Benefit of polyhexamethylene biguanide in Fusarium keratitis.

PURPOSE: To report a series of 3 patients with soft contact lens-related Fusarium keratitis. Two of them were treated with the antiamoebic polyhexamethylene biguanide 0.02% (PHMB) in combination with antifungal drugs, and 1 patient was treated with PHMB as sole antifungal regimen. METHODS: Chart review of 3 patients treated with PHMB in Fusarium keratitis. Two of them were refractory to the commonly used therapy. The antifungal power of PHMB and propamidine isethionate was tested against the patients' isolates as well as against the clinical isolates from another 9 patients with ocular mould infections. RESULTS: An excellent outcome could be achieved in 2 patients with Fusarium solani keratitis refractory to common antifungal treatment by the additional use of PHMB 0.02%. In another patient PHMB alone was sufficient to resolve Fusarium proliferatum infection. The drug was well tolerated. In all patients repeated abrasion was done for better penetration of the drugs. PHMB revealed a marked in vitro antifungal activity for the three Fusarium isolates as well as for another 9 isolates of ocular infections from other patients including also the genera Scedosporium, Aspergillus and Rhizopus giving minimal inhibitory concentrations ranging from 1.56 to 3.12 µg/ml. CONCLUSIONS: Fusarium keratitis is a severe ocular infection. We report on the use of PHMB in 3 patients given additionally or as sole antifungal drug. We emphasize the benefit of PHMB 0.02% in Fusarium keratitis which might be considered as a therapeutic option especially in cases refractory to common antifungal therapy and possibly in keratitis due to other fungi.

[Management of complex thrombocytopenia with thrombelastometry : a case of simultaneous posttransfusion purpura and heparin-induced thrombocytopenia]. / Management einer komplexen Thrombozytopenie mithilfe der Thrombelastometrie : Koinzidenz von posttransfusioneller Purpura und Heparin-induzierter Thrombozytopenie.

The case presented describes the combined onset of heparin-induced thrombocytopenia II (HIT) and post-transfusion purpura (PTP) 5-10 days following exposure to heparin and blood transfusion during aortic dissection repair. On day 4 the platelet count decreased by 40% and D-dimers started to increase again. Despite a low clinical probability for HIT-II at this time (4T score of 3) serological testing was done the next day and yielded a negative test result. Following a transient rise after platelet transfusion another 40% decrease in platelet count occurred on day 8. To increase precision of the 4T score, screening ultrasonography was performed and identified a clinically unapparent jugular vein thrombosis. As this increased the 4T score to 6 points, serological testing was repeated and now showed the presence of HIT-II antibodies. Despite switching from heparin to argatroban the platelet count continued to decrease to <5×10(3)/µl. Conventional clotting tests showed a prolonged prothrombin time and severe hypofibrinogenemia. Because of the female sex, age >50 years, history of pregnancy and transfusion 8 days before, PTP was suspected. The alteration of the plasmatic coagulation, however, could not be explained by PTP. Therefore, disseminated intravascular coagulation (DIC) and interference of argatroban with conventional clotting tests were considered as alternative diagnoses. In order to differentiate between the two alternatives rotational thrombelastometry (ROTEM®) was performed and revealed an increased functional fibrinogen level without signs of hyperfibrinolysis. This argued for an interference of argatroban with the Clauss method of fibrinogen measurement and rendered DIC unlikely. Under suspicion of PTP, treatment with immunoglobulin was initiated and blood transfusions were avoided. Detection of PTP antibodies 1 day later confirmed the combined presence of PTP and HIT-II. As hyperfibrinogenemia compensated for the effects of thrombocytopenia on clot firmness in ROTEM®, anticoagulation with lepirudin was started at 9×10(3) platelets/µl only. The next day the platelet count increased to 32×10(3)/µl and clot firmness returned to normal. No thromboembolic complications and no relevant bleeding were observed. In summary, this case shows for the first time that HIT-II and PTP can occur in parallel in patients with simultaneous exposure to heparin and blood transfusions. Confounding effects of argatroban on conventional clotting tests may mimic DIC under these circumstances and make diagnosis difficult. Careful evaluation of the time-related magnitude in platelet decrease, patient history, course of D-dimers, screening ultrasonography and ROTEM® seem to be helpful to initiate early appropriate therapy before serological test results become available. In contrast to the Clauss method of fibrinogen measurement, assessment of clot firmness in ROTEM® is not influenced by argatroban. Moreover, ROTEM® reveals the compensatory effects of increased functional fibrinogen on clot firmness during severe thrombocytopenia as an important variable for anticoagulation therapy during thrombocytopenia with increased thromboembolic risk.

