Gliotoxin as putative virulence factor and immunotherapeutic target in a cell culture model of cerebral aspergillosis.

The mycotoxin gliotoxin is an important metabolite produced by Aspergillus fumigatus, but its precise role in the pathogenesis of cerebral aspergillosis is not yet determined. We could demonstrate that growth in cerebrospinal fluid (CSF) induced the production and secretion of significant amounts of gliotoxin by A. fumigatus. These concentrations of 590-720nM were sufficient to reduce the viability of astrocytes and neurons, as well as of primary microglia, already after few hours of incubation. Annexin staining and electron microscopy revealed the induction of apoptosis rather than necrosis as the relevant mode of gliotoxin action in the brain. Furthermore, even a low gliotoxin concentration of 100nM, which was subtoxic for astrocytes, was able to significantly down-modulate the phagocytic capacity of astrocytes. In order to improve the current antimycotic therapy of cerebral aspergillosis by supporting innate immunity in the fight against Aspergillus, we aimed to neutralize the toxic potency of gliotoxin towards different brain cell types. Compounds such as dithiothreitol (DTT) or glutathione that reduce the internal disulfide bond of gliotoxin were shown here to be able to interfere with the gliotoxin-induced decrease of cell viability and to save the cells from induction of apoptosis. Thus, exploration of these substances may lead to novel approaches for adjunctive treatment of cerebral aspergillosis.

Secretion of a fungal protease represents a complement evasion mechanism in cerebral aspergillosis.

Complement represents a central immune weapon in the brain, but the high lethality of cerebral aspergillosis indicates a low efficacy of the antifungal complement attack. Studies with cerebrospinal fluid (CSF) samples derived from a patient with cerebral aspergillosis showed a degradation of complement proteins, implying that Aspergillus might produce proteases to evade their antimicrobial potency. Further investigations of this hypothesis showed that Aspergillus, when cultured in CSF to simulate growth conditions in the brain, secreted a protease that can cleave various complement proteins. Aspergillus fumigatus, the most frequent cause of cerebral aspergillosis, destroyed complement activity more efficiently than other Aspergillus species. The degradation of complement in CSF resulted in a drastic reduction of the capacity to opsonize fungal hyphae. Furthermore, the Aspergillus-derived protease could diminish the amount of complement receptor CR3, a surface molecule to mediate eradication of opsonized pathogens, on granulocytes and microglia. The lack of these prerequisites caused a significant decrease in phagocytosis of primary microglia. Additional studies implied that the complement-degrading activity shares many characteristics with the previously described alkaline protease Alp1. To improve the current therapy for cerebral aspergillosis, we tried to regain the antifungal effects of complement by repressing the secretion of this degrading activity. Supplementation of CSF with nitrogen sources rescued the complement proteins and abolished any cleavage. Glutamine or arginine are of special interest for this purpose since they represent endogenous substances in the CNS and might be included in a future supportive therapy to reduce the high lethality of cerebral aspergillosis.

Complement induction and complement evasion in patients with cerebral aspergillosis.

Cerebral aspergillosis is a mostly lethal infection of the central nervous system. Former results identified low cerebral complement levels as one cause for insufficient immune reaction. Therefore we studied cerebral complement expression after fungal invasion and investigated putative mechanisms of Aspergillus spp to cope with the complement-induced selection pressure. Brain tissue derived from patients with cerebral aspergillosis or non-infected individuals was analyzed immunohistochemically for complement synthesis. Correlations between expression levels were determined statistically. Increased complement synthesis, a prerequisite for strengthened antifungal potency, was visible in resident astrocytes, neurons, oligodendrocytes as well as in infiltrating macrophages after fungal infection. Surprisingly, microglia, although regarded as major immune cells, only marginally participated in synthesis of most complement proteins. Several evasion mechanisms were found that help the fungus to establish a cerebral infection even in the presence of complement: Fungal hyphae limit the surface deposition of C3 and thus interfere with complement-dependent phagocytosis. Furthermore, the "sealing off" in brain abscesses not only inhibits fungal spreading but also forms protection shields against complement attack. Complement indeed seems to represent an important selection pressure and evokes the development of fungal evasion mechanisms. Counteractions for these evasion processes might represent interesting therapeutic approaches.

