[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.

Are low inflammatory reactions involved in exudative age-related macular degeneration? Morphological and immunhistochemical analysis of AMD associated with basal deposits.

PURPOSE: Basal laminar and linear deposits (BLD) are associated with the development of choroidal neovascularization (CNV). Therefore, analysis of BLD composition may provide further information concerning the pathogenesis of BLD and CNV in age-related macular degeneration (AMD). METHODS: BLD in 25 specimens of surgically removed CNV were examined, using histochemical and immunohistochemical methods, for extracellular matrix proteins and their modulating enzymes, and for cell markers and proteins involved in inflammatory processes. In addition, ultrastructural electron microscopic analysis (EM) was performed. RESULTS: The chemical and structural composition of all the BLD was similar. Only the inner aspect of the BLD contained laminin and collagen IV, which corresponded to a new RPE basal lamina upon EM analysis. The extracellular matrix protein predominantly found in all layers of BLD was vitronectin, which was seen as a homogeneous material within the BLD upon EM analysis. The metalloproteinases MMP-2 and MMP-9 could only be detected in the inner aspect, while MMP-7 and TIMP-3 were observed predominantly in the outer aspect of BLD. In this area, staining for phospholipids and less intensely for neutral lipids was also visible. The labelling of complement complexes C3 and C5b-9 was intensely positive, and vascular endothelial growth factor (VEGF) was detected in all BLDs. CONCLUSIONS: Diffuse deposits such as BLD appear consistently with the development of CNV in AMD. They consist of extracellular matrix components and predominantly vitronectin. However, activated complement and VEGF could also be detected. The results of the current study may support the hypothesis that inflammatory processes are involved in the pathogenesis of BLD and CNV in AMD.

Endoparasite KC5/2 encloses large areas of sol-like cytoplasm within Acanthamoebae. Normal behavior or aberration?

In addition to the characteristic development of the rod-shaped or tube-like endocytobionts replicating within the sol-like cytoplasm of acanthamoebae which was published recently (Michel et al. 2003) we observed the strange behavior of the endoparasites described as follows. The material normally forming the characteristic cell walls of KC5/2 organisms was found to form a long array along the borderline between the granuloplasm and the central homogenous sol-like cytoplasm, so that about one third of the sol plasm had already been surrounded. We do not know whether this process belongs to the normal developmental repertoire of the endocytobiont that was interrupted by fixation for electron microscopy, or whether it is an aberrant behavior.

Endocytobiont KC5/2 induces transformation into sol-like cytoplasm of its host Acanthamoeba sp. as substrate for its own development.

New investigations of a novel, recently described, non-cultivable endocytobiont of Acanthamoeba sp. reveal at least three hitherto unobserved developmental stages which shed some light on the nature of this peculiar organism. The development of the endocytobiont is closely connected with conspicuous changes in the host amoeba, inducing the transformation from gel to sol-like cytoplasm which bulges like a balloon inside the host cell. Young and transitory developmental stages were found within the homogenous, sol-like cytoplasm. The infectious stages, with their voluminous cell wall and a conspicuous ostiole, could be observed within all parts of the cytoplasm with the exception of the nucleus. It is a remarkable adaptation for this parasite to be able to induce this gel-sol transformation in order to facilitate its own development. The fate of the heavily infected host amoebae is death by rupture or lysis after being overcrowded with parasites. As no structures could be observed within the endoparasites that were comparable to other bacteria, the real nature and taxonomic position of these peculiar organisms remain obscure.

Members of the Cytophaga-Flavobacterium-Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'.

Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host (Acanthamoeba sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as 'Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence analysis of the host of 'Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.

Fungal colonization and invasive fungal infections following allogeneic BMT using metronidazole, ciprofloxacin and fluconazole or ciprofloxacin and fluconazole as intestinal decontamination.