Release of S(+) enantiomers in breath samples after anaesthesia with isoflurane racemate.

BACKGROUND AND OBJECTIVE: Isoflurane is a chiral volatile anaesthetic, routinely administered as racemate. It has a low metabolic rate and is mostly eliminated via respiration. In blood samples, S(+) enantiomers are found in greater proportion in the days immediately after administration of isoflurane racemate whereas the ratio in breath samples is unknown. METHODS: Breath and blood samples were drawn immediately after recovery and daily up to 19 days after operation from patients undergoing anaesthesia with isoflurane racemate. The percentage of isoflurane enantiomer was determined by gas chromatography mass spectrometry in blood and thermodesorption gas chromatography mass spectrometry in breath samples. RESULTS: In breath samples, there were significant differences in S(+) enantiomers at all time points compared to the racemate. During the early postoperative phase, the percentage of S(+) enantiomers were significantly enhanced whereas 5 days after surgery predominantly R(-) enantiomers (50.41%) were detected in the breath samples. Also in blood samples a statistical significant accumulation of the S(+) enantiomer was noted between days 1 and 5 compared to isoflurane racemate blood control. S(+) enantiomers were significantly higher in blood compared to breath samples and was most evident on the third day after surgery (51.43%). CONCLUSIONS: During the first days after application of isoflurane racemate, the percentage of S(+) enantiomers are higher in breath and blood samples of patients. We suggest that resorption and/or redistribution of enantiomers are responsible for the different kinetics of isoflurane enantiomers.

Accumulation of S(+) enantiomer in human beings after general anaesthesia with isoflurane racemate.

BACKGROUND AND OBJECTIVE: Isoflurane is a chiral, volatile anaesthetic with low metabolic rate (0.17%) that is routinely administered in its racemic form. Knowledge about the distribution of the enantiomers in human beings may give some important information about the understanding of the mechanisms of volatile anaesthetics. METHODS: Blood samples were drawn immediately after tracheal extubation and daily up to 8 days postoperatively from patients undergoing general anaesthesia with isoflurane racemate. The enantiomer enrichment of isoflurane was determined by headspace gas chromatography-mass spectrometry. RESULTS: At all time points, there was a statistically significant accumulation of the S(+) enantiomer in blood, especially at days 2 (52.01%) and 7 (52.1%). Separate analysis of obese patients or in a small group of patients with co-existing lung disease did not show any difference to the total population. In addition, duration of anaesthesia did not influence the enantiomer concentrations. CONCLUSIONS: We suggest that a slower association and dissociation rate is responsible for the S(+) enrichment.

Multiple cis regulatory elements control RANTES promoter activity in alveolar epithelial cells infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normally T-cell expressed and presumably secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study, we analyzed the mechanism of RSV-induced RANTES promoter activation in human type II alveolar epithelial cells (A549 cells). Promoter deletion and mutagenesis experiments indicate that RSV requires the presence of five different cis regulatory elements, located in the promoter fragment spanning from -220 to +55 nucleotides, corresponding to NF-kappaB, C/EBP, Jun/CREB/ATF, and interferon regulatory factor (IRF) binding sites. Although site mutations of the NF-kappaB, C/EBP, and CREB/AP-1 like sites reduce RSV-induced RANTES gene transcription to 50% or less, only mutations affecting IRF binding completely abolish RANTES inducibility. Supershift and microaffinity isolation assays were used to identify the different transcription factor family members whose DNA binding activity was RSV inducible. Expression of dominant negative mutants of these transcription factors further established their central role in virus-induced RANTES promoter activation. Our finding that the presence of multiple cis regulatory elements is required for full activation of the RANTES promoter in RSV-infected alveolar epithelial cells supports the enhanceosome model for RANTES gene transcription, which is absolutely dependent on binding of IRF transcription factors. The identification of regulatory mechanisms of RANTES gene expression is fundamental for rational design of inhibitors of RSV-induced lung inflammation.

Induction of astrocyte differentiation by endothelial cells.

Here we have investigated the mechanisms that control astrocyte differentiation within the developing rat optic nerve. Astrocytes are normally generated by astrocyte precursor cells within the embryonic optic nerve. We show that there is a close temporal and spatial correlation between endothelial and astrocyte differentiation. We tested the potential role of endothelial cells in inducing astrocyte differentiation by developing an immunopanning method to highly purify endothelial cells from developing optic nerves. We show that the purified endothelial cells, but not other embryonic optic nerve cell types, strongly induce the differentiation of purified astrocyte precursor cells into astrocytes in vitro. Leukemia inhibitory factor (LIF) and LIF receptors have been implicated previously in astrocyte differentiation in vivo. We show that purified endothelial cells express LIF mRNA and that their ability to induce astrocyte differentiation is prevented by a neutralizing anti-LIF, but not anti-ciliary neurotrophic factor, antiserum. These findings demonstrate a role for endothelial cells in inducing astrocyte differentiation. The induction of astrocyte differentiation by endothelial cells makes sense phylogenetically, anatomically, and functionally, because astrocytes evolved concurrently with brain vasculature and ensheathe capillaries throughout the brain. The ability to purify and culture astrocytes and endothelial cells should provide an excellent model system for future studies of blood-brain barrier development.

Inducible expression of inflammatory chemokines in respiratory syncytial virus-infected mice: role of MIP-1alpha in lung pathology.

Lower respiratory tract disease caused by respiratory syncytial virus (RSV) is characterized by profound airway mucosa inflammation, both in infants with naturally acquired infection and in experimentally inoculated animal models. Chemokines are central regulatory molecules in inflammatory, immune, and infectious processes of the lung. In this study, we demonstrate that intranasal infection of BALB/c mice with RSV A results in inducible expression of lung chemokines belonging to the CXC (MIP-2 and IP-10), CC (RANTES, eotaxin, MIP-1beta, MIP-1alpha, MCP-1, TCA-3) and C (lymphotactin) families. Chemokine mRNA expression occurred as early as 24 h following inoculation and persisted for at least 5 days in mice inoculated with the highest dose of virus (10(7) PFU). In general, levels of chemokine mRNA and protein were dependent on the dose of RSV inoculum and paralleled the intensity of lung cellular inflammation. Immunohisthochemical studies indicated that RSV-induced expression of MIP-1alpha, one of the most abundantly expressed chemokines, was primarily localized in epithelial cells of the alveoli and bronchioles, as well as in adjoining capillary endothelium. Genetically altered mice with a selective deletion of the MIP-1alpha gene (-/- mice) demonstrated a significant reduction in lung inflammation following RSV infection, compared to control littermates (+/+ mice). Despite the paucity of infiltrating cells, the peak RSV titer in the lung of -/- mice was not significantly different from that observed in +/+ mice. These results provide the first direct evidence that RSV infection may induce lung inflammation via the early production of inflammatory chemokines.

Regulation of the mucosal immune response.