Immune response to retroviral infections of the brain.

Various neurological manifestations of retroviral infections have been reported, including peripheral neuropathy, encephalopathy and neuronal degeneration. After penetration into the central nervous system (CNS) the invading retroviruses meet a unique immunological situation that differs significantly from that in the periphery. Due to the blood-brain barrier with its general access restrictions peripheral T-cells, monocytes and B-cells are only "guests" in the brain; instead the immune balance is shifted in favour of the local innate immunity with microglia, astrocytes, cytokines/chemokines and complement forming the dominating defence network. The present article focuses on the most important retroviral infections and highlights the immunological aspects of the neuropathogenesis induced by selected retroviruses. These aspects include: (i) local and infiltrated immune cells as targets of retroviral infection; (ii) stimulation of the cerebral immunity network by retroviruses and subsequent steps of antiviral defence; and (iii) immune activation products as potential contributors to neural damage in the sensitive brain tissue.

Effectiveness and handling characteristics of a three-layer polymer dura substitute: a prospective multicenter clinical study.

OBJECT: The goal of this study was to assess the effectiveness and handling characteristics of a dura substitute composed of two outer layers of expanded polytetrafluoroethylene (PTFE) and a middle layer consisting of an elastomeric fluoropolymer. METHODS: In a prospective multicenter study, the dura substitute was implanted using a standard technique in 119 patients undergoing cranial or spinal surgery requiring duraplasty. Intraoperative assessments of the dura patch consisted of testing for cerebrospinal fluid (CSF) leakage employing the Valsalva maneuver and a surgeon's standard evaluation of the handling characteristics of the device. Postoperative assessments conducted during a mean follow-up time of 15.7 months (range 0.3-45.6 months) consisted of physical examinations, routine computed tomography (CT) or magnetic resonance (MR) imaging studies, and histological studies of any removed dura patches. The mean age of the 119 patients was 40 years (range < 1-81 years). The dura substitute was implanted cranially in 102 patients and spinally in 17. Intraoperative assessment including the Valsalva maneuver led to application of additional sutures in 17 patients. Handling features were rated very good to excellent. Postoperative clinical evaluation resulted in 79 excellent and 18 good results. Imaging studies (MR imaging studies in 69 patients and CT studies in 34 patients) showed no adhesions in 87 patients and minimal adhesions in seven patients (the dura was not visualized in nine patients). Postoperative complications occurred in 12 patients. There were six cases of CSF leakage, three cases of extradural hematoma, one case of arachnoid fibrosis after decompression of a Chiari malformation Type I, and two cases of infection. Eight (7%) of these complications were potentially related to the dura patch. CONCLUSIONS: In a large, multicenter clinical study of the use of an expanded-PTFE-containing dura substitute, the device was found to be easy to handle and implant. No serious dura patch-related intraoperative adverse events were observed. Postoperatively, there were no major sealing problems or long-term complications. In two cases the patch had to be removed due to fibrosis and infection. The three-layer polymer dura substitute appears to be safe and effective in minimizing CSF leakage and adhesion formation, and its use avoids any risk of prion disease transmission.

Antifungal activity of the local complement system in cerebral aspergillosis.

Dissemination of aspergillosis into the central nervous system is associated with nearly 100% mortality. To study the reasons for the antifungal immune failure we analyzed the efficacy of cerebral complement to combat the fungus Aspergillus. Incubation of Aspergillus in non-inflammatory cerebrospinal fluid (CSF) revealed that complement levels were sufficient to obtain a deposition on the surface, but opsonization was much weaker than in serum. Consequently complement deposition from normal CSF on fungal surface stimulated a very low phagocytic activity of microglia, granulocytes, monocytes and macrophages compared to stimulation by conidia opsonized in serum. Similarly, opsonization of Aspergillus by CSF was not sufficient to induce an oxidative burst in infiltrating granulocytes, whereas conidia opsonized in serum induced a clear respiratory signal. Thus, granulocytes were capable of considerably reducing the viability of serum-opsonized Aspergillus conidia, but not of conidia opsonized in CSF. The limited efficacy of antifungal attack by cerebral complement can be partly compensated by enhanced synthesis, leading to elevated complement concentrations in CSF derived from a patient with cerebral aspergillosis. This inflammatory CSF was able to induce (i) a higher complement deposition on the Aspergillus surface than non-inflammatory CSF, (ii) an accumulation of complement activation products and (iii) an increase in phagocytic and killing activity of infiltrating granulocytes. However, levels and efficacy of the serum-derived complement were not reached. These data indicate that low local complement synthesis and activation may represent a central reason for the insufficient antifungal defense in the brain and the high mortality rate of cerebral aspergillosis.

