Two cases demonstrate an association between Tropheryma whipplei and pulmonary marginal zone lymphoma.

BACKGROUND: Marginal zone lymphomas of mucosa-associated lymphatic tissues (MZL of MALT) are a group of indolent B-cell neoplasms, which are thought to arise from chronic antigenic stimulation of B-cells either due to underlying chronic infection or autoimmune disease. Little is known about potential causative pathogens in pulmonary MZL (PMZL), although some data suggests a potential role of Achromobacter (A.) xylosoxidans. METHODS: An index case of chronic pulmonary colonisation with Tropheryma (T.) whipplei and subsequent development of PMZL was identified by T. whipplei specific PCR and metagenomic next genome sequencing (mNGS). This case prompted a retrospectively conducted analysis of T. whipplei-specific PCRs in lung tissue from PMZL patients (n = 22), other pulmonary lymphomas, and normal controls. Positive results were confirmed by mNGS. A systematic search for T. whipplei and A. xylosoxidans in our in-house mNGS dataset comprising autopsy lungs, lung biopsies and lung resection specimens (n = 181) was subsequently performed. RESULTS: A 69-year-old patient presented with weight loss and persistent pulmonary consolidation. Subsequent mNGS analysis detected T. whipplei in the resected lung specimen. An antibiotic regimen eventually eliminated the bacterium. However, the consolidation persisted, and the diagnosis of PMZL was made in a second lung resection specimen. A second case of T. whipplei-associated PMZL was subsequently detected in the retrospectively analysed PMZL cohort. Both cases showed comparatively few mutations and no mutations in genes encoding for NF-κB pathway components, suggesting that T. whipplei infection may substitute for mutations in these PMZL. None of the samples in our in-house dataset tested positive for T. whipplei. In contrast, A. xylosoxidans was frequently found in both autopsy lungs and lung biopsy / resection specimens that were not affected by PMZL (> 50%). CONCLUSIONS: Our data suggests that T. whipplei colonisation of lungs may trigger PMZL as a potential driver. Systematic analyses with larger cohorts should be conducted to further support this hypothesis. The frequent detection of A. xylosoxidans in lung tissue suggests that it is a common component of the pulmonary microbiome and therefore less likely to trigger lymphomas.

CALCIUM HYDROXYLAPATITE MICROSPHERES - BIOCOMPATIBILITY AND CLINICAL EFFECTS.

Soft tissue fillers are used in aesthetic medicine for volumizing and for contouring. Calcium hydroxylapatite (CaHA) is a fully biodegradable biostimulatory filler. The study presents results of in vitro biocompatibility and cytotoxicity investigations performed on CaHA and illustrates its clinical effects. We used a human cell culture model for in vitro studies with HaCaT keratinocytes and human dermal fibroblasts, known to be a sensitive cell type for biocompatibility and cytotoxicity. Cells were exposed to different concentrations of CaHA (Radiesse®). Cell proliferation was calculated by luminometric adenosine triphosphate (ATP) measurement using the ATPLiteTM-M Assay. Possible cytotoxic effects were detected by the calorimetric Cytotoxicity Detection Kit. Clinical data were obtained from our own treatment files. CaHA did neither inhibit cell proliferation nor cause cytotoxicity. Clinical data suggest an excellent tolerability and long-term efficacy superior to hyaluronic acid-based fillers. CaHA is a versatile and well-tolerated biodegradable and biostimulatory filler.

A novel native collagen dressing with advantageous properties to promote physiological wound healing.

OBJECTIVE: Chronic hard-to-heal wounds generate high costs and resource use in western health systems and are the focus of intense efforts to improve healing outcomes. Here, we introduce a novel native collagen (90 %):alginate (10 %) wound dressing and compare it with the established oxidised dressings Method: Matrices were analysed by atomic force microscopy (AMF), scanning electron microscopy (SEM), and immunoelectron microscopy for collagen types I, III and V. Viability assays were performed with NIH-3T3 fibroblasts. Matrix metalloproteinase (MMP) binding was analysed, and the effect of the wound dressings on platelet-derived growth factor B homodimer (PDGF-BB) was investigated. RESULTS: Unlike oxidised regenerated cellulose (ORC)/collagen matrix and ovine forestomach matrix (OFM), the three-dimensional structure of the native collagen matrix (NCM) was found to be analogous to intact, native, dermal collagen. Fibroblasts seeded on the NCM showed exponential growth whereas in ORC/collagen matrix or OFM, very low rates of proliferation were observed after 7 days. MMP sequestration was effective and significant in the NCM. In addition, the NCM was able to significantly stabilise PDGF-BB in vitro. CONCLUSION: We hypothesise that the observed microstructure of the NCM allows for an effective binding of MMPs and a stabilisation and protection of growth factors and also promotes the ingrowth of dermal fibroblasts, potentially supporting the re commencement of healing in previously recalcitrant wounds. DECLARATION OF INTEREST: This work was supported by BSN Medical, Hamburg, Germany.

Typha capensis (Rohrb.)N.E.Br. (bulrush) extract scavenges free radicals, inhibits collagenase activity and affects human sperm motility and mitochondrial membrane potential in vitro: a pilot study.

The biodiversity in South Africa provides more than 30,000 higher plants, of which more than 3000 are used by traditional healers to treat diseases. Typha capensis (bulrush) is one of the medicinal plants used in South Africa to treat male fertility problems. Considering that South African traditional healers have been recognised by Law and the health benefits of T. capensis have not been scientifically investigated yet, this study aimed at investigating the in vitro effects of aqueous extracts from this plant on male reproductive functions. Both leaves and rhizomes of T. capensis were dried, infused with distilled water and freeze-dried. Motile sperm from 50 men were isolated by swim-up and incubated with 1 µg ml(-1) aqueous extract of Typha rhizome for 1 h at 37 °C. Vitality, motility, sperm production of reactive oxygen species and mitochondrial membrane potential were analysed in the test sample, a control and in the pellet from the swim-up. Results showed that the rhizome extract had significant (P < 0.0001) negative effects on all parameters. The extracts from the leaves and rhizomes revealed dose-dependent inhibitory activity for collagenase and free radical formation. No inhibitory activity for elastase was found. The inhibitory activity for collagenase might indicate possible anti-cancer effects.

Both copy number and sequence variations affect expression of human DEFB4.

Copy number variations (CNVs) were found to contribute massively to the variability of genomes. One of the best studied CNV region is the beta-defensin cluster (DEFB) on 8p23.1. Individual DEFFB copy numbers (CNs) between 2 and 12 were found, whereas low CNs predispose for Crohn's disease. A further level of complexity is represented by sequence variations between copies (multisite variations, MSVs). To address the relation of DEFB CN and MSV to the expression of beta-defensin genes, we analyzed DEFB4 expression in B-lymphoblastoid cell lines (LCLs) and primary keratinocytes (normal human epidermal keratinocyte, NHEK) before and after stimulation with lipopolysaccharide, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). Moreover, we quantified one DEFB4 MSV in DNA and mRNA as a marker for variant-specific expression (VSE) and resequenced a region of approximately 2 kb upstream of DEFB4 in LCLs. We found a strong correlation of DEFB CN and DEFB4 expression in 16 LCLs, although several LCLs with very different CNs exhibit similar expression levels. Quantification of the MSV revealed VSE with consistently lower expression of one variant. Costimulation of NHEKs with TNF-alpha/IFN-gamma leads to a synergistic increase in total DEFB4 expression and suppresses VSE. Analysis of the DEFB4 promoter region showed remarkably high density of sequence variabilities (approximately 1 MSV/41 bp).

Molecular-weight-dependent toxic effects of chitosans on the human keratinocyte cell line HaCaT.

The cationic polysaccharide chitosan possesses bioactive properties such as antimicrobial activity, antitumor effects, hemostatic assets and positive effects on wound healing. The influence of polycations like chitosan on human cells has been reported to depend on their molecular weight. However, the mechanism of cytotoxicity caused by polycations is not yet fully understood. In the study presented, the influence of two chitosans with a similar degree of deacetylation but different molecular weight, chitosan 1130 (120 kDa) and chitosan oligosaccharide (5 kDa), on the human keratinocyte cell line HaCaT was analyzed. The results obtained indicate that chitosans exhibit a molecular-weight-dependent negative effect on HaCaT cell viability and proliferation in vitro. The chitosans tested also stimulated the release of inflammatory cytokines by HaCaT cells depending on incubation time and concentration. Chitosan 1130 and chitosan oligosaccharide induced apoptotic cell death which was mediated by activation of the effector caspases 3/7. At least for chitosan 1130, the involvement of both, extrinsic and intrinsic signal pathways, was shown by activation of caspases 8 and 9.

Measurement of intracranial pressure in children: a critical review of current methods.

Assessment of intracranial pressure (ICP) is essential in the management of acute intracranial catastrophe to limit or actively reduce ICP. This article provides background information and reviews the current literature on methods of measuring ICP in children. Indications for ICP measurement are described for children with traumatic brain injury, shunt insertion or malfunction, arachnoid cyst, craniosynostosis, and prematurity. Various methods of ICP monitoring are detailed: non-invasive, indirect (lumbar puncture, visual-evoked potentials, fontanelle compression, and optic nerve sheath), and direct assessment (ventricular cannulation, and epidural, subdural, and intraparenchymal devices). Normal levels of ICP will depend on the age and position of the child during monitoring. This article provides clinical and research-based evidence in this area where there is currently limited guidance.

Mutants of tobacco mosaic virus with temperature-sensitive coat proteins induce heat shock response in tobacco leaves.

We analyzed, with respect to heat shock proteins (HSPs), systemically reacting tobacco leaves inoculated with Tobacco mosaic virus (TMV), wild-type vulgare, and temperature-sensitive coat protein (CP) mutants Ni 118 (P20L) and flavum (D19A), kept at 23 or 30 degrees C. HSP18 and HSP70 mRNAs and proteins were induced with temperature-sensitive CP mutants after 1 to 2 days at 30 degrees C. After 4 to 6 days, HSP70 was also induced at 23 degrees C. The induction of HSPs paralleled the amount of insoluble TMV CP in leaf extracts, indicating that denatured TMV CP by itself induces a heat-shock response.

Uptake, effects, and metabolism of cyanobacterial toxins in the emergent reed plant Phragmites australis (cav.) trin. ex steud.

The commonly occurring cyanobacterial toxin microcystin-LR (MC-LR) was rapidly taken up by the emergent reed plant Phragmites australis with clear distribution in the different cormus parts of the plant. Highest uptake was detected in the stem, followed by the rhizome. Enzyme extracts of the rhizome system, the stem, and the leaf revealed the presence of soluble glutathione S-transferases (sGST) measured with the model substrate 1-chloro-2,4-dinitrobenzene. A significant elevation of sGST activity in the rhizome and stem parts of P. australis was detected after a 24-h exposure to 0.5 microg/L MC-LR. Rhizome, stem, and leaf tissues were also able to conjugate several microcystin toxins. However, no conjugation, either chemical nor enzymatic, was detected using the related cyanobacterial toxin nodularin as substrate. Highest glutathione S-transferase activity for the toxin substrates was detected in the pkat/mg range in the stem of P. australis. For MC-LR, a complete metabolism from the formation of a glutathione conjugate to the degradation of a cysteine conjugate in all cormus parts of the plant is reported. The stepwise degradation of the MC-LR-glutathione conjugate to a gamma-glutamylcysteine and a cysteine conjugate was demonstrated by comparison with chemically formed reference compounds and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This is the first evidence for the uptake and metabolism of cyanobacterial toxins by an emergent aquatic macrophyte.