Infectious diseases continue to exact an extensive toll on populations living closest to the equatorial regions of the globe. A substantial proportion of these infections gain access to the host via the mucosal tissues. Thus, the development of new vaccines that enhance mucosal immunity is considered to be of paramount importance in order to prevent or limit the impact of these infections. Mucosal immune responses must discriminate between commensal flora within the lumen and potential pathogens. These responses are highly adapted to induce protection without excessive amounts of inflammation. The balances that regulate mucosal immune and inflammatory responses have to be understood if effective mucosal immunity is to be induced through local immunization. This review will summarize some of the unique properties of mucosal immune responses and focus on recent advances that have significantly influenced our understanding of the regulation of immune and inflammatory responses following infection.

The effect of class II major histocompatibility complex expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells: a mechanism for T helper cell type 1-mediated damage.

Helicobacter pylori infection is associated with gastric epithelial damage, including apoptosis, ulceration, and cancer. Although bacterial factors and the host response are believed to contribute to gastric disease, no receptor has been identified that explains how the bacteria attach and signal the host cell to undergo apoptosis. Using H. pylori as "bait" to capture receptor proteins in solubilized membranes of gastric epithelial cells, class II major histocompatibility complex (MHC) molecules were identified as a possible receptor. Signaling through class II MHC molecules leading to the induction of apoptosis was confirmed using cross-linking IgM antibodies to surface class II MHC molecules. Moreover, binding of H. pylori and the induction of apoptosis were inhibited by antibodies recognizing class II MHC. Since type 1 T helper cells are present during infection and produce interferon (IFN)-gamma, which increases class II MHC expression, gastric epithelial cell lines were exposed to H. pylori in the presence or absence of IFN-gamma. IFN-gamma increased the attachment of the bacteria as well as the induction of apoptosis in gastric epithelial cells. In contrast to MHC II-negative cell lines, H. pylori induced apoptosis in cells expressing class II MHC molecules constitutively or after gene transfection. These data describe a novel receptor for H. pylori and provide a mechanism by which bacteria and the host response interact in the pathogenesis of gastric epithelial cell damage.

Differential stimulation of interleukin-12 (IL-12) and IL-10 by live and killed Helicobacter pylori in vitro and association of IL-12 production with gamma interferon-producing T cells in the human gastric mucosa.

The objective of these experiments was to examine the ability of Helicobacter pylori to stimulate interleukin-10 (IL-10) or IL-12 and select for either Th1 or Th2 cells. Gastric biopsy specimens were collected from patients who were categorized with respect to the presence of H. pylori and gastric disease as well as their age, gender, medications, and other factors. As Th1 and Th2 cells are selected by IL-12 and IL-10, respectively, biopsy specimens were screened for mRNA and protein for these cytokines. Although mRNA for IL-12 and IL-10 was detected in biopsy specimens obtained from both infected and uninfected patients, IL-12 protein predominated. Levels of IL-10 and IL-12 in gastric tissue did not change in response to infection. Moreover, gamma interferon (IFN-gamma)-producing T cells were found in both the infected and the uninfected gastric mucosa. Stimulation of peripheral blood leukocytes from either infected or uninfected donors with various concentrations of live or killed H. pylori induced immunoreactive IL-12 and IL-10. After stimulation with live H. pylori, IL-12 levels increased more than 30-fold, whereas IL-10 levels increased only 2- to 5-fold, compared to cells stimulated with medium alone. Interestingly, killed H. pylori induced significantly more IL-10 (P < 0.05) than live H. pylori, while recombinant urease only induced IL-10. These results demonstrate that live H. pylori selectively stimulates the induction of IL-12 and Th1 cells that produce IFN-gamma, whereas preparations used in oral vaccines induce more IL-10 and may favor Th2 cell responses.

Respiratory syncytial virus induces selective production of the chemokine RANTES by upper airway epithelial cells.

