Dirk Theegarten

Evaluation of peptide nucleic acid-fluorescence in situ hybridization for identification of clinically relevant mycobacteria in clinical specimens and tissue sections.

ABSTRACT With fluorescently labeled PNA (peptide nucleic acid) probes targeting 16S rRNA, we established a 3-h fluorescence in situ hybridization (FISH) procedure for specific visualization of members of the Mycobacterium tuberculosis complex, M. leprae, M. avium, and M. kansasii. Probe specificity was tested against a panel of 25 Mycobacterium spp. and 10 gram-positive organisms. After validation, probes were used to identify 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplification-based methods. All isolates (M. tuberculosis complex, n = 24; M. avium, n = 7; M. kansasii, n = 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for species-specific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology.

Eosinophilic gastroenteritis in a young girl – long term remission under Montelukast

BackgroundEosinophilic gastrointestinal disorders are an emerging disease entity characterized by eosinophilic infiltration of the intestinal wall. Oral steroids can be still considered as first line treatment. Unfortunately relapses are quite common. Usually long term low-dose prednisone or immunosuppressive therapy is required, which is especially problematic in young patients. Thus a reliable steroid sparing agent with low side effects suitable for long term use is needed. There are strong hints to a similar pathophysiology of eosinophilic gastrointestinal disorders to that of asthma. Indeed leukotriene D4 plays an important role in the recruitment of eosinophils into the intestinal tissue causing damage. This patho-mechanism provides the rationale for the treatment with a leukotriene D4 receptor antagonist. Recently there have been first reports about successful short term use of Montelukast in eosinophilic gastrointestinal disorders.Case presentationWe report the case of a 17 year old girl with a long history of severe abdominal complaints leading to several hospitalizations in the past. Mimicking the picture of an intestinal tuberculosis she received an anti mycobacterial treatment without any success. Marked eosinophilia in blood, ascites and tissue samples of the intestinal tract finally lead to the diagnosis eosinophilic gastroenteritis. Tapering off prednisone caused another severe episode of abdominal pain. At that point leukotriene antagonist Montelukast was started at a dose of 10 mg once daily. Steroids could be tapered off completely within six weeks. The patient has been free of symptoms for over two years by now. Routine examinations, blood tests and endoscopy have rendered regular results. So far no side effects were noted.ConclusionHere report about successful long term remission of eosinophilic gastroenteritis under Montelukast. Further randomized control trials are required to asses the full benefits of Montelukast therapy in the whole spectrum of eosinophilic gastrointestinal disorders.

Respiratory chlamydial infection based on experimental aerosol challenge of pigs with Chlamydia suis

An experimental study of aerogeneous challenge in pigs was conducted in order to reveal characteristic features of porcine respiratory chlamydiosis. Eight conventionally raised pigs were exposed to a pathogenic strain of Chlamydia (C.) suis, four controls were mock infected. Besides pathological changes, the acute-phase and humoral immune responses, as well as the dissemination and transmission of the challenge strain was monitored in the course of infection. The data from clinical investigations, LPS-binding protein assay, antibody ELISAs, confocal laser scanning and light microscopy, immunohistochemical staining and PCR provided extensive evidence of the pathogenic potential of C. suis for the porcine respiratory system. This model appears suitable for further pathophysiological and immunological investigations of chlamydial respiratory infections and can also be recommended for studies of Chlamydia-associated infections of the human lung.

The role of chlamydia in the pathogenesis of pulmonary emphysema. Electron microscopy and immunofluorescence reveal corresponding findings as in atherosclerosis.

Abstractu2002 Chlamydia pneumoniae has been detected in atherosclerotic plaques by various means. Chlamydiae are able to cause persistent infections. Serologically elevated antibody titers are found in severe chronic obstructive pulmonary disease. In atherosclerosis and pulmonary emphysema, inflammatory reactions can be seen by means of light microscopy. Specimens from patients with obliterative arteriosclerosis undergoing thrombendarteriectomy and with advanced emphysema undergoing lung volume reduction surgery were examined using scanning (SEM) and transmission (TEM) electron microscopy, and using immunofluorescence with monoclonal antibodies and antiserum against chlamydiae. SEM shows spherical bodies (SBs) with a diameter from 0.3 µm to 0.6 µm on the surface of the alveoli and bronchioles, as well as in atherosclerotic plaques. In atherosclerosis and emphysema, SBs reveal a double membrane, adherence to collagen fibers, tissue destruction, as well as intracellular and interstitial localization in TEM. They show in parts a densely packed central structure. SBs are seen both in alpha-1-antitrypsin deficiency emphysema and smoker’s emphysema. Using immunofluorescence microscopy, spots are seen in corresponding distributions to the SBs. Morphological findings are typical for aberrant chlamydiae seen in persistent infections. Chronic infection and bacterial colonization associated with progressive disease seems to be relevant not only in atherosclerosis but also in pulmonary emphysema.