Complement synthesis and activation in the brain of SIV-infected monkeys.

Complement is one of the most critical defence tools against cerebral infections, but uncontrolled complement biosynthesis and activation can induce profound brain tissue damage. To clarify the role of complement in the pathogenesis of AIDS-associated neurological disorders, we analysed the synthesis of complement in the brains of SIV-infected rhesus macaques. Using immunohistochemical staining we could show that the cerebral synthesis of complement factors C1q and C3 was strongly upregulated in SIV-infected monkeys compared to the spontaneous synthesis in uninfected control monkeys. Astrocytes, neurons, microglia, infiltrating macrophages and multinuclear giant cells all contribute to the high amounts of C1q and C3 in the brain. Secreted C1q and C3 are also deposited on the membrane of neurons, a prerequisite for formation of the membrane-driven lytic membrane attack complex. The membrane deposition thus might suggest complement-induced lysis of bystander neurons as a potential mechanism for cell damage during viral infection of the brain.

HIV-1 induces complement factor C3 synthesis in astrocytes and neurons by modulation of promoter activity.

Virus-induced complement expression and activation in the brain is hypothesized to contribute to the process of neurodegeneration in AIDS-associated neurological disorders. Previous experiments have shown that the human immunodeficiency virus (HIV) upregulates the low basal production of complement factor C3 in astrocytes and neurons. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analysed the mechanism of the HIV-induced modulation of C3 expression. Detailed studies using different C3 promoter constructs revealed that HIV activates the synthesis of C3 by stimulation of the promoter. This HIV-induced promoter activation could be measured both in different astrocytic cell lines and in neurons. Deletion constructs of the C3 promoter defined the IL-6/IL-1beta responsive element within the promoter region as a central element for the responsiveness of the C3 promoter towards the influence of HIV. A binding site for the transcription factor C/EBPdelta was identified as important regulatory domain within the IL-6/IL-1beta responsive element, since a point mutation which eliminates the binding capacity of C/EBPdelta to this site also abolishes the induction by HIV-1. Similarly, the viral proteins Nef and gp41 which had also been shown to stimulate the synthesis of C3, exert their effect via the IL-6/IL-1beta responsive element with binding of the transcription factor C/EBPdelta representing the critical step. Our experiments clearly define the mechanism for the induction of complement factors in the HIV-infected brain and reveal a decisive role of the regulator protein C/EBPdelta for the HIV-induced increase in C3 expression.

Neurochemical monitoring using intracerebral microdialysis during cardiac resuscitation.

OBJECTIVE: After cardiac resuscitation, hypoxic encephalopathy often is the limiting factor regarding outcome due to the oxygen sensitivity of the brain. Intracerebral microdialysis is a highly sensitive technique to monitor cerebral energy metabolism and for the early detection of cerebral hypoxia. PATIENT AND INTERVENTIONS: A 34-year-old male patient who had to be craniectomized due to a mass demanding middle cerebral artery infarct. A microdialysis catheter was inserted into the left frontal lobe and, as control, into the abdominal subcutaneous adipose tissue. Pulmonary embolism and asystolia leading to cardiac resuscitation occurred. MEASUREMENTS AND RESULTS: The cerebral chemical markers of energy metabolism glucose, lactate, pyruvate, and the marker of cell membrane damage glycerol were measured. Except for subcutaneous glucose, all markers showed a sudden and significant increase during resuscitation and a prolonged period afterwards. After some hours all values returned to normal. CONCLUSION: This is the first reported case of monitoring neurochemical markers using intracerebral microdialysis during cardiac resuscitation. The findings indicate the importance of early and efficient resuscitation and demonstrate that deviations in cerebral energy metabolism are reversible.