The presence of histamine and eosinophil cationic protein in nasopharyngeal secretions of infants with respiratory syncytial virus (RSV)-induced bronchiolitis implies the activation of basophil and eosinophil leukocytes, but the specific mechanism of their recruitment has not been elucidated. Chemokines are potent and selective leukocyte chemotactic molecules that are also expressed by airway epithelial cells. Therefore, the pattern of chemokines produced in response to RSV infection was investigated in primary cultures of human nose- and adenoid-derived epithelial cells. Interleukin-8, growth-related peptide-alpha, and monocyte chemotactic protein-1 were constitutively released by uninfected epithelial cells and were not further enhanced by infection with RSV. RANTES (regulated upon activation, normal T cell-expressed and -secreted), which was present in negligible concentrations in uninfected cultures, was strongly induced by RSV infection, in a dose- and time-dependent manner. Through the release of RANTES, epithelial cells may control the selective concentration and activation of basophils and eosinophils in RSV-infected airway mucosa.

Respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class I MHC expression through the induction of IFN-beta and IL-1 alpha.

CD8+ T cells mediate some of the damage to the lung epithelium following respiratory syncytial virus (RSV) infection. Since CD8+ T cells recognize antigen-laden class I MHC molecules on the target cells, we examined in this study the expression of class I MHC by RSV-infected respiratory epithelial cells. Respiratory epithelial cell lines and bronchial epithelial cells from normal human tissue responded to RSV infection with an increased expression of class I MHC as determined by flow cytometry and immunoprecipitation of class I MHC from metabolically radiolabeled cells. The increase in class I MHC expression was dependent on infectious, replicating virus. UV-irradiated culture supernatants from RSV-infected A549 cells, when added to fresh A549 cell cultures, induced an increase in class I MHC expression by those cells. The class I MHC increasing activity within supernatants from A549 cells was due, in large part, to IFN-beta, and to a lesser extent to IL-1 alpha. The addition of neutralizing Abs to both cytokines completely blocked the increase in class I MHC expression by cells treated with the above-mentioned supernatants. These results demonstrate that RSV infection elicits IFN-beta production by respiratory epithelial cells, which in turn leads to an increase in their synthesis of class I MHC, which would facilitate their recognition and lysis by RSV-specific CD8+ T cells.

[The postoperative MR tomographic findings following the removal of a hypophyseal adenoma]. / Postoperative kernspintomographische Befunde nach Hypophysenadenomentfernung.

36 patients with pituitary adenomas were examined via MRI to describe physiological changes and to visualise a residual tumour. Pre- and postoperative examinations included T1- and T2-weighted SE sequences. T1-weighted images were obtained in sagittal and coronal orientation pre- and post-Gd-DTPA application and T2-weighted images in coronal orientation. In 12 cases a residual tumour was found. Its signal intensity and contrast enhancement were similar to those of the primary tumour. Implanted material could be distinguished by localisation, decrease in volume and different signal intensity. The behaviour of contrast enhancement was helpful, since implanted material showed a rim enhancement. In our experience a sensitive imaging protocol in the follow-up of operated pituitary adenomas would be an early examination three months postoperatively followed by a control examination after one year. Information on the size and localisation of the primary tumour and the performed operative procedure is essential.

[Dye laser coagulation of diabetic maculopathy]. / Farbstofflaserkoagulation der diabetischen Makulopathie.

Eighty eyes of 48 patients were followed after laser photocoagulation (615 nm) of the macula because of diabetic macular edema. The follow-up time was at least 6 months (average 10.2 months). The morphological status improved in more than 60% of eyes with focal edema but in only 40% of eyes with diffuse macula edema. Improvement of distance visual acuity was obtained in 35% of the eyes, mostly in those with good baseline acuity (greater than or equal to 0.7). The fraction with improved visual acuity decreased to 13% in eyes with a baseline acuity of 0.6 or less. Similar results were found for near visual acuity. The discrepancies between morphological and functional improvement are still unclear.