Induction of cytochrome P450 1A1 in multiple organs of minipigs after oral exposure to soils contaminated with polycyclic aromatic hydrocarbons (PAH).

Abstract. We have used the minipig as a prospective animal model for human risk characterization to study primary biochemical alterations upon oral contaminant intake. The effects of three orally administered soils containing polycyclic aromatic hydrocarbons (PAH) on the expression pattern of the cytochrome P450 enzyme CYP1A1 in various organs have been analyzed. Dependent on the soil sample, subchronic daily oral PAH doses ranged from 0.38 to 1.90xa0mg PAHEPA/kg body weight. In all cases, soil administration lead to significant CYP1A1 induction in several organs of minipigs to a different extent, following the order liver ≈ duodenum >lung >kidney ≈ spleen. Hepatic ethoxyresorufin-O-deethylase activities were elevated to 310, 1250 and 1780 compared with a background level of 200xa0pmol resorufin/mg protein per min. Induced duodenal activities appear to be even higher than in the liver, namely 405, 1280 and 2500 compared with a basal activity of 11xa0pmol resorufin/mg protein per min. CYP1A1 induction in several organs is clear evidence for successful contaminant mobilization and absorption in the duodenum and subsequent distribution of contaminant into diverse body compartments. As is shown in one case, impairment of CYP1A1 induction in the liver and thus breakdown of its PAH-metabolizing activity appears to have no effect on induced CYP1A1 levels in other organs. It appears important with respect to risk assessment that induction of CYP1A1 is particularly sensitive in the duodenum of minipigs and is achieved with soil doses which are in the range of amounts ingested by playing children due to hand-to-mouth activities. Induced duodenal CYP1A1 activities obtained in minipigs by oral exposure to PAH largely exceed maximal duodenal activities so far observed in rats. This is equally relevant for risk assessment and for selection of a suitable animal model that reflects effects of PAH exposure in humans.

Infection of Murine Precision Cut Lung Slices (PCLS) with Respiratory Syncytial Virus (RSV) and Chlamydophila pneumoniae Using the Krumdieck Technique

The Krumdieck technique allows the investigation of the so-called precision cut lung slices (PCLS) with a special microtome. It is thus possible to evaluate morphologic changes over a longer period of time using only a small group of animals. Chlamydophila pneumoniae (Cp) and respiratory syncytial virus (RSV) proved to be important causes of pneumonia, rhinitis and exacerbations of asthma bronchiale, as well as of lower respiratory tract infections in young children. PCLS should be tested for their suitability as an in vitro model for these infections. The PCLS were infected with Cp and RSV over different periods of time. Investigations were carried out by light and transmission electron microscopy (TEM). Furthermore, immunofluorescence (IF) studies with antibodies against bacterial or viral proteins and cell-specific markers were done using confocal laser scanning microscopy (CLSM). Non-infected and infected PCLS showed a well-preserved morphology up to 72 hours. After short infection intervals, typical inclusions of Cp or RSV were detected in vacuoles of different cell types. Infection and cell types could be verified using IF. Cytopathic effects were not prominent. Ciliary beat was detectable up to 96 hours after infection. This in vitro technique offers the possibility of studying mechanisms and effects of bacterial and viral infections on viable tissue complexes.

A comparative ultrastructural and molecular biological study on Chlamydia psittaci infection in alpha-1 antitrypsin deficiency and non-alpha-1 antitrypsin deficiency emphysema versus lung tissue of patients with hamartochondroma

