Quantitative proteomic analysis of cerebrospinal fluid from patients with idiopathic facial nerve palsy.

BACKGROUND AND PURPOSE: Idiopathic facial palsy (IFP) accounts for over 60% of peripheral facial palsy (FP) cases. The cause of IFP remains to be determined. Possible etiologies are nerve swelling due to inflammation and/or viral infection. In this study, we applied an integrative mass spectrometry approach to identify possibly altered protein patterns in the cerebrospinal fluid (CSF) of IFP patients. METHODS: We obtained CSF samples from 34 patients with FP. In four patients, varicella-zoster virus was the cause (VZV-FP). Among the 30 patients diagnosed with IFP, 17 had normal CSF parameters, five had slightly elevated CSF cell counts and normal or elevated CSF protein, and eight had normal CSF cell counts but elevated CSF protein. Five patients with primary headache served as controls. All samples were tested for viral pathogens by PCR and subjected to liquid chromatography tandem mass spectrometry and bioinformatics analysis and multiplex cytokine/chemokine arrays. RESULTS: All CSF samples, except those from VZV-FP patients, were negative for all tested pathogens. The protein composition of CSF samples from IFP patients with normal CSF was comparable to controls. IFP patients with elevated CSF protein showed dysregulated proteins involved in inflammatory pathways, findings which were similar to those in VZV-FP patients. Multiplex analysis revealed similarly elevated cytokine levels in the CSF of IFP patients with elevated CSF protein and VZV-FP. CONCLUSIONS: Our study revealed a subgroup of IFP patients with elevated CSF protein that showed upregulated inflammatory pathways, suggesting an inflammatory/infectious cause. However, no evidence for an inflammatory cause was found in IFP patients with normal CSF.

In vivo proteomics identifies the competence regulon and AliB oligopeptide transporter as pathogenic factors in pneumococcal meningitis.

Streptococcus pneumoniae (pneumococci) is a leading cause of severe bacterial meningitis in many countries worldwide. To characterize the repertoire of fitness and virulence factors predominantly expressed during meningitis we performed niche-specific analysis of the in vivo proteome in a mouse meningitis model, in which bacteria are directly inoculated into the cerebrospinal fluid (CSF) cisterna magna. We generated a comprehensive mass spectrometry (MS) spectra library enabling bacterial proteome analysis even in the presence of eukaryotic proteins. We recovered 200,000 pneumococci from CSF obtained from meningitis mice and by MS we identified 685 pneumococci proteins in samples from in vitro filter controls and 249 in CSF isolates. Strikingly, the regulatory two-component system ComDE and substrate-binding protein AliB of the oligopeptide transporter system were exclusively detected in pneumococci recovered from the CSF. In the mouse meningitis model, AliB-, ComDE-, or AliB-ComDE-deficiency resulted in attenuated meningeal inflammation and disease severity when compared to wild-type pneumococci indicating the crucial role of ComDE and AliB in pneumococcal meningitis. In conclusion, we show here mechanisms of pneumococcal adaptation to a defined host compartment by a proteome-based approach. Further, this study provides the basis of a promising strategy for the identification of protein antigens critical for invasive disease caused by pneumococci and other meningeal pathogens.

Combined therapy with ceftriaxone and doxycycline does not improve the outcome of meningococcal meningitis in mice compared to ceftriaxone monotherapy.

BACKGROUND: Meningococcal meningitis (MM) is a life-threatening disease associated with approximately 10% case fatality rates and neurological sequelae in 10-20% of the cases. Recently, we have shown that the matrix metalloproteinase (MMP) inhibitor BB-94 reduced brain injury in a mouse model of MM. The present study aimed to assess whether doxycycline (DOX), a tetracycline that showed a neuroprotective effect as adjuvant therapy in experimental pneumococcal meningitis (PM), would also exert a beneficial effect when given as adjunctive therapy to ceftriaxone (CRO) in experimental MM. METHODS: BALB/c mice were infected by the intracisternal route with a group C Neisseria meningitidis strain. Eighteen h post infection (hpi), animals were randomised for treatment with CRO [100 mg/kg subcutaneously (s.c.)], CRO plus DOX (30 mg/kg s.c.) or saline (control s.c.). Antibiotic treatment was repeated 24 and 40 hpi. Mouse survival and clinