[The NTP in allergy research : open questions regarding nasal provocation tests using allergens]. / Der NPT in der allergologischen Forschung : Offene Fragen bei der nasalen Provokationstestung mit Allergenen.

The nasal provocation test (NPT) is a simple procedure with high specificity and sensitivity that is used in the investigation of allergic and nonallergic diseases. Uniform standards are of particular importance in the clinical setting and for the comparability of clinical and basic allergy research. These standards should cover the composition, dosage and pharmacological formulation of provocative substances (e.g. allergen extracts), the necessity of titration, allergen application methods and the evaluation criteria for a positive NPT reaction. Detection of various mediators and cytokines in nasal discharge can be very useful in the late phase reactions. NPT finds specific applications in studies of local IgE secretion in the nasal mucosa, the diagnosis of analgesic intolerance and in assessments of the efficacy of specific immunotherapies. Additional parameters warranting further evaluation include provocation with cold dry air in nasal hyperreactivity patients and nasal nitric oxide formation. Determination of nasal blood flow during NPT provides an additional clinical parameter.

Eicosanoid imbalance correlates in vitro with the pattern of clinical symptoms of Samter's triad.

BACKGROUND: Hypersensitivity to non-steroidal anti-inflammatory drugs (NSAIDs) is often associated with chronic rhinosinusitis (CRS), nasal polyps (CRSwNP) and asthma, together known as Samter's triad. The disease is characterised by eicosanoid imbalance. In our study, we determined clinical and laboratory parameters in respect of three groups of patients: 1) CRSwNP, 2) CRSwNP and asthma (CRSwNP-A), and 3) CRSwNP with asthma and NSAID-triggered hypersensitivity (CRSwNP-AA). Our main goal was to improve the characterisation of the stages of development in Samter's triad, pointing to the homogeneous or heterogeneous course of disease. METHODOLOGY: Forty-three patients (10 CRSwNP, 14 CRSwNP-A, 19 CRSwNP-AA) and 10 control subjects were included in the study. Nasal assessment using the CRS visual analogue score, endoscopy- and computer tomography scores, allergy tests, analysis of sinus surgeries, asthma severity and in vitro functional eicosanoid tests (FET) with peripheral blood leucocytes were performed. RESULTS: The scores reflecting CRS symptoms such as nasal congestion, nasal discharge and smell impairment differed between the patients groups reflecting the severity of disease (CRSwNP-AA > CRSwNP-A > CRSwNP). Eicosanoid imbalance correlated with nasal congestion, nasal discharge and loss of smell. CONCLUSION: The data presented support the hypothesis of the continuous development of NSAID-triggered hypersensitivity, culminating in Samter's triad.

[Diagnostics and exclusion of hereditary angioedema : a standarized approach for the practice]. / Diagnostik und Ausschluss des hereditären Angioödems : Ein standardisierter Ansatz für die Praxis.

The differentiation between mast cell mediator-mediated and bradykinin-mediated forms of angioedema can be difficult. Bradykinin-mediated hereditary angioedema is a rare autosomal dominant hereditary disease which is characterized by recurrent edema attacks of varying magnitude. The edema occurs in the skin and mucous membranes and can be temporarily disfiguring, very painful and life-threatening by attacks in the laryngeal region. Because of the multitude of differential diagnoses, a final diagnosis is only achieved after an average duration of more than 10 years. The anamnestic and laboratory diagnostic algorithm presented here is designed to assist a simpler differentiation of the various forms of angioedema and to reach the correct diagnosis more quickly.

[Conservative therapy of the chronic rhinosinusitis]. / Konservative Therapie der chronischen Rhinosinusitis.

Chronic inflammation of nasal mucosa and sinuses are of increasing prevalence. Patients suffering from chronic rhinosinusitis (CRS) are characterised by nasal obstruction and secre- tion, impaired sense of smell, head and facial pain, causing impact on quality of life as well as tremendous socioeconomic effects. Therefore, effective and specific diagnostics as well as therapies are essential, which have to be selected from state of the art, evidence based guidelines. According to EP3OS guidelines from 2007 the CRS is defined as chronic inflammation of the nose and nasal sinuses, with or without nasal polyps (CRSwNP/CRSsNP). Upon shown diagnostic criteria of CRS first choice of therapy should be topical glucocorticoids causing anti-inflammatory and curative effects. Improvement of nasal symptoms can be achieved by hypertonic salt solutions. Median to severe symptoms of CRSsNP might be improved by longterm-treatment with oral macrolides. Patients suffering additionally from allergies will benefit from antihistamines, whereas those suffering from analgesic-intolerance (AI) will show improvement upon adaptive desensitisation. Leukotriene receptor antagonists, anti-IgE-antibodies (Omalizumab) or anti-interleukin-5- antibodies (Mepolizumab) are new therapeutic options for the treatment of CRS. This paper reviews recent pharmacologic and non-pharmacologic therapeutic options for conservative treatment of CRS. In addition, evidence based therapeutic options of CRS treatment are evaluated.

[Analgesic intolerance (AI). Key position of ENT physicians for early detection of this condition]. / Analgetika-Intoleranz. Schlüsselposition von HNO-Arzten bei der Früherkennung des Krankheitsbildes.

The clinical manifestation of analgesic intolerance (AI) is frequently associated with symptoms examined by ear-nose-throat (ENT) specialists. The prevalence of AI is reported in the literature to be 0.6-2.5%. Even though there are no concluding results concerning its pathogenesis, an altered arachidonic acid metabolism is most likely the underlying pathomechanism. The symptoms include chronic rhinosinusitis with nasal polyps, asthma, gastrointestinal ulcers, angioedema, and urticaria. Clinical reactions after ingestion of nonsteroidal anti-inflammatory-drugs (NSAIDs) are often obvious in the progress of disease. In order to initiate early therapy and therefore prevent the progress of disease, the diagnosis of AI should occur before the complete picture of AI is obvious. Adaptive desensitization is currently the single causal therapy. Frequency of endonasal revision surgery is reduced after desensitization; severe asthma and reactions after ingestion of NSAIDs are avoided. ENT specialists are particularly in a key position for early detection of AI.

Eosinophilic nasal polyps are a rich source of eotaxin, eotaxin-2 and eotaxin-3.

INTRODUCTION: The CC-chemokine eotaxin plays a key role in the pathologic mechanism of tissue eosinophilia in nasal polyposis. In this study, we investigated a possible role of eotaxin-2 and eotaxin-3, the recently discovered members of the eotaxin family. METHODS: Nasal polyps from 24 patients (non allergic/allergic/aspirin-intolerant patients) and turbinate tissue from 8 controls were investigated. Chemokine protein content (eotaxin, eotaxin-2, and -3) of tissue homogenates was measured by ELISA. Paraffin sections of samples were stained to determine the extent of eosinophilia. RESULTS: Protein expression of eotaxin, eotaxin-2 and eotaxin-3 was significantly higher in nasal polyps than in controls. There was a direct correlation between the protein concentrations of all three eotaxins. Further, protein levels of all chemokines were significantly correlated to the amount of eosinophilia. In aspirin-sensitive polyps the number of eosinophils was significantly higher than in the other patient groups and they had significantly higher eotaxin, eotaxin-2, and -3 protein levels than non-allergic and significantly higher amounts of eotaxin-3 compared with allergic patients. CONCLUSIONS: Our findings suggest, that all members of the eotaxin family are involved in the pathogenesis of nasal polyposis. The results are more likely indicative of a complex cooperation between all members of the eotaxin family than of a specific role in the development of eosinophilia and nasal polyposis.

[Neuroendocrine tumors of the larynx]. / Neuroendokrine Tumoren des Larynx.

BACKGROUND: The location of neuroendocrine tumors in the larynx (NETL) is atypical and relatively rare. It has become possible to determine increasing numbers of these tumors in recent years owing to improvements in immunohistological methods. Extensive recommendations were made with regard to diagnostics and treatment by the "European Neuroendocrine Tumor Group" in 2004. These recommendations relate mainly to the very much more frequent gastroenteropancreatic neuroendocrine tumors and cannot be applied directly to NET of the larynx. This is why precise scientific investigations of NETL and publications of new cases are important. METHODS: Retrospective review of all laryngeal malignancies treated from 1994 to 2004 revealed a NETL in three of about 1000 patients. These patients were evaluated with regard to clinical symptoms, tumor locations, immunohistological findings and the clinical courses. RESULTS: Evaluation of the patients (incidence 0.23%: average age 58 years: female to male 1:2) revealed a poorly differentiated neuroendocrine carcinoma in two patients and a moderately differentiated neuroendocrine carcinoma in one patient. After surgical treatment and radiochemotherapy, the patients with a poorly differentiated carcinoma survived for seven and 17 months respectively and the patient with a moderately differentiated carcinoma survived for 30 months. The patients investigated showed findings consistent with those of previously published cases with regard to the parameters investigated. CONCLUSIONS: NETL require more extensive staging investigations and a specific treatment adapted to the subtyping. Treatment at specialized centers with publication of individual cases is desirable to extend and deepen knowledge.

Acute trichinellosis increases susceptibility to Giardia lamblia infection in the mouse model.

The intestinal protozoan parasite Giardia lamblia causes diarrhoea in humans and animals. In the present study, we used the C57BL/6 inbred mouse model to assess the impact of a nematode (Trichinella spiralis) infection on the course of a G. lamblia (clone GS/M-83-H7) infection. Acute trichinellosis coincided with transient intestinal inflammation and generated an intestinal environment that strongly promoted growth of G. lamblia trophozoites although the local anti-Giardia immunoglobulin (Ig) A production was not affected. This increased G. lamblia infection intensity correlated with intestinal mast cell infiltration, mast cell degranulation, and total IgE production. Furthermore, a G. lamblia single-infection investigated in parallel also resulted in intestinal mast cell accumulation but severe infiltration was triggered in the absence of IgE. Recently, intestinal mast cells emerging during a G. lamblia infection were reported to be involved in those immunological mechanisms that control intestinal proliferation of the parasite in mice. This anti-giardial activity was assumed to be related to the capacity of mast cells to produce IL-6. However, this previous assumption was questioned by our present immunohistological findings indicating that murine intestinal mast cells, activated during a G. lamblia infection were IL-6-negative. In the present co-infection experiments, mast cells induced during acute trichinellosis were not able to control a concurrent G. lamblia infection. This observation makes it feasible that the T. spiralis infection created an immunological and physiological environment that superimposed the anti-giardial effect of mast cells and thus favoured intestinal growth of G. lamblia trophozoites in double-infected mice. Furthermore, our findings raise the possibility that intestinal inflammation e.g. as a consequence of a 'pre-existing' nematode infection is a factor which contributes to increased susceptibility of a host to a G. lamblia infection. The phenomenon of a 'pre-existing' nematode infection prior to a G. lamblia infection is a frequent constellation in endemic areas of giardiasis and may therefore have a direct impact on the epidemiological situation of the disease.

Immune dysregulation in flea allergy dermatitis--a model for the immunopathogenesis of allergic dermatitis.

BACKGROUND: Flea allergy dermatitis (FAD) is a common skin disease in dogs and can be induced experimentally. It often coexists with other allergic conditions. So far no studies have investigated the quantitative production of cytokine mRNA in skin biopsies and peripheral blood mononuclear cells (PBMC) in flea allergic dogs. OBJECTIVE: The aim of our study was to improve the understanding of the immunopathogenesis of allergic dermatitis as a response to fleabites. MATERIAL AND METHODS: Allergic and non-allergic dogs were exposed to fleas. Before and after 4 days of flea exposure mRNA was isolated from biopsies and PBMC. Production of chymase, tryptase, IL-4, IL-5, IL-13, TNF-alpha and IFN-gamma mRNA was measured by real-time RT-PCR. The inflammatory infiltrate in the skin was scored semi-quantitatively. The number of eosinophils, mast cells (MC) and IgE+ cells/mm2 was evaluated to complete the picture. RESULTS: FAD was associated with a higher number of MC before flea exposure and with a significant increase of eosinophils after flea exposure as compared to non-allergic dogs. The number of IgE+ cells was higher in allergic dogs before and after flea exposure. In allergic dogs mRNA for most cytokines and proteases tested was higher before flea exposure than after flea exposure. After exposure to fleas an increased mRNA production was only observed in non-allergic dogs. In vitro stimulation with flea antigen resulted in a decreased expression of most cytokines in allergic dogs before flea exposure. In contrast, in PBMC, only increased levels of IL-4 and IL-5 mRNA were observed in allergic dogs before flea exposure. However, after flea exposure and additional stimulation with flea antigen the production of mRNA for all cytokines tested was significantly increased in allergic dogs. CONCLUSION: We demonstrated that the response in biopsies and PBMC is different and that FAD is associated with a TH2 response.

PCR-based detection of canine Leishmania infections in formalin-fixed and paraffin-embedded skin biopsies: elaboration of a protocol for quality assessment of the diagnostic amplification reaction.

Diagnosis of the cutaneous form of canine leishmaniosis is mostly performed by histological or immunohistological examination of skin biopsies. In modern histology, the polymerase chain reaction (PCR) has gained increasing importance as a complementary tool to directly demonstrate the presence of parasite DNA in the tissue sections. For the present study, a previously described Leishmania-PCR has been further developed and optimised in view of its practicability for routine histological application. Since formalin-fixation of histological specimens causes partial DNA-destruction, which may hamper diagnostic PCR analysis, primers specific for the highly conserved alpha-actin gene sequences were used to pre-diagnostically assess the isolated sample-DNA for its functionality in a PCR-reaction. This alpha-actin-specific PCR detects DNA from a large variety of mammalian species and thus exhibits relevance for both human and veterinary medical application. A recombinant internal positive control was introduced to monitor possible sample-related inhibitory effects during the amplification reaction. We performed a retrospective evaluative study with 18 formalin-fixed samples from dogs with suspected or proven leishmaniosis. Six samples were PCR-incompatible. In turn, 9 of the other 12 samples were PCR-positive, and immunohistochemical results matched these findings. Based on these technical achievements, the Leishmania-PCR proved to be a valuable tool to complement conventional histological and immunohistological methods for diagnosis of cutaneous leishmaniosis in formalin-fixed, paraffin-embedded skin biopsies.

[Langerhans cell histiocytosis: petrosal remodelling after chemotherapy--case report and review of the literature]. / Langerhans-Zell-Histiozytose: Petrosale Remodellierung nach Chemotherapie--Fallbericht und Literaturübersicht -

We report the case of a child presented by her parents to the ENT outpatient service for swelling of the right temporal bone. The child had a history of recurrent bilateral inflammation of the middle ear. Tympanometry revealed a reduced compliance. Due to conductive hearing loss it was impossible to measure otoacustic emissions. Otherwise a normal ENT status was found. Imaging (MRI/CT) demonstrated bitemporal soft-tissue changes with extensive osseous destruction, but no typical imaging signs of an inflammatory, dysplastic or expansive process. The tentative diagnosis of Langerhans' cell histiocytosis (LCH) made on the basis of the clinical and imaging findings was confirmed by biopsy. After exclusion of disseminated LCH, chemotherapy was initiated, and the child underwent follow-up imaging after 3 months. CT showed clear signs of bitemporal reossification. The case reported here illustrates the problems encountered in diagnosing LCH which may present with unspecific clinical symptoms despite advanced osseous destruction. ENT specialists should be familiar with this very heterogeneous entity and think of LCH especially in children presenting with therapy-refractory otitis media, otitis externa, or mastoiditis in order to ensure a timely diagnosis and to thus improve the chances of successful therapy. Imaging modalities (CT, MRI) have a role in the early diagnosis and follow-up of this disorder.

The role of in vitro-induced lymphocyte apoptosis in feline immunodeficiency virus infection: correlation with different markers of disease progression.

Human immunodeficiency virus infection is characterized by a progressive decline in the number of peripheral blood CD4(+) T lymphocytes, which finally leads to AIDS. This T-cell decline correlates with the degree of in vitro-induced lymphocyte apoptosis. However, such a correlation has not yet been described in feline AIDS, caused by feline immunodeficiency virus (FIV) infection. We therefore investigated the intensity of in vitro-induced apoptosis in peripheral blood lymphocytes from cats experimentally infected with a Swiss isolate of FIV for 1 year and for 6 years and from a number of long-term FIV-infected cats which were coinfected with feline leukemia virus. Purified peripheral blood lymphocytes were either cultured overnight under nonstimulating conditions or stimulated with phytohemagglutinin and interleukin-2 for 60 h. Under stimulating conditions, the isolates from the infected cats showed significantly higher relative counts of apoptotic cells than did those from noninfected controls (1-year-infected cats, P = 0.01; 6-year-infected cats, P = 0.006). The frequency of in vitro-induced apoptosis was inversely correlated with the CD4(+) cell count (P = 0. 002), bright CD8(+) cell count (P = 0.009), and CD4/CD8 ratio (P = 0. 01) and directly correlated with the percentage of bright major histocompatibility complex class II-positive peripheral blood lymphocytes (P = 0.004). However, we found no correlation between in vitro-induced apoptosis and the viral load in serum samples. Coinfection with feline leukemia virus enhanced the degree of in vitro-induced apoptosis compared with that in FIV monoinfected cats. We concluded that the degree of in vitro-induced apoptosis was closely related to FIV-mediated T-cell depletion and lymphocyte activation and could be used as an additional marker for disease progression in FIV infection.

[Alternative methods: development and use of two "in vitro" models for endocrine studies]. / Méthodes alternatives: développement et utilisation de deux modèles'in vitro' pour des études endocriniennes.

Most endocrinological studies are normally performed on several animal groups: one group for the determination of hormonal levels in tissues and plasma under basal conditions, a second group for the same determinations after "in vivo" treatment of the animals (stimulation or inhibition of the endocrine activities) and two additional groups for morphological investigations. Surgical approaches belong not rarely to the "in vivo" treatment of the animals. Moreover, the results from the different animal groups have to be extrapolated. In order to overcome these drawbacks, we have developed two "in vitro" models by means of which we can study on the tissues from the same animal: 1. function and structure of the hypothalamus and 2. function and structure of isolated pituitary cells. By using these models we can considerably reduce the number of animals needed for the studies, replace the "in vivo" by the "in vitro" experiments and refine the methods avoiding, among others, the extrapolation of results.

[Vaginal Streptococcus B colonization in pregnancy]. / Vaginale B-Streptokokken-Kolonisation in der Schwangerschaft.

500 women in late pregnancy (III. trimenon) have been examined for colonization of group B streptococci by means of vaginal swabs. Identification of bacteria was effected with modified Wallerströmtest, CAMP-test and coagglutination. In 19 pregnant women (3.8%) B-streptococci could be found. In most cases type I was predominant, followed by type II and III. One newborn child died with symptoms of early onset disease. In mother and infant the same serotype could be found (III/R). The titre of maternal antibody, performed in B-streptococci ELISA was too low. A carrier with B-streptococci had a stillbirth. There existed a congenital abnormality. Both in mother and infant the serotype Ib/c was present. The titre of maternal antibody in B-streptococci ELISA was lowered.

Relative rates of Aujeszky's disease virus attenuation, as assessed in mice.

Field strains of Aujeszky's disease virus (ADV) were attenuated by heat treatment and serial passage at sub-optimal growth temperatures in chicken embryo fibroblasts (CEF). At chosen passage levels, virus was titrated in cell culture and in mice. For each strain, the pathogenicity was expressed as a mouse lethal index (MLI), defined as the inverse of the log10 (CCID50:LD50). MLIs determined for field strains displayed a wide range of comparatively high values. The attenuation of field strains was accompanied by a rapid fall in MLI values, particularly in the initial stages. Heat-treated ADV attenuated faster than untreated ADV, when passaged at 30 degrees C. Passage at 27 degrees C resulted in considerably accelerated attenuation compared to passage at 30 degrees C, in the case of both untreated and heat-treated ADV. MLIs were determined for attenuated ADV strains that had been tested in 6-day-old piglets. Low MLI values were found to correlate with low virulence in piglets and high MLI values with virulence.

Induction of monooxygenase and UDP-glucuronosyltransferase activities in primary cultures of rat hepatocytes.

The inducibility of two monooxygenase and UDP-glucuronosyltransferase (GT) forms was studied in primary cultures of adult rat hepatocytes. The following enzyme activities were determined: cytochrome P-448-dependent ethoxyresorufin O-deethylase (ERDE) and cytochrome P-450-dependent aldrin epoxidase (AE), and, furthermore, the GT form(s) metabolizing 3-hydroxybenzo(a)pyrene (GT1) and the GT form(s) metabolizing 4-hydroxybiphenyl (GT2). The results were as follows. The activity of AE and GT2 decreased markedly during the first days of culture, whereas ERDE and GT1 remained stable or even increased slightly. The maintenance of ERDE activity was dependent on the presence of dexamethasone. Pregnenolone-16 alpha-carbonitrile (PCN), phenobarbital (PB), and benz(a)anthracene (BA) induced the activity of ERDE in hepatocytes cultured in HM 84 medium by a factor of 4, 8, and 12, respectively. Similar factors of induction were obtained at the fifth day of culture using a modified Leibovitz L-15 medium. However, the time course of induction differed greatly in the two media. BA and PB had an additive effect on ERDE activity, suggesting different mechanisms of action for the two inducers. Monoclonal antibodies directed against cytochrome P-448 inhibited ERDE activities induced by BA and PB to a similar extent. Neither PB nor PCN significantly increased AE activity. However, these compounds induced GT2. BA did not affect GT2 but induced GT1. The present results show that the culture of adult rat hepatocytes changes the relative distribution of monooxygenase and GT forms. The response to inducers resembles only partially that observed in vivo.

Application, limitations and research requirements of in vitro test systems in toxicology.

There is increasing application of in vitro-test systems for toxicological evaluation of chemicals, which became possible by increasing understanding of the biological endpoints present in such systems and their capability for metabolic activation and inactivation. This communication focuses on the capacities of metabolic activation and inactivation in mutagenicity test systems, using bacteria, mammalian cells in culture and isolated hepatocytes. Bacterial test procedures with S-9-fraction are specific metabolic activation systems with low inactivation capacity. Mammalian cells are either deficient in metabolic activities or contain only limited activation capacity although special cell lines derived from hepatoma cells express certain metabolic activation as well as inactivation reactions. Isolated hepatocytes contain enzymatic activities similar to those in the intact liver, which however decrease at different rates. It is the goal of present research to construct cell lines with defined and sufficient activities of these enzymes. In isolated hepatocytes chemically induced DNA repair can be readily detected when a clear discrimination between mitochondrial, semiconservative and repair synthesis is provided. In such systems genotoxicity of reactive oxygen species is decreased by physiological concentrations of alpha-keto-acids, pyruvate possessing the highest antioxidant activity. It is concluded that in vitro test systems provide a suitable tool for detecting genotoxic and toxic effects of chemicals. However, many biological parameters such as metabolic activity, degree of differentiation of the cells, cofactor requirement, and composition of the medium affect the reliability of the test system. Thus, only a detailed understanding of the biology and biochemistry of such test allow production of reliable and reproducible results.