BackgroundChlamydiales are familiar causes of acute and chronic infections in humans and animals. Human pulmonary emphysema is a component of chronic obstructive pulmonary disease (COPD) and a condition in which chronic inflammation manifested as bronchiolitis and intra-alveolar accumulation of macrophages is common. It is generally presumed to be of infectious origin. Previous investigations based on serology and immunohistochemistry indicated Chlamydophila pneumoniae infection in cases of COPD. Furthermore, immunofluorescence with genus-specific antibodies and electron microscopy suggested involvement of chlamydial infection in most cases of pulmonary emphysema, but these findings could not be verified by PCR. Therefore, we examined the possibility of other chlamydial species being present in these patients.MethodsTissue samples from patients having undergone lung volume reduction surgery for advanced alpha-1 antitrypsin deficiency (AATD, n = 6) or non-alpha-1 antitrypsin deficiency emphysema (n = 34) or wedge resection for hamartochondroma (n = 14) were examined by transmission electron microscopy and PCR.ResultsIn all cases of AATD and 79.4% of non-AATD, persistent chlamydial infection was detected by ultrastructural examination. Intra-alveolar accumulation of macrophages and acute as well as chronic bronchiolitis were seen in all positive cases. The presence of Chlamydia psittaci was demonstrated by PCR in lung tissue of 66.7% AATD vs. 29.0% non-AATD emphysema patients. Partial DNA sequencing of four positive samples confirmed the identity of the agent as Chlamydophila psittaci. In contrast, Chlamydophila pneumoniae was detected only in one AATD patient. Lung tissue of the control group of non-smokers with hamartochondroma was completely negative for chlamydial bodies by TEM or chlamydial DNA by PCR.ConclusionsThese data indicate a role of Chlamydophila psittaci in pulmonary emphysema by linking this chronic inflammatory process to a chronic infectious condition. This raises interesting questions on pathogenesis and source of infection.

[Unanticipated outbreak of Q fever during a study using sheep, and its significance for further projects].

Effective measures for protection of laboratory staff against infections due to experiments with pathogens are a challenge for safeguarding against hazards. Investigations on limited epidemic infections and their causes are of high relevance. During an experiment with pregnant sheep 5 of 7 persons who took part in the operation procedures developed Q fever 10 to 24 days after exposure. One sheep aborted spontaneously 3 days after the third experiment. Examinations of the aborted material revealed Coxiella burnetii, Chlamydophila psittaci, and Staphylococcus chromogenes. Coxiella burnetii and Chlamydophila psittaci were also detected in some of the other placentae. This outbreak of Q fever during an experiment with infected sheep affected more than 70% of the people involved with this experiment and shows the high risk of working with the zoonotic agent Coxiella burnetii. Hence, technical, organizational, and personnel measures have to be taken, and laboratory animals are to be monitored to prevent further infections.

The human eye (retina): a site of persistent HCMV infection?

BackgroundHuman cytomegalovirus (HCMV) retinitis frequently occurs in severely naturally and iatrogenically immunocompromised patients. It has been shown that the immune-privileged retina is a major site of HCMV infection in AIDS patients. It is conceivable either that during the immunosuppression HCMV infection reactivates in various other organs viremically affecting the retina or that HCMV persisting in the retina may locally reactivate and result in HCMV retinitis.MethodsAs there is still controversy about the sites of HCMV latency and persistence we investigated 75 eyes of HIV-seronegative patients undergoing enucleation due to a variety of malignant and non-viral benign ophthalmic disorders for the retinal presence of HCMV antigen and DNA.ResultsNone of the analyzed patients had symptoms of HCMV retinitis. Immunohistologic staining as well as TaqMan DNA PCR analysis showed all samples to be free of HCMV.ConclusionsOur data suggest that the human eye is rather unlikely to be a site of productive or latent HCMV persistence.

The Role of Persisting Infections in the Pathogenesis of Pulmonary Emphysema: Electron Microscopy Reveals a Probable Bacterial Colonization of the Alveolar Space and the Bronchioles

Lung volume reduction surgery (LVRS) yields resection specimens from patients with advanced pulmonary emphysema. Regarding the development of lung function parameters, recent results obtained by light microscopy revealed an unfavorable prognosis in patients with remarkable inflammation, particularly in the bronchioli. Tissue from ten patients (alpha1-antitrypsin level in the normal range) was furthermore investigated by electron microscopy. Scanning electron microscopy shows 0.4-0.6 micron spherical bodies variably densely arranged in the whole alveolar space and in the bronchioles of all patients. These bodies are mostly seen on the microvilli of type II pneumocytes. An immunological reaction with activation of macrophages and granulocytes occurs simultaneously. Macrophages show cytoplasmic extensions to the spherical bodies, which exhibit a cellular membrane but no cellular wall. This favors the diagnosis of bacterial colonization of the alveolar space and the bronchioles by mycoplasmas or L-forms of other bacteria. As patients undergoing lung volume reduction surgery are under optimal medical treatment and without any infection clinically, these findings appear to be relevant for the pathogenesis and/or progression of pulmonary emphysema.