Invasive fungal infections (IFI) are increasingly diagnosed in patients undergoing allogeneic BMT. We have previously shown that the addition of metronidazole to ciprofloxacin for gastrointestinal bacterial decontamination significantly reduces the incidence of grades II-IV aGVHD by reduction of the anaerobic intestinal bacterial flora. Here, we found that the combined use of ciprofloxacin, metronidazole and fluconazole as antifungal prophylaxis increased intestinal yeast colonization when compared to ciprofloxacin and fluconazole alone (P < 0.01). Based on the EORTC criteria, a total of 18 out of 134 study patients developed IFI: seven of 68 (10%) patients who received metronidazole compared to 11 of the 66 (17%) patients decontaminated without metronidazole developed IFI (log-rank P = 0.36). Lethal IFI occurred in two of seven patients receiving metronidazole and in four of 11 patients without anaerobic decontamination. In conclusion, bacterial intestinal decontamination using metronidazole as an antibiotic with activity against most anaerobic intestinal bacteria significantly increases the intestinal yeast burden without influencing the incidence of IFI in patients undergoing allogeneic BMT.

A self-perpetuating vicious cycle of tissue damage in human hibernating myocardium.

Recently, we proposed the hypothesis that a vicious cycle exists in human hibernating myocardium (HM) between the progression of myocyte degeneration and the development of fibrosis. We now investigated the pathomechanism of this cycle in more detail and established a correlation between the severity of the morphological changes and the degree of postoperative functional recovery of HM. HM was diagnosed by dobutamine echocardiography, thallium-201 scintigraphy and radionuclide ventriculography. Functional recovery was present at 3 months after coronary bypass surgery but remained unchanged at 15 months. Forty patients were subdivided into 2 groups: A with complete and B with incomplete recovery. Biopsies taken during surgery and studied by electron microscopy, immunocytochemistry, rt-PCR, and morphometry revealed myocyte degeneration and inflammatory and fibrinogenic changes in a widened interstitial space. We report here for the first time an upregulation of TGF-beta1 evident by a 5-fold increase of fibroblasts and macrophages exhibiting a TGF-beta1 content 3-fold larger than in control, and a > 3-fold increase in TGF-beta1 mRNAby rt-PCR. The number of angiotensin converting enzyme (ACE) containing structures was increased (n/mrm2: control-11.4, A-17.6, B-19.2, control vs. A and B, p < 0.05). Fibrosis was more severe in group B than A or control (%: C-10.1; A-21.2; B-40.6; p < 0.05). Capillary density was significantly reduced (n/mm2: C-1152; A-782; B-579, p < 0.05) and intercapillary distance was widened (microm: C-29.5, A-36.1, B-43.3, p < 0.05). The number of CD 3 (n/mm2: C-5.0; A-9.6; B-9.4, ns) and CD 68 positive cells (n/mm2: C-37.2; A-80.7; B-55.0, C vs. A p < 0.05) was elevated in HM as compared to control indicating an inflammatory reaction. Cut-off points for functional recovery are fibrosis > 32%, capillary density < 660/mm2 and intercapillary distance > 39.0 microm. In HM a self-perpetuating vicious cycle of tissue alterations leads to progressive replacement fibrosis and continuous intracellular degeneration which should be interrupted by early revascularization.

Vannella sp. harboring Microsporidia-like organisms isolated from the contact lens and inflamed eye of a female keratitis patient.

Viable Hartmannella sp. and two strains of Vannella sp.--but no Acanthamoebae--multiplied on NN-agar inoculated with pieces of the contact lens from a female keratitis patient. Within the cytoplasm of one Vannella isolate, intracellular parasites could be observed whose earliest stages were developing within the nucleus, resembling those Microsporidia-like parasites seen within Vannella isolated recently from a warm tapwater system. This assumption was also confirmed by electron microscopy. In swabs taken directly from the cornea, Pseudomonas aeruginosa were identified, but they did not yield any growth of amebas in culture. However, cocultivation of parasite-free Vannella strains with the above-mentioned swab matter resulted in infected amebas harboring the same intracellular parasites seen before. This infection could be established only if the corresponding spores were present as infective agents in the swab matter. The successful treatment of the patient with antibiotics supports the assumption that P. aeruginosa was the main cause of the corneal ulceration. The extent to which the Microsporidia-like organisms may have been involved in the development of keratitis remains a matter of discussion.

