.</p>

The diagnosis and treatment of patients with advanced tumors in the head and neck is an interesting challenge where there is a need for new approaches in diagnostics and adjuvant treatment. Differences in antigen expression between tumors and normal tissues provide a means for application of antibody-based targeting techniques. By targeting a structure that is abundant on tumor cells and limited on normal cells, radioactivity can be delivered. The use of positron emission tomography (PET) in patients with head and neck tumors is evaluated in this thesis. PET using the tracer fluorodeoxyglucose (FDG) is found to play an important diagnostic role and often has a direct clinical impact on planned surgery or other treatment. Possible targeting structures are also investigated in this thesis, and it is concluded that the EGFR and CD44v6 stand out as possible antigens for targeting approaches of squamous cell carcinomas in the head and neck (HNSCC). A radioimmunoassay for quantification of EGFR and CD44v6 is validated and concluded to be a valuable complement to immunohistochemistry for the analysis of tumors and for the planning of radioimmunotherapy. Finally, promising results of radioimmunotherapy in tumor bearing mice with the monoclonal antibody U36 labeled with the alpha emitter astatine-211 are presented. These results demonstrate how differences between tumors and normal tissues can be used to improve diagnostic outcomes and indicate that radioimmunotherapy can be a future adjuvant therapy or treatment of residual disease in HNSCC.

Tonsillectomy is one of the most frequently performed surgical procedures in children and youths. The aim of this thesis was to study children and youths in relation to tonsil surgery with the goal of improving the care, and to describe partial tonsillectomy/tonsillotomy (TT) using radiofrequency technique (RF) (Ellman International) in comparison with the more commonly used total tonsillectomy (TE). The thesis covers studies of wo age-groups with obstructive problems, with or without recurrent tonsillitis. Randomization to surgery was done from the existing waiting list; 92 children, 5-15 years old to 49/TT and 43/TE, (I-III) and 76 youths, 16-25 years old to 32/TT and 44/TE (IV-V). The first purpose (I, IV) was to compare the two surgical techniques with respect to pain and postoperative morbidity. Pain measures were for the children the Face Pain Scale and for the youths and parents and staff a verbal-pain-rating-scale. From the first day, the TT-groups scored significantly less pain than the TE-groups. The doses of pain-killing drugs (paracetamol and diclofenac) taken were significantly less for the children and youths receiving the TT-surgery, they could stop taking pain-killers sooner, and were back to normal activity three (5-15yrs) or four (16-25yrs) days earlier compared with TE-groups. Paper II focused on the child’s behavior (Child Behavior Checklist/CBCL), experience of pain, anxiety (State-Trait-Anxiety Inventory for Children /STAIC), previous experiences of surgery/tonsillitis, and the management of pain. The children scored higher on CBCL than a normative group before surgery, but no connection was observed between CBCL rating and experience of pain reported post surgically. There was no relation between preoperative anxiety and reported pain, but the postoperative anxiety level correlated with pain. The Egroup scored higher anxiety after surgery. Previous experience of surgery or tonsillitis did not influence the postoperative pain. The nurses scored pain lower than the parents/children and under-medicated. The second purpose was to compare the long-term effects of TT and TE-surgery after one and three years (5-15yrs) and one year (16-25yrs) (III, IV). The effect on snoring was the same for both TT and TE-groups and the rate of recurrence of throat infections was low after both surgical techniques. After one year, all children (TT/TE) showed improvements on CBCL to the same degree and there was no longer a difference between total behavior and normative values. They also scored improvements in health-related quality of life (HRQL) with Glasgow-Children-Benefit-Inventory. For both TT and TE, the older group reported lower HRQL preoperatively on all dimensions of Study-Short-Form (SF-36) compared with a normal population. After one year, a large improvement was found in HRQL in both groups and there were no differences compared with a normal population. Conclusion: Preoperative obstructive problems, in combination with recurrent tonsillitis have a negative impact on HRQL. Both after TE and TT there are large improvements in HRQL, infections, obstructive, and behavior problems one to three years after surgery, indicating that both surgical methods are equally effective. With fewer postoperative complications, less pain, shorter recovery time, and lower cost, TT with RF should be considered as method of choice.

Antibody-based targeting techniques play an increasingly important role in cancer research. By targeting a structure that is abundant in tumour cells, but rare in healthy tissues, an antibody can mediate the delivery of radioactivity specifically to tumour cells in the body. This idea is particularly appealing for head and neck squamous cell carcinoma (HNSCC), as the advanced stages have a large fraction of spread disease that is difficult to treat with procedures available today. In this thesis, we have investigated possible radioimmunotargeting structures for HNSCC, and found that CD44v6 is a suitable target for antibody-based radiotherapy and diagnostics in this patient group. We have identified radiohalogens as attractive nuclides for such use, and have investigated the possibility of radiohalogenating the anti CD44v6 chimeric monoclonal antibody (cMAb) U36. Several feasible labelling methods were identified, using both direct and indirect labelling. The cMAb U36 was then successfully labelled with 211At and 131I, and preclinically evaluated for therapeutic use. Results proved the astatinated conjugate to be most efficient in this context, demonstrating a specific and dose-dependent cytotoxicity. The cMAb U36 was then evaluated for diagnostic use in thyroid anaplastic carcinoma, using 124I as the diagnostic nuclide. Results in tumour-bearing mice were promising, with all of the tumours identified in micro-PET studies. These results demonstrate how antibody-based radionuclide targeting can provide more sensitive and specific methods for identifying and treating head and neck cancer, and hopefully help improve long-term survival rates for this patient group in the future.

The healing of tympanic membrane (TM) perforations is a complex wound healing process including inflammation, migration of keratinocytes and tissue remodelling. Most TM perforations in human heal spontaneously, however some perforations become chronic, and the reason to why is still largely unknown. In cutaneous wound healing plasminogen (plg) has been shown to play an important role. Plg is converted into the protease plasmin regulated by two plasminogen activators (PA), urokinase type PA (uPA) and tissue-type PA (tPA). The aim of the present thesis was to evaluate the role of plg in healing of TM perforations, both in vivo and in vitro. The main objectives were to determine the healing capacity of the TM, the involvement of keratinocytes, fibrin(ogen) and inflammatory cells in the healing process. The studies were performed in plg deficient and uPA deficient mice, with littermate wild type (wt) mice as controls It was shown that myringotomies of the TMs in plg deficient mice still remained open 143 days following a perforation. The wound area was characterized by an abundant recruitment and accumulation of inflammatory cells; mainly macrophages and neutrophils, an arrested keratinocyte migration and a fibrin deposition covering the surface of the TM. The TM perforations in the wt mice all healed within 11 days. Interestingly, the myringotomies of the plg deficient mice could be closed by reconstitution with systemic injections of plg, whereas injections of PBS had no affect on the healing. To characterize mechanisms involved in the development of persistent TM perforations in plg deficient mice after a myringotomy the early inflammatory response during the first 48 hours was studied. The recruitment and accumulation of inflammatory cells in the perforated TMs was found to be similar between the plg deficient and the wt mice. Myringotomized TMs in uPA deficient mice healed similar to perforations of wt controls. Neither did the keratinocyte migration nor the occurrence of inflammatory cells differ between these genotypes. In the in vitro experiments TMs from plg deficient and wt mice, were dissected out, perforated and cultured in absence or surplus of plg. A decrease in perforation size was seen in all groups regardless of genotype or amount of plg in the medium. In conclusion, the present studies show: • Plg is essential for the healing of TM perforations in mice. • The altered healing process after a myringotomy in plg deficient mice involves a disturbed keratinocyte migration, a massive deposition of fibrin and an abundant accumulation of inflammatory cells in the wound area. • Plasminogen deficiency does not alter the early inflammatory response, following a myringotomy. • Deficiency of uPA does not influence the healing of TM perforations. • During in vitro conditions healing of TM perforations is initiated irrespectively of genotype of the explant (plg deficient or wt) or supply of plg. The increased knowledge of the involvement of plg in the healing of TM perforations may open therapeutical possibilities in the treatment of chronic TM perforations in humans.