[Diagnosis of pulmonary tuberculosis using Ziehl-Neelsen stain and polymerase chain reaction]. / Diagnostik der Lungentuberkulose mit Ziehl-Neelsen-Färbung und Polymerasekettenreaktion.

BACKGROUND: Definitive diagnosis of unclear pulmonary lesions is mainly based on morphological methods. In addition to a neoplasm, inflammatory reactions, in particular tuberculosis (TB), have to be considered in most cases. Therefore, the aim of this work was to determine whether established methods used in general pathology can be efficiently used with cytological material. MATERIALS AND METHODS: An established polymerase chain reaction (PCR) protocol for the detection of Mycobacterium tuberculosis complex (Mtc) DNA in fixed specimens was conducted on fixed material available as an assay for liquid-based cytology (LBC). CytoLyt®-fixed material of 45 patients with clinically suspected TB or other mycobacteriosis were selected and were initially tested cytologically. In cases of absent tumor cells, PCR for detection of Mtc DNA and Ziehl-Neelsen stain (ZN) were performed. RESULTS: In 9 patients (20 %), Mtc DNA was found by PCR. The following methods were used to obtain material: catheter biopsy (5), needle biopsy (2), transbronchial needle aspiration (1), and bronchoalveolar lavage (1). Cytologically an inflammatory reaction was observed in all cases. In 2 patients, a history of TB, in 2 further cases either silicosis or a posttransplant situation was known. In cases with a positive PCR, 7 patients (78 %) were positive in ZN and 3 patients (33.3 %) in TB culture (15.5 % vs. 6.7 % of the total cohort); however, the material used for investigation was not always from identical sources, respectively. In 36 out of 45 patients, both PCR and ZN were negative for the detection of Mtc DNA. CONCLUSION: The material intended for LBC can be used for detection of TB with ZN and Mtc PCR.

[Infections in organ transplantations]. / Infektionen bei Organtransplantationen.

Infections play a crucial role in organ transplantations as possible complications. Viruses, bacteria, fungi and parasites are potential agents. The relevance of individual diseases depends on the organ transplanted. Morphology of the inflammatory reaction is given by the agent involved, but often several reactions can be caused by the same agent and different agents can also lead to the same reaction. Histology therefore provides concrete identification of the causal agent only in some cases, such that additional microbiological diagnostics are necessary. Results from these investigations should be transferred to the pathologist to distinguish between infection-associated changes and transplant rejection.

Antigen dose, type of antigen-presenting cell and time of differentiation contribute to the T helper 1/T helper 2 polarization of naive T cells.

Antigenic encounter by T cells induces immunological synapse formation and T-cell activation. Using different concentrations of toxic shock syndrome toxin-1 (TSST-1) as stimulus, we examined the capacities of dendritic cells (DC) and macrophages (Mphi) to prime syngeneic naive T cells. DCs were, under all experimental settings, more efficient than Mphi at clustering T cells. Translocation of the T-cell receptor (TCR) to the contact area was found to be induced by DCs, as well as by Mphi, in an antigen-dependent manner, although Mphi were less efficient at inducing TCR translocation. Capping of protein kinase C theta (PKCtheta) was also antigen dependent but induced exclusively by DCs. Likewise, DCs were found to be more potent inducers of interleukin-2 (IL-2) production and proliferation of naive T cells than Mphi. After 3 days of culture, DCs presenting 100 ng/ml TSST-1 induced interferon-gamma (IFN-gamma)-secreting cells, whereas Mphi did not. After 7 days of culture, DCs presenting 0.1 ng/ml TSST-1, and Mphi presenting high (as well as low) doses of TSST-1, induced IL-4-producing cells. We therefore provide evidence to show that antigen dose, type of antigen-presenting cell and time of differentiation can contribute to T-cell differentiation.

Large-scale isolation of immature dendritic cells with features of Langerhans cells by sorting CD34+ cord blood stem cells cultured in the presence of TGF-beta1 for cutaneous leukocyte antigen (CLA).

Epidermal Langerhans cells (LCs) are a subset of immature dendritic cells (DCs) and play a key role in the initiation and regulation of T cell responses. Upon antigenic stimulation, LCs differentiate into mature DCs undergoing profound morphologic and functional changes. Studies of the biological details of this conversion process have been hampered by difficulties in generating immature dendritic cells of a defined lineage. We propose a new method of purifying homogenous immature DCs in large numbers by sorting for CLA (Langerhans-like cells) from cord-blood-derived haematopoietic progenitor cells (HPCs). Established protocols describe the generation of LCs from CD34(+) HPCs by sorting for CD1a after 5 days of culture in the presence of GM-CSF and TNF-alpha. However, the numbers of LCs obtained by this method remain within the low range. Furthermore, CD1a is also expressed on interstitial DCs. LCs but not interstitial DCs express the cutaneous leukocyte antigen (CLA). The expression of CLA by cells stimulated with TNF-alpha and GM-CSF peaks on day 10. This expression can be raised further by stimulating the cells with TGF-beta1 and omitting TNF-alpha from day 6 onwards. CLA(+) cells were isolated on day 10 by AutoMACS. Their LC phenotype was established by the presence CD207. The immaturity of Langerhans-like cells was shown by the lack of CD83 and CD208 expression as well as their lower ability to activate allogeneic naive T cells as compared to maturing dendritic cells. However, CLA(+) cells cannot be termed Langerhans cells as they do not express Birbeck granules. Compared to sorting for CD1a (on day 6), sorting for CLA (on day 10) results in isolates of higher purity (80% vs. 50%) and a yield eight times higher (4.9x10(6) vs. 6.5x10(5) cells) when using identical numbers of input cells (5x10(5) cells). This novel method guarantees large numbers of pure and functionally active immature dendritic cells.

Detection of Chlamydia pneumoniae in unexplained pulmonary hypertension.

The pathogenesis of primary pulmonary hypertension is still unclear. The case of a 68-yr-old female patient who complained of recurrent dizzy spells and collapses over a period of 6 weeks and died of global cardiac failure is presented. Autopsy revealed severe pulmonary hypertension, slight chronic bronchitis, and bronchiolitis as well as intra-alveolar accumulation of macrophages. Chlamydiae were detected within the pulmonary arteries and in intramural and intra-alveolar macrophages by immunofluorescence, confocal laser scanning microscopy, scanning and transmission electron microscopy. Nested-polymerase chain reaction (PCR) and nonradioactive deoxyribonucleic acid (DNA) hybridization of PCR products from pulmonary arteries revealed Chlamydia pneumoniae DNA. Chlamydia pneumoniae has already been detected in atherosclerosis and in pulmonary emphysema. It can induce proliferation of smooth muscle cells. Chlamydia pneumoniae might be relevant in aggravation of primary pulmonary hypertension and might perhaps be a trigger factor in some cases.

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.

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

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 microm to 0.6 microm 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.