Intracellular bacteria of Acanthamoebae resembling Legionella spp. turned out to be Cytophaga sp.

Acanthamoeba sp. isolated from the drinking water system of a hospital harboured gram-negative bacteria multiplying inside phagosomes and within the cytoplasm of their host cells. According to their morphology demonstrated by electron microscopy they resembled Llaps (Legionella-like amoebal pathogens) but turned out to be Cytophaga sp. as shown by a typical profile of cellular fatty acids obtained by means of gas-liquid chromatography.

Influence of intestinal bacterial decontamination using metronidazole and ciprofloxacin or ciprofloxacin alone on the development of acute graft-versus-host disease after marrow transplantation in patients with hematologic malignancies: final results and long-term follow-up of an open-label prospective randomized trial.

In a single-center open-label prospective study, a total of 134 marrow transplant recipients with hematologic malignancies were randomly assigned to a bacterial decontamination medication using metronidazole and ciprofloxacin (n = 68) or ciprofloxacin alone (n = 66) during 5 weeks posttransplant. The development of grades II to IV acute graft-versus-host disease (GVHD) was defined as the primary study endpoint. According to the intention-to-treat, 17 patients (25%) randomized to the combined decontamination medication and 33 patients (50%) randomized to ciprofloxacin alone developed grades II to IV GVHD (P <.002). The higher frequency of grades II to IV acute GVHD in patients randomized to ciprofloxacin alone resulted from a more than twofold increased number of patients developing liver or intestinal involvement with acute GVHD compared with patients randomized to the combined decontamination medication (P <.003). The influence of the study medication on grades II to IV acute GVHD was significant only in recipients of transplants from genotypically HLA-identical sibling donors (n = 80), whereas in recipients of transplants from donors other than HLA-identical siblings (n = 54), grades II to IV acute GVHD frequencies between the study arms were not significantly different. The combined decontamination was associated with a significant reduction of culture growth of intestinal anaerobic bacteria during 5 weeks posttransplant (P <. 00001). In addition, the number of cultures with growth of anaerobic bacteria (P <.005) as well as the median concentrations of anaerobic bacteria in the posttransplant period (P <.0001) were higher in patients contracting grades II to IV acute GVHD. Neither chronic GVHD nor overall survival was significantly different between the two study arms. In patients with HLA-identical sibling donors who were treated in early disease stages, the 5-year survival estimate was slightly, but not significant, higher after the combined decontamination medication (60% +/- 11%) compared with ciprofloxacin alone (46% +/- 9%). In conclusion, the present study provides evidence that antimicrobial chemotherapy targeted to intestinal anaerobic bacteria in marrow transplant recipients significantly reduces the severity of acute GVHD and supports the theory that the intestinal anaerobic bacterial microflora plays a role in the pathogenesis of acute GVHD after human marrow transplantation.

Ascomycin macrolactam derivative SDZ ASM 981 inhibits the release of granule-associated mediators and of newly synthesized cytokines in RBL 2H3 mast cells in an immunophilin-dependent manner.

Mast cells play an important role in the pathological development of many inflammatory and allergic diseases and inhibition of mast cell activation is a potential target for therapeutic intervention. Therefore, the effect of the novel ascomycin macrolactam derivative SDZ ASM 981 on Fc epsilonRI-mediated activation of rat basophilic leukemia (RBL) cells, as a model for mast cell activation, was investigated. First, the ability to inhibit different mast cell immunophilins in vitro was tested. Using recombinant macrophilin-12 (FKBP-12), inhibition of rotamase activity with an IC50 of approximately 6 nM was observed. The rotamase activity of cyclophilin A (18 kDa) was not affected. Secondly, the effect of SDZ ASM 981 on Fc epsilonRI-mediated mast cell activation was investigated in the RBL cell model. SDZ ASM 981 inhibited exocytosis of preformed mediators (e.g. serotonin) with an IC50 of approximately 30 nM. Transcription and release of newly synthesized mediators (e.g. TNF-alpha) was inhibited with an IC50 of approximately 100 nM. The inhibitory effect of SDZ ASM 981 was antagonized by rapamycin. We conclude that SDZ ASM 981 is a potent inhibitor of Fc epsilonRI-mediated activation of mast cells in vitro. The mechanism of action involves formation of (calcineurin) inhibitory complexes with macrophilins. We suggest that this inhibitory action on mast cells might contribute to the antiinflammatory effect of SDZ ASM 981 observed in vivo (e.g. in aptopic dermatitis and psoriasis).

