Andreas Schaffner

Selective Protection against Conidia by Mononuclear and against Mycelia by Polymorphonuclear Phagocytes in Resistance to Aspergillus: OBSERVATIONS ON THESE TWO LINES OF DEFENSE IN VIVO AND IN VITRO WITH HUMAN AND MOUSE PHAGOCYTES

By comparing natural immunity to Aspergillus fumigatus (AF) in vivo with the action of human or mouse phagocytes against AF in vitro, we delineated two sequential lines of defense against AF. The first line of defense was formed by macrophages and directed against spores. Macrophages prevented germination and killed spores in vitro and rapidly eradicated conidia in vivo, even in neutropenic and athymic mice. The second was the neutrophilic granulocyte (PMN), which protected against the hyphal form of AF. Human and mouse PMN killed mycelia in vitro. Normal, but not neutropenic mice, stopped hyphal growth, and eradicated mycelia. Either line of defense acting alone protected mice from high challenge doses. Natural immunity collapsed only when both the reticuloendothelial system and PMN were impaired. These findings are in keeping with the clinical observation that high doses of cortisone and neutropenia are the main risk factors for invasive aspergillosis. Cortisone inhibited the conidiacidal activity of mouse macrophages in vivo and of human or mouse mononuclear phagocytes in vitro. Cortisone damaged this first line of defense directly and not through the influence of T lymphocytes or other systems modifying macrophage function as shown in athymic mice and in vitro. In addition, daily high doses of cortisone in mice reduced the mobilization of PMN so that the second line of defense was also impaired. Thus, cortisone can break down natural resistance on its own. Myelosuppression rendered mice susceptible only when the first line of defense was overpowered by high challenge doses, by activated spores that cannot be killed by macrophages, or by cortisone suppression of the conidiacidal activity of macrophages. The host, thus, can call upon two independent phagocytic cell lines that form graded defense systems against aspergillus. These lines of defense function in the absence of a specific immune response, which seems superfluous in the control and elimination of this fungus.

Safety of stringent prophylactic platelet transfusion policy for patients with acute leukaemia

Early studies suggested that the risk of haemorrhagic complications become unacceptable when platelet counts drop below 20 x 10(9)/l. Because there are insufficient data to define 20 x 10(9)/l as the threshold for prophylactic platelet transfusions, the practicability of a more restrictive transfusion policy has been assessed prospectively in 102 consecutive patients being treated for acute leukaemia. Besides platelet count, the transfusion protocol took into consideration factors such as presence of bleeding, fever, coagulation disorders, and intention to do therapeutic procedures. 31 major bleeding episodes occurred on 1.9% of the study days when platelet counts were 10 x 10(9)/l or less and on 0.07% of study days when counts were 10-20 x 10(9)/l. The findings indicate that the threshold for prophylactic transfusions can safely be set at 5 x 10(9)/l in patients without fever or bleeding manifestations and at 10 x 10(9)/l in patients with such signs. For patients with coagulation disorders or anatomical lesions, or for those on heparin, the threshold should be at least 20 x 10(9)/l. Such a restrictive platelet transfusion policy, which is applicable not only to thrombocytopenia associated with acute leukaemia but also to other forms of hypoproliferative thrombocytopenia, reduces exposure of such patients to blood donors and results in substantial health-care savings.

Infection imaging using whole-body FDG-PET

Abstract.The purpose of this study was to evaluate fluorine-18 fluorodeoxyglucose positron emission tomography (FDG-PET) for the detection of soft tissue and bone infections. Forty-five PET examinations in 39 patients (26 male, 13 female, age range 27–86 years) with suspected infectious foci were examined with whole- or partial-body PET scans using FDG. Twenty-seven scans were done in patients with soft tissue and 18 in patients with bone infections. Corrected and uncorrected transaxial PET images were acquired. Seven hundred and twelve body regions in these 45 PET scans were evaluated. Pathological findings were graded using a confidence scale from A to E (A, definitive infection; E, no infection). Disease status was defined in all patients by culture, biopsy or surgery and clinical follow-up. In 45 PET scans there were 40 true-positive, four false-positive and one false-negative findings. Twelve foci suspected to be infectious in nature on the basis of other imaging examinations were identified as negative by PET, thus representing true-negative findings. Sensitivities for the patients with soft tissue (STI) and bone infections (BI) and for the pooled data were 96%, 100% and 98%, respectively. As the calculation of specificity is not straightforward, it was calculated on a per lesion as well as on a per body region basis to permit estimation of an upper and a lower limit. On a per lesion basis, specificities were 70% (STI), 83% (BI) and 75% for the pooled data and on a per body region basis (dividing the body into 22 regions) they were 99% (STI), 99% (BI) and 99% for the pooled data. One false-negative result was found in a patient with cholangitis. It is concluded that PET appears to be a highly sensitive method to detect infectious foci. Specificity is more difficult to estimate, but is probably in the range from 70% to above 90%.