Monoclonal antibody MS13 identifies a plasmatocyte membrane protein and inhibits encapsulation and spreading reactions of Manduca sexta hemocytes.

Lepidopterans generally can successfully defend themselves against a variety of parasites or parasitoids. One mechanism they use is to encapsulate the invader in many layers of hemocytes. For encapsulation to occur, the hemocytes must attach to the foreign material, spread, and adhere to each other. The molecules that mediate these processes are not known. One method to identify proteins potentially necessary for adhesion, spreading, and, thus, encapsulation is to use monoclonal antibodies that interfere with these functions. In this paper, we report that a monoclonal antibody against Manduca sexta plasmatocytes effectively inhibited encapsulation of synthetic beads in vitro and in vivo. Furthermore, it inhibited plasmatocyte spreading in vitro. Other anti-hemocyte antibodies did not have these effects. The plasmatocyte-specific monoclonal antibody, mAb MS13, recognized a protein of approximately 90,000 daltons as indicated by Western blot analysis of hemocyte lysate proteins. The epitope recognized by mAb MS13 was present on the exterior surface of plasmatocytes. Using indirect immunohistochemistry with hemocyte-specific antibodies, we also determined that during encapsulation plasmatocytes were the first cells bound to latex beads and later layers consisted of both plasmatocytes and granular cells. Arch.

Identification of an enzymatically formed glutathione conjugate of the cyanobacterial hepatotoxin microcystin-LR: the first step of detoxication.

Cyanobacterial toxins have adverse effects on mammals, birds and fish and are being increasingly recognised as a potent stress factor and health hazard factor in aquatic ecosystems. Microcystins, cyclic heptapeptides and a main group of the cyanotoxins are mainly retained within the producer cells during cyanobacterial bloom development. However, these toxins are released into the surrounding medium by senescence and lysis of the blooms. Any toxin present could then come into contact with a wide range of aquatic organisms including phytoplankton grazers, invertebrates, fish and aquatic plants. Recent studies showed the conversion of microcystin in animal liver to a more polar compound in correlation with a depletion of the glutathione pool of the cell. The present study shows the existence of a microcystin-LR glutathione conjugate formed enzymatically via soluble glutathione S-transferase in various aquatic organisms ranging from plants (Ceratophyllum demersum), invertebrates (Dreissena polymorpha, Daphnia magna) up to fish eggs and fish (Danio rerio). The main derived conjugate was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry yielding a mass of m/z 1302, which is equivalent to the mass assumed for a glutathione microcystin-LR conjugate. This conjugate appears to be the first step in the detoxication of a cyanobacterial toxin in aquatic organisms.

The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts.

Vasoactive agents which elevate either cGMP or cAMP inhibit platelet activation by pathways sharing at least one component, the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP). VASP is stoichiometrically phosphorylated by both cGMP-dependent and cAMP-dependent protein kinases in intact human platelets, and its phosphorylation correlates very well with platelet inhibition caused by cGMP- and cAMP-elevating agents. Here we report that in human platelets spread on glass, VASP is associated predominantly with the distal parts of radial microfilament bundles and with microfilaments outlining the periphery, whereas less VASP is associated with a central microfilamentous ring. VASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, VASP is concentrated at focal contact areas, along microfilament bundles (stress fibres) in a punctate pattern, in the periphery of protruding lamellae, and is phosphorylated by cGMP- and cAMP-dependent protein kinases in response to appropriate stimuli. Evidence for the direct binding of VASP to F-actin is also presented. The data demonstrate that VASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix.

The upright position of brush border-type microvilli depends on myosin filaments.

We have studied the correlation between the actomyosin organization and microvillar position in an epithelial cell line derived from the proximal pig kidney tubule (LLC-PK1). When grown on glass, these cells are approximately 5-6 microns in height and develop numerous microvilli that project from the dorsal membrane. A fairly homogeneous distribution of microvilli was achieved by synchronization of the cell cycle. These microvilli are of the brush border type, as defined by their content of villin and their anchorage in a myosin-rich terminal web-like structure. When LLC-PK1 cells were injected with two monoclonal antibodies against pig brain nonmuscle myosin, in concentrations yielding a 1:1 ratio of antibody to myosin, neither microvillar number nor length was affected. However, when we examined the cells by scanning electron microscopy 1-3 h after microinjection, we found that one of the antibodies (a-PBM 4) had a profound effect on microvillar position: more than 50% were seen tilted or lying prone on the plasma membrane. The microvilli of cells injected with the other antibody (a-PBM 9) were not significantly different from those of cells injected with control antibodies. This difference correlates with in vitro properties of the antibodies: a-PBM 4 decreases the actin-activated Mg(2+)-ATPase of pig brain nonmuscle myosin quite substantially, while a-PBM 9 affects it only moderately. These differential effects are probably a consequence of the different epitope location as determined for both antibodies, not of differences in antibody affinity. Our data are compatible with the hypothesis that a-PBM 4 also interferes with the actomyosin interaction in situ, thus decreasing the effective cross-linking of microvillar rootlets by myosin filaments in the terminal web. On the basis of this model, we suggest that myosin filaments are essential for the upright position of brush-border type microvilli.

[Fulminant malignant hyperthermia during the 6th general anesthesia using volatile anesthetics]. / Fulminante maligne Hyperthermie während der sechsten Vollnarkose mit volatilen Anästhetika.

We report on the fulminant crisis of malignant hyperthermia occurring in a 30-year-old female during kidney transplantation. In the past, she had been anaesthetised repeatedly without complications. Anaesthesia was induced with thiopental and vecuronium and continued with isoflurane/N2O/O2. After an initially normal course of anaesthesia, the patient developed symptoms of a fulminant malignant hyperthermia (MH) including excessive increase in end expiratory CO2, hyperkalaemia, tachycardia and hyperpyrexia. The patient was saved by the timely administration of dantrolene. A surgical revision required the next day because of bleeding was done under dantrolene cover and took an uncomplicated course. The patient was extubated 7.5 hours after the second intervention and transferred to a normal ward after 4 days. A subsequently performed in vitro contracture test clearly revealed susceptibility to malignant hyperthermia.

Characterization of the coated vesicle uncoating ATPase: tissue distribution, association with and activity on intact coated vesicles.

We have analyzed the uncoating process of clathrin-coated vesicles (CV) performed by an ATPase (UA; apparent molecular mass 70 kDa) prepared from various mammalian tissues. Our data show that this enzyme removes the clathrin coat from isolated, intact coated vesicles, as seen by sedimentation analysis on gels and also by electron microscopy. The isolated UA does not discriminate between CV from homologous or heterologous tissues. This finding implies that the brain-specific insertion in clathrin light chains cannot be essential for the binding of brain UA to target vesicles. Polyclonal antibodies were raised against UA and were found to inhibit UA activity. Immunoblotting of purified CV and immunoblotting of CV in situ indicate that a subpopulation of CV contains bound UA. However, most of the uncoating enzyme is not associated with coated structures in mammalian tissue culture cells. Our data support the hypothesis that the 70 kDa uncoating ATPase is responsible for the in vivo uncoating of coated vesicles.

[Cannabinoid intake by passive smoking]. / Cannabinoid-Aufnahme durch Passivrauchen.

The presence of cannabinoids in the urine can be produced by passive inhalation of hashish resp. marihuana smoke. The height depends on the intensity of exposure. Under extreme conditions concentrations between 40 to 50 ng/ml of cannabinoids had been found in the urine. In regard to the maximum variation-coefficient of the immunoassay methods, which is about 30%, we think it is useful to define a threshold value of 65 ng/ml of cannabinoids to distinguish between active and passive inhalation.