The extracellular matrix in hibernating myocardium--a significant factor causing structural defects and cardiac dysfunction.

Recently, we described chronic intracellular degeneration accompanied by fibrosis as typical structural features of hibernating myocardium and we concluded that cellular degeneration as a sign of the incomplete adaptation to the reduced blood flow is characteristic of hibernation. This study has been extended by analyzing the composition of the extracellular matrix proteins of the diseased myocardium. Areas of hibernating myocardium were identified in 38 patients by angiography, multigated radionuclide ventriculography, thallium scintigraphy with reinjection and low-dose dobutamine echocardiography. These areas were biopsied at cardiac surgery and were studied by electron microscopic and immunofluorescence techniques. Electron microscopy showed an enlarged extracellular space containing numerous particles of cellular debris, macrophages, fibroblasts, homogeneous matrix material and collagen fibrils. The basement membrane of the cardiomyocytes was thickened by an augmentation of laminin, fibronectin and collagen VI, but these proteins also were present in the matrix itself. Collagen fibrils were numerous and macrophages (CD68) and fibroblasts (vimentin) were increased. In situ hybridization showed an increase in mRNA for laminin, fibronectin and collagen. This observation is consistent with the conclusion that fibrotic scar formation was occurring continuously. It is postulated that fibrosis is the consequence of myocyte loss due to chronic underperfusion in the hibernating tissue. This will further injure myocytes so that a vicious cycle is established that leads to progressive loss of structural integrity and functional capacity. Since these changes are progressive, revascularization should be performed at the earliest time point possible in patients with areas of hibernating myocardium.

Assessment of myocardial viability: Dobutamine echocardiography and thallium-201 single-photon emission computed tomographic imaging predict the postoperative improvement of left ventricular function after bypass surgery.

The aim of this study was to evaluate the usefulness of dobutamine echocardiography and quantitative thallium-201 single photon emission computed tomography myocardial scintigraphy with reinjection in the detection of viable myocardium in patients with coronary artery disease and reduced left ventricular function, which will improve after aortocoronary bypass surgery. Forty-eight patients (47 men, aged 61 +/- 6 years) with angiographically documented reduced left ventricular function (ejection fraction 35 +/- 14, 63% with chronic transmural myocardial infarction) were examined by dobutamine two-dimensional echocardiography (before and during low dosage), 201Tl, and gated radionuclide ventriculography before and 3 +/- 2 months after aortocoronary bypass surgery. Four of 55 areas classified viable before operation were revascularized inadequately and discarded. Global left ventricular ejection fraction at rest rose from 35% +/- 14% before operation to 40% +/- 13% (p < 0.05) after operation. Stress-induced perfusion defects involved 40% +/- 19% of the left ventricle circumference after stress and showed a significant reduction of size to 23% +/- 14% (p < 0.01) at rest, 4 hours later, and after reinjection. This value fell to 16% +/- 12% (p < 0.05) 3 months after aortocoronary bypass surgery. We conclude that both dobutamine echocardiography (sensitivity 95%, specificity 80%, positive predictive value 87%, negative predictive value 88%) and 201Tl studies (sensitivity 87%, specificity 98%, positive predictive value 97%, negative predictive value 93%) are suitable and comparable accurate methods for predicting improvement in systolic function 3 months after revascularization in a selected population with a high prevalence of viable but hypokinetic or akinetic myocardium.

Characterization of the periodontal microflora by the fatty acid profile of the broth-grown microbial population.