Therapeutic concentrations of glucocorticoids suppress the antimicrobial activity of human macrophages without impairing their responsiveness to gamma interferon.

By exposing human blood-derived macrophages and alveolar macrophages in vitro to dexamethasone, we showed in these studies that glucocorticoids markedly suppress the antimicrobial activity of macrophages but not macrophage activation by lymphokines. As little as 2.5 X 10(-8) mol/liter of dexamethasone prevented macrophages from inhibiting germination of Aspergillus spores or from eliminating ingested bacteria such as Listeria, Nocardia, or Salmonella. Damage to macrophage function was inhibited by progesterone and appeared to be receptor-mediated. In accordance with in vivo observations, dexamethasone required 24-36 h to suppress antimicrobial activity. While glucocorticoids interfered with base-line activity of macrophages, dexamethasone concentrations comparable to drug levels in patients had no effect on macrophage activation. Proliferating lymphocytes and gamma-interferon thus increased the antimicrobial activity of phagocytes exposed to glucocorticoids over that of control cells. Macrophage activation and correction of the dexamethasone effect by gamma-interferon, however, was dependent on the pathogen. The lymphokine enhanced the antimicrobial activity of dexamethasone-treated macrophages against Listeria and Salmonella but not against Aspergillus or Nocardia. Dexamethasone-induced damage to the antimicrobial activity of human macrophages in vitro parallels observations that glucocorticoids render laboratory animals susceptible to listeriosis and aspergillosis by damaging resident macrophages. Suppression of macrophage antimicrobial activity should thus be considered when treating patients with glucocorticoids; its prevention by gamma-interferon might be beneficial for some but not all pathogens.

Deactivation of Macrophages with Interleukin-4 Is the Key to the Isolation of Tropheryma whippelii

Whipples disease is a systemic illness caused by a specific agent. Despite recognition of bacteria in lesions, efforts to isolate the causative agent remained futile. A novel strategy was devised to culture Whipple bacilli in deactivated mononuclear phagocytes. Infected tissue was inoculated into human phagocytes deactivated with interleukin (IL)-4, IL-10, and dexamethasone. Within 8-10 days, diastase-resistant periodic acid-Schiff-positive inclusions appeared, corresponding to intact and degenerating bacteria shown to be Tropheryma whippelii by electron microscopy and molecular analyses. T. whippelii was passaged several times in deactivated monocytes and a monoblastic cell line. Time-kinetics growth studies and comparative polymerase chain reaction analysis documented multiplication of T. whippelii in deactivated macrophages. Complementary studies showed that IL-4 rendered phagocytes permissive for T. whippelii, a strong indication that host factors contribute to the pathogenesis of Whipples disease. The propagation of T. whippelii will permit microbiologic, immunologic, seroepidemiologic, and therapeutic studies of this pathogen.

Soluble hemoglobin-haptoglobin scavenger receptor CD163 as a lineage-specific marker in the reactive hemophagocytic syndrome.

Abstract:  Reactive hemophagocytic syndrome (RHS) is a disease of overwhelming macrophage activity triggered by infection, malignancy or autoimmune disorders. Currently used laboratory markers for the quantitative assessment of monocyte/macrophage activation lack lineage‐restricted expression patterns and thus specificity. Serum levels of the macrophage specific scavenger receptor CD163 were detemined by enzyme‐linked immunosorbent assay (ELISA) and were found to be highly increased in patients with RHS (median 39.0 mg/L). Significantly lower levels were determined in patients with sepsis (median 9.1 mg/L), acute mononucleosis (median 8.2 mg/L), Leishmania infection (median 6.7 mg/L) and healthy controls (median 1.8 mg/L). Follow‐up of patients with a relapsing course of the disease revealed close correlations of sCD163 with clinical disease activity, serum ferritin and other markers of macrophage activity. Large sinusoidal accumulations of CD163 expressing macrophages actively engaged in phagocytosis of blood cells were detected in spleen sections of RHS patients. Our data suggests sCD163 to be a macrophage‐specific marker in patients with disorders of inappropriate macrophage activation.

In vitro susceptibility of fungi to killing by neutrophil granulocytes discriminates between primary pathogenicity and opportunism.

