<p>Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer and yearly include 500 000 new cases worldwide. The outcomes for these patients have not been significantly improved over the last decades and the five year survival is still around 50 %. Establishing predictive markers of treatment response will have great impact on the clinical management of this disease.</p><p>The aim of this thesis was to elucidate markers of intrinsic response to radiotherapy and cisplatin. Combining expression patterns of 14 proteins and identifying mutations in the p53 gene, we were able to incorporate both protein and genetic changes to create a predictive model termed Number of Negative Points (NNP). We used the NNP model to statistically calculate the combination of factors that had the best correlation to intrinsic radiosensitivity (IR). We established that a panel of three markers, epidermal growth factor receptor (EGFR), survivin and splice site/missence mutations of p53, had the best correlation to IR (R=0.990, p<0.0001).</p><p>We also conducted gene expression analysis to investigate what genes and gene ontologies that are different between cell lines with varying IR. Furthermore, we wanted to identify key regulator genes with central positions of molecular networks, which were generated from the transcripts included in the deregulated gene ontologies. A transcriptional profile of 28 key regulator genes was generated. Immunoblot analysis supported deregulation at the protein level of three markers implicated from the transcriptional profile. We propose that these proteins, notch1, thrombospondin 1, and pai‐1 are predictive markers of IR.</p><p>Finally, on a subset of cell lines with sensitivity or resistance to cisplatin, we performed gene expression analysis. Markers of intrinsic cisplatin sensitivity (ICS) such as gene ontologies and key regulators of molecular networks were proposed and five genes, APOE, CTNNB1, MMP7, MMP13, and THBS1 were selected for further analysis. Quantitative polymerase chain reaction (qPCR) analysis of these genes in 25 cell lines established that MMP7 (p=0.0013) and MMP13 (p=0.058) are possible predictive markers of ICS.</p><p>The markers of IR and ICS presented here could, if confirmed in a clinical setting, guide clinicians in the choice of treatment and thus give a more individualized and effective therapy for patients with HNSCC.</p>

<p>Idiopathic Sudden Sensorineural Hearing Loss (ISSNHL) is a rapid loss of hearing caused by damage to the cochlea (inner ear) or auditory nerve. Spontaneous recovery has been seen in 32% - 81%. The incidence of the ISSNHL has been estimated to be between 5 and 20 per 100,000 per year. Different theories (infections, vascular catastrophes, immunologic damage or intracochlear membrane break) about the etiology have resulted in different treatment policies. The effect of therapy is difficult to evaluate for a single physician who sees just a few patients annually.</p><p>The aim of the present thesis was to analyze the management and treatment of ISSNHL patients in Sweden with regard to outcome.</p><p>A national database was developed for Sweden with half of all ENT clinics in Sweden participating by submitting a questionnaire for each patient with SSNHL. The questionnaire covered the patient’s background, current disorder, past and family history of different diseases, examinations and treatment. Audiograms at the onset of SSNHL and after three months were requested.</p><p>All results were analyzed using ordinal logistic regression looking for interactions with hearing recovery and remaining hearing loss as dependent variables. Independent of treatment or no therapy heredity for hearing loss (I, II), older age (I, II) and presence of vertigo (II) was significantly associated with negative outcome. 40% of all patients had an MRI or CT, where 3 – 4% had acoustic neuroma. 24% of patients with ISSNHL who had hematological tests taken had one or more pathological findings. Blood screening varied from simple routine tests to a complete analysis with such tests as HSP70, Anti-Neutrophilic Cytoplasmic Antibodies (ANCA) and Borrelia tests. There was no association between any of these laboratory tests and either hearing improvement or remaining hearing loss evaluating the tests separately (I, II) or after categorization in comparison with those who had normal laboratory findings (II). Patients with hearing loss in the mid-frequency region had significantly better odds for hearing improvement compared to the other three frequency regions (low, high and “flat loss”). Almost 60% of patients with ISSNHL were medically treated, of which nearly 90% got corticosteroids. The medication had no association with either hearing improvement or remaining hearing loss. However, patients who were prescribed rest or sick leave had higher odds for hearing improvement regardless of other treatment. Those patients who did not receive any treatment at all also came significantly later to the ENT clinics than those treated medically and consequently had worse prognosis.</p><p><em>Conclusion:</em> There is no standard program for management or treatment of ISSNHL in Sweden. The diagnostic protocol varies. MRI is an underused resource to get specific diagnoses for the condition especially acoustic neuromas. Regardless of pathological findings, treatment is mainly limited to corticosteroids or no medication with no difference in outcome. A randomized placebo controlled study is necessary to evaluate whether there is an effect of corticosteroids on ISSNHL.</p>

The aim of this thesis was two-fold: first, to evaluate four different functional aspects of the speech and language spectrum; oral-motor function, voice, /s/-articulation and phonology in preschool children with tonsillar hypertrophy before and after surgical treatment. The second aim was to investigate weather the outcome of surgery was equal for two surgical techniques; tonsillectomy or tonsillotomy combined with adenoidectomy when necessary. In all included publications (I-IV), 67 children on waiting list for tonsil surgery and randomized to either tonsillectomy (33) or tonsillotomy (34) participated. The children were assessed and audio-recorded within a month before surgery and six months postoperatively. Results were compared to age-matched control groups. In the first study, oral motor function was assessed using the Nordic Orofacial Test-Screening, NOT-S, consisting of a structured interview and a clinical examination. Before surgery, the children in the study group differed in all domains of the structured interview in comparison to age-matched controls and in the clinical examination regarding the parameters deviant lip position and trouble nose-breathing. Postoperatively oral motor functions were normalized in both surgical groups and no differences to age matched controls were observed. In study two, recordings of three sustained vowels (/α , u, i/) and 14 words elicited by picture naming were analysed both perceptually and acoustically. Compared to the controls, significant differences were found in the study group preoperatively with higher ratings on Visual Analogue Scales (VAS) for the voice quality parameters “hyponasality” and “compressed/throaty” and also lower for pitch. Significantly higher values on all studied perturbation measures (jitter, shimmer and Noise to Harmonics Ratio) were found. Regarding center frequencies of formants, the study groups had lower F3 values for /u / and also lower F2 and F3 for / i / compared to age-matched controls. After surgery there were no significant differences between the perceptual ratings of voice quality of the two surgical groups and there were no significant differences between the children in the surgical groups and the corresponding controls. The acoustic analyses showed a decrease in all the measures of perturbation for the study group after surgery with a slight difference between the two surgical groups. The children in the tonsillotomy group had higher shimmer value for /u/ and higher NHR for /α/. In comparison to the older controls significantly higher values were found an all perturbation measures and the difference seen regarding formant frequencies for the /i/-sound in comparison to controls still remained. The significantly lower third formant (F3) of the /u/-sound also remained. When comparing pre- versus postoperative results for the surgical group as a whole, a decrease was found on all perturbation measures postoperatively, however the differences were not statistically significant. A significant increase was found in formant 3 for /α/ and /u/ was found. The material used in the third study were speech samples containing the /s/-sound and elicited by picture naming and sentence repetition. Before surgery the study group was rated to have more indistinct /s/-sounds than agematched controls. The acoustic analyses showed that the study group had lower spectral peak values for the /s/-sound than controls. After surgery the operated children’s /s/-production did not differ perceptually from the older controls, neither as a whole group nor when divided according to surgical methods. Regarding the acoustic analyses however, the study groups differed from the age-matched control group showing that noise duration was longer and the peak location higher in the study groups. In study four, a Swedish phonology test was performed and transcribed phonetically. The transcription of each child was analyzed in terms of phonological processes and categorized into one of six developmental stages according to the model developed by Nettelbladt (1983) and adapted by Sahlén, Reuterskiold-Wagner, Nettelbladt &amp; Radeborg (1999). A majority of the children in the study group (62.7 %) showed a slowed phonological development preoperatively (developmental stages 0-4), compared to the age-matched control group. Postoperatively the children in both surgical groups had improved their phonological skills. However, they were still behind in comparison to age-matched controls and the difference was even larger than before surgery. The results of this thesis project have clinical relevance for both speech and language pathologists (SLP’s) and ear-nose and throat-surgeons (ENT-surgeons). SLP’s must be aware of the potential impact of tonsillar hypertrophy on oral-motor function and the speech and language spectrum to be able to help affected children adequately and ENT- surgeons should include oral motor and speech and language problems as additional indications for tonsillar surgery.

<p>Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. </p><p>In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate.</p><p>In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36.</p><p>In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice.</p><p>In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.</p>

Radioimmunotherapy (RIT) has been shown to be a practicable way to treat head and neck squamous cell carcinoma. A specific antibody recognizes the charasteristic structure of tumour cells when loaded with cytotoxic agents (toxins, drugs, radionuclides, etc). But RIT kills not only tumour cells with attached radionuclides but also adjacent tumour cells due to the “cross fire effect”. To be efficacious, RIT depends closely on suitable monoclonal antibody, on the properties of the chosen radionuclides, and on a suitable labelling method for attaching radionuclide to antibody. In this study we initially used radionuclide-labelled cMAB U36, via linker DABI in order to improve the retention of radio-conjugates in the tumour cells. Improved retention is important because the longer the radionuclide remains in tumour cells, the more effective will the tumour cells be eradicated. In the investigation, both normal mice and HNSCC-bearing nude mice were used to compare our form of treatment against other radio-iodination methods. In the biodistribution study, normal mice showed that radioactive uptake in organs diminished with time, irrespectively of whether the conjugate was directly or indirectly labelled. But in thyroid, there was a tenfold greater accumulation of direct-labelled than of indirectly labelled conjugate. In tumour-bearing nude mice, by contrast, the results showed promising uptake of radioactivity, but little uptake in direct-labelled conjugate in thyroid. Significant differences were observed on comparing tumour: organ ratios between 131I-cMAb U36 vs. 125I-DABI-cMAb U36. In the present study, cMAb U36 labelled with 211Astatine was initially used to treat HNSCC in nude mice. The biodistribution of 211At-cMAb U36 did not reveal any significant difference between an antibody-blocked group and a non-blocked group. But it did highlight the characteristics of a successful targeting conjugate in HNSCC-bearing nude mice. In the subcutaneous therapy experiment, most of the treated tumours (n=18) had disappeared by the 26th day, in both U36-blocked and non-blocked groups. Treatment in the intravenous therapy experiment had also proved effective. In the antibody non-blocked group, the smallest tumour volume was 25 mm3 (average 111 mm3) vis-á-vis 65 mm3 (average 145 mm3) in the blocked group. None of tumours grew again following treatment.

Objective: To follow a cohort of children from age 4-6-12 with respect to sleep disordered breathing (SDB) and orofacial development. Questionnaires were completed about sleep, snoring, apneas, enuresis, sucking habits, and adenotonsillar surgery and, from age 12, about allergies, asthma, and general health. Children snoring regularly had an ENT- examinations including sleep studies (at ages 4 and 12) and an orthodontic evaluation. Development of biometric data in snoring children and not snoring controls was studied in relation to adenotonsillar surgery. Result: Of the original group of 615 children, 509 (83%) participated at age 6 and 393 (64%) at age 12. 27 snored regularly and 231 did not snore at age 12. Differences between groups were seen on all answers. From age 4–12 the prevalence of OSA decreased from 3.1% to 0.8%, and the minimum prevalence of snoring regularly from 5.3% to 4.2%. The odds for a child who snored regularly at four or six to be snoring regularly at age 12 was 3.7 times greater than for a not snoring child in spite of surgery (OR 3.7, 95% CI 2.4-5.7). 63 children were operated for snoring by age 12, of them 14 never snored and 17 snored regularly at age 12. Cross-bite was more common among snoring children at ages 4, 6 and 12 as was a narrower maxilla. In most cases, surgery cured the snoring temporarily, but the maxillar width was still smaller by age 12—even when nasal breathing was attained. Children snoring regularly at age 12, operated or not operated, showed long face anatomy and were oral breathers; the seven cases who were not operated and the five who were still snoring in spite of surgery, did not have reduced maxillary arch width. Conclusion: The prevalence of children snoring regularly is about the same from age four to twelve in a cohort where adenotonsillar surgery has been performed on obstructed cases, but the prevalence of OSA decreases considerably. The children snoring regularly have a more narrow maxilla compared to children not snoring—a condition that is not changed by adenotonsillar surgery regardless of symptom relief.

<p>Acute otitis media (AOM) is a common disease in childhood and is one of the most common causes for outpatient antibiotic treatment. The major aetiological agents of AOM have varied over the decades. Now the three most common pathogens are <i>Streptococcus pneumoniae</i>, <i>Haemophilus influenzae</i> and <i>Moraxella catarrhalis</i>. The resistance patterns of these organisms have also varied from the beginning of the antibiotic era to the situation we have today with an increasing incidence of penicillin-resistant <i>S. pneumoniae</i> and a moderate to high frequency of beta-lactamase production in <i>H. influenzae</i> and <i>M. catarrhalis</i>. In Sweden we have continued to use the Scandinavian treatment policy of penicillins as the first-line antibiotic treatment of AOM, which has been implemented with good results in the past. The question is if this policy will continue to have acceptable treatment results.</p><p>In order to investigate aspects of treatment, protection and structural changes in AOM, an animal model was used.</p><p>Amoxicillin treatment of AOM caused by <i>H. influenzae</i> was studied. Amoxicillin treatment was shown to shorten the duration of the infection and to reduce the morphological changes normally observed after an untreated AOM. The influence of antibiotic treatment on recurrent AOM was evaluated. Amoxicillin treatment did not lead to less protection against reinfection. Abstaining from antibiotics did not improve the levels of serum IgG antibodies. The IgG levels were significantly higher in treated animals after rechallenge. AOM caused by <i>H</i>. <i>influenzae</i> with a non-beta-lactamase-mediated resistance to beta-lactams was investigated and it was observed that during amoxicillin treatment the chromosomal changes mediating resistance were possibly advantageous for the bacterium. In cultures from children with AOM, there is sometimes growth of several bacteria. The possibility of a sheltering effect of beta-lactamase-producing <i>H. influenzae</i> on a penicillin-sensitive <i>S. pneumoniae</i> in a mixed infection was investigated, and amoxicillin was shown to eradicate the pneumococci from the middle ear despite the presence of beta-lactamase. An increasingly cultured bacterium in nasopharynx and in AOM is <i>M. catarrhalis</i>. It is now beta-lactamase-producing in almost 100% of cases and is thus not eradicated by penicillins. An animal model of AOM caused by beta-lactamase-producing <i>M. catarrhalis</i> was established to study the course of this infection with the possibility of evaluating aspects of virulence between AOM pathogens. The AOM observed was a self-limiting disease.</p><p>The results obtained in this study in a rat model support the continuing use of penicillins as first-line drugs in the treatment of AOM. Penicillins are not sufficient to treat all causative agents, but the majority of pathogens including the most virulent bacteria are eradicated from the middle ear. </p>

<p>Our knowledge of the fine structure of the Human Spiral Ganglion (HSG) is still inadequate and new treatment techniques for deafness using electric stimulation, call for further information and studies on the neuronal elements of the human cochlea. This thesis presents results of analyses of human cochlear tissue and specimens obtained during neurosurgical transpetrosal removal of life-threatening meningeomas. The use of surgical biopsies produced a well-preserved material suitable for ultrastructural and immunohistochemical studies on the human inner ear. The SG was studied with respect to fine structure, using TEM technique and the immunostaining pattern of synaptophysin, which is an integral membrane protein of neuronal synaptic vesicles. The immunostaining patterns of the tight junctional protein ZO-1 and the gap junctional proteins Cx26 and Cx43 in the human cochlea were also studied. The ultrastructural morphology revealed an absence of myelination pattern in the HSG, thus differing from that in other species. Furthermore, formation of structural units as well as signs of neural interaction between the type 1 neurons could be observed. Type 1 cells were tightly packed in clusters, sharing the ensheathment of Schwann cells. The cells frequently made direct physical contact in Schwann cell gaps in which membrane specializations appeared. These specializations consisted of symmetrically or asymmetrically distributed filamentous densities along the apposed cell membranes. The same structures were also present between individual unmyelinated nerve fibres and the type 1 cells. Synapses were observed on the type 2 neurons, with nerve fibres originating from the intraganglionic spiral bundle. Such synapses, though rare, were also observed on the type 1 cells. The ultrastructural findings were confirmed by the synaptophysin study. A 3-D model of a Schwann cell gap between two type 1 cells was constructed, describing the distribution pattern of membrane specializations. In the immunohistochemical studies on the human cochlea, ZO-1 was expressed in tissues lining scala media, thus contributing to the formation of a closed compartment system, important for the maintenance of the specific ionic composition of the endolymph. Protein Cx26 could be identified in non-sensory epithelial cells of the organ of Corti, in connective tissue cells of the spiral ligament and spiral limbus, as well as in the basal and intermediate cell layers of stria vascularis. Cx26 in this region may be involved in the recycling of potassium. Protein Cx43 stained weakly in the spiral ligament, but intense staining in the SG may indicate that gap junctions exist between these neurons. A different functional role for the HSG can be assumed from the morphological characteristics and the signs of a neural interaction between the SG cells. This might be relevant for neural processing mechanisms in speech coding and could have implications for cochlear implant techniques.</p>

Acute otitis media (AOM) is a common disease in childhood and is one of the most common causes for outpatient antibiotic treatment. The major aetiological agents of AOM have varied over the decades. Now the three most common pathogens are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. The resistance patterns of these organisms have also varied from the beginning of the antibiotic era to the situation we have today with an increasing incidence of penicillin-resistant S. pneumoniae and a moderate to high frequency of beta-lactamase production in H. influenzae and M. catarrhalis. In Sweden we have continued to use the Scandinavian treatment policy of penicillins as the first-line antibiotic treatment of AOM, which has been implemented with good results in the past. The question is if this policy will continue to have acceptable treatment results. In order to investigate aspects of treatment, protection and structural changes in AOM, an animal model was used. Amoxicillin treatment of AOM caused by H. influenzae was studied. Amoxicillin treatment was shown to shorten the duration of the infection and to reduce the morphological changes normally observed after an untreated AOM. The influence of antibiotic treatment on recurrent AOM was evaluated. Amoxicillin treatment did not lead to less protection against reinfection. Abstaining from antibiotics did not improve the levels of serum IgG antibodies. The IgG levels were significantly higher in treated animals after rechallenge. AOM caused by H. influenzae with a non-beta-lactamase-mediated resistance to beta-lactams was investigated and it was observed that during amoxicillin treatment the chromosomal changes mediating resistance were possibly advantageous for the bacterium. In cultures from children with AOM, there is sometimes growth of several bacteria. The possibility of a sheltering effect of beta-lactamase-producing H. influenzae on a penicillin-sensitive S. pneumoniae in a mixed infection was investigated, and amoxicillin was shown to eradicate the pneumococci from the middle ear despite the presence of beta-lactamase. An increasingly cultured bacterium in nasopharynx and in AOM is M. catarrhalis. It is now beta-lactamase-producing in almost 100% of cases and is thus not eradicated by penicillins. An animal model of AOM caused by beta-lactamase-producing M. catarrhalis was established to study the course of this infection with the possibility of evaluating aspects of virulence between AOM pathogens. The AOM observed was a self-limiting disease. The results obtained in this study in a rat model support the continuing use of penicillins as first-line drugs in the treatment of AOM. Penicillins are not sufficient to treat all causative agents, but the majority of pathogens including the most virulent bacteria are eradicated from the middle ear.

Our knowledge of the fine structure of the Human Spiral Ganglion (HSG) is still inadequate and new treatment techniques for deafness using electric stimulation, call for further information and studies on the neuronal elements of the human cochlea. This thesis presents results of analyses of human cochlear tissue and specimens obtained during neurosurgical transpetrosal removal of life-threatening meningeomas. The use of surgical biopsies produced a well-preserved material suitable for ultrastructural and immunohistochemical studies on the human inner ear. The SG was studied with respect to fine structure, using TEM technique and the immunostaining pattern of synaptophysin, which is an integral membrane protein of neuronal synaptic vesicles. The immunostaining patterns of the tight junctional protein ZO-1 and the gap junctional proteins Cx26 and Cx43 in the human cochlea were also studied. The ultrastructural morphology revealed an absence of myelination pattern in the HSG, thus differing from that in other species. Furthermore, formation of structural units as well as signs of neural interaction between the type 1 neurons could be observed. Type 1 cells were tightly packed in clusters, sharing the ensheathment of Schwann cells. The cells frequently made direct physical contact in Schwann cell gaps in which membrane specializations appeared. These specializations consisted of symmetrically or asymmetrically distributed filamentous densities along the apposed cell membranes. The same structures were also present between individual unmyelinated nerve fibres and the type 1 cells. Synapses were observed on the type 2 neurons, with nerve fibres originating from the intraganglionic spiral bundle. Such synapses, though rare, were also observed on the type 1 cells. The ultrastructural findings were confirmed by the synaptophysin study. A 3-D model of a Schwann cell gap between two type 1 cells was constructed, describing the distribution pattern of membrane specializations. In the immunohistochemical studies on the human cochlea, ZO-1 was expressed in tissues lining scala media, thus contributing to the formation of a closed compartment system, important for the maintenance of the specific ionic composition of the endolymph. Protein Cx26 could be identified in non-sensory epithelial cells of the organ of Corti, in connective tissue cells of the spiral ligament and spiral limbus, as well as in the basal and intermediate cell layers of stria vascularis. Cx26 in this region may be involved in the recycling of potassium. Protein Cx43 stained weakly in the spiral ligament, but intense staining in the SG may indicate that gap junctions exist between these neurons. A different functional role for the HSG can be assumed from the morphological characteristics and the signs of a neural interaction between the SG cells. This might be relevant for neural processing mechanisms in speech coding and could have implications for cochlear implant techniques.