The applicability of fatty acid analysis to the characterization of periodontal microflora was investigated using gas-liquid chromatography (GLC) and the software of the Sherlock Microbial Identification System (MIS) from MIDI Inc. Sulcus fluid was collected with paper points and anaerobically cultured in broth at 35 degrees C for four days. The broth-grown microbial population was extracted and the fatty acid methyl esters (FAME) were separated by GLC. The investigation of 67 specimens from asymptomatic sulci and of 32 specimens from inflamed sulci showed that the patterns of FAME profiles, the clustering of FAME profiles by computerized 2-D plot procedure, and the determination of the peak area index (PAI) of the FAME profiles differentiate between normal and pathological sulcus flora. Comparison of the clinical sulcus rating and the FAME data indicated that a pathological FAME profile may precede manifest periodontitis, and the normalization of the FAME profile may precede healing. It is concluded that the FAME analysis of sulcus fluid is a diagnostic aid for periodontological surveillance, for the initiation of preventive treatment of periodontitis, and for controlling the antimicrobial efficiency of therapeutic measures.

Hibernating myocardium: an incomplete adaptation to ischemia.

BACKGROUND: We tested the hypothesis that hibernating myocardium represents an incomplete adaptation to a reduced myocardial oxygen supply. METHODS AND RESULTS: In 38 patients, areas of hibernating myocardium were identified by angiography, multigated radionuclide ventriculography, thallium scintigraphy with reinjection, and low-dose dobutamine echocardiography. Biopsies removed at cardiac surgery showed structural degeneration characterized by a reduced protein and mRNA expression and disorganization of the contractile and cytoskeletal proteins myosin, actin, desmin, titin, alpha-actinin, and vinculin by electron microscopy, immunohistochemistry, and in situ hybridization. Additionally, an increased amount of extracellular matrix proteins resulting in a significant degree of reparative fibrosis was present. Dedifferentiation, ie, expression of fetal proteins, was absent. Apoptosis indicating suicidal cell death was found by the terminal deoxynucleotidyl transferase end-labeling method and electron microscopy. Radionuclide ventriculography showed improvement of regional function at 3 months postoperatively compared with preoperative values (mean values, 23.5% and 48%, respectively), and the echocardiographic wall-motion score index decreased from 3.4 to 1.8. The degree of severity of the morphological changes (three stages) correlated well with the extent of postoperative functional recovery: more advanced clinical improvement was observed in patients with slight and moderate morphological degeneration (stages 1 and 2), but recovery was only partial in severe degeneration (stage 3). CONCLUSIONS: Cellular degeneration rather than adaptation is present in hibernating myocardium. The consequence is progressive diminution of the chance for complete structural and functional recovery after restoration of blood flow. The practical consequence from this study should be early revascularization in patients showing areas of hibernating myocardium.

A comparison of methods of phenotypic and genotypic fingerprinting of Exophiala dermatitidis isolated from sputum samples of patients with cystic fibrosis.

Phenotypic and genotypic characteristics of 11 strains of Exophiala dermatitidis were investigated. Ten strains (including three reference strains) were isolated from sputum samples of six patients with cystic fibrosis (CF) in Germany, and one reference strain was isolated from a patient with phaeohyphomycosis in Japan. The strains showed differences in their ability to assimilate sorbitol, palatinose, rhamnose, gluconate and melezitose, leading to the differentiation of seven auxotypes. The IC30 of amphotericin B, and ketoconazole and itraconazole, respectively, indicated susceptibility, whereas the IC30 of fluconazole and 5-fluorocytosine indicated resistance in all strains. Protein patterns in SDS-PAGE revealed no major differences. The glycoconjugate patterns distinguished the Japanese strain from the other strains. Cluster analysis of whole-cell fatty acid methyl ester (FAME) profiles with the Microbial Identification System (MIS) revealed two major clusters separating a reference strain and the Japanese strain from the other strains. Analysis of patterns resulting from random amplification of polymorphic DNA (RAPD) with two arbitrary primers showed four genotypes. Comparison of the results revealed no agreement between the different fingerprinting methods, except the separation of the Japanese strain from the European CF strains. As the results of assimilation tests seem to vary between different laboratories, the analysis of FAME profiles and RAPD analysis are recommended for typing E. dermatitidis.