Pathogenic fungi, according to their propensity to cause infection of apparently normal individuals, can be grouped into either primary pathogens (e.g., Coccidioides, Histoplasma, Paracoccidioides, Blastomyces, and Sporothrix) or opportunists (e.g., Candida, Mucoraceae, Aspergillus spp., Petriellidium, and Trichosporon). There is, however, no unifying concept explaining the difference between the virulence of the two fungal categories. Previously we have speculated that neutrophils are the common denominator of the high natural resistance to opportunistic fungi. Accordingly, we then compared the susceptibility to killing by neutrophil granulocytes of Histoplasma, Blastomyces, Paracoccidioides, and Sporothrix with that of 14 opportunistic fungi. We found the four virulent dimorphic yeasts, in contrast to opportunistic fungi, to be resistant to killing by neutrophils. Virulent dimorphic yeasts were ingested by neutrophils, and triggered a respiratory burst comparably to opportunists but were less susceptible to hydrogen peroxide, suggesting that differences in the susceptibility to microbicidal products of leukocytes may explain the difference in virulence.

Induction of the CD163-dependent haemoglobin uptake by macrophages as a novel anti-inflammatory action of glucocorticoids.

Summary.  Highly efficient systems remove toxic and pro‐inflammatory haemoglobin (Hb) from the circulation and local sites of tissue damage. Macrophages are major Hb‐clearing cells; CD163 was recently recognized as the specific haemoglobin–haptoglobin scavenger receptor (HSR). We show that dexamethasone strongly induced the specific uptake of haemoglobin–haptoglobin complexes, CD163 mRNA transcription (13‐fold) and cell surface expression (10‐fold) by human macrophages. In contrast, the TH2‐cytokine interleukin 4 (IL‐4) completely suppressed functional CD163 expression. The range of functional receptor modulation reached a factor of 100 after 4 h of macrophage–ligand interaction. Based on these results, we propose the augmentation of Hb clearance as a novel anti‐inflammatory action of glucocorticoids.

Continuous Infusion of Escalated Doses of Amphotericin B Deoxycholate: An Open-Label Observational Study

Amphotericin B deoxycholate (AmB-d) remains a mainstay of antifungal therapy for immunocompromised patients, despite being associated with significant therapy-related toxicity. Because continuous infusion of AmB-d is better tolerated than traditional administration over 2-6 hours, we evaluated escalation of the AmB-d dose in 33 patients (31 of whom were neutropenic), for whom the initial dosage of AmB-d (1 mg/kg/day) was gradually increased to 2.0 mg/kg/day when renal function remained stable and the drug was tolerated. Dose escalation was possible without delay in 28 patients. Median duration of AmB-d therapy was 16 days (range, 7-72 days). Infusion-related reactions accompanied <18% of AmB-d infusions. Twenty-seven patients had a decrease in creatinine clearance while receiving AmB-d therapy. A >2-fold decrease in creatine clearance was observed in 5 patients, and the decrease was dose-limiting in only 1 patient; no dialysis was required. In conclusion, continuous infusion of AmB-d escalated to 2.0 mg/kg/day seems not to cause additional impairment of vital organ functions and to be well tolerated by most patients.

Gene Expression Profiling of Inflamed Human Endothelial Cells and Influence of Activated Protein C

Background—During systemic inflammation, activation of vascular endothelium by proinflammatory cytokines leads to hypotension, microvascular thrombosis, and organ damage. Recent data suggest a link between coagulation and inflammation through the activated protein C (APC) pathway. We studied gene expression profiles in human coronary artery endothelial cells (HCAECs) exposed to proinflammatory stimuli and the influence of APC on expression of candidate genes regulated by these stimuli. Methods and Results—HCAECs were stimulated with interleukin-1&bgr;, interferon-&ggr;, and tumor necrosis factor-&agr;. In gene expression profiling, 400 of 8400 genes were regulated >2-fold. Verification of selected candidate genes was achieved by measuring expression of mRNA species by real-time polymerase chain reaction, cytokine secretion by ELISA, and metabolites of tetrahydrobiopterin (BH4) biosynthesis by high-performance liquid chromatography. BH4 synthesis, interleukin-6, interleukin-8, monocyte chemotactic protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) were downregulated by APC at the transcriptional and protein level. Endothelial nitric oxide synthase, endothelial adhesion molecule, and vascular cell adhesion molecule-1 were not affected by APC. Activities of transcription factors c-Fos, FosB, and c-Rel were inhibited by APC in inflamed HCAECs. Conclusions—Our study revealed a novel antiinflammatory mechanism of APC-dependent gene regulation in HCAECs since c-Fos–dependent induction of MCP-1 and ICAM-1 was suppressed. APC downregulates expression and activity of genes related to inflammation, most pronounced under intermediate or mild inflammatory conditions. (Circulation. 2004;110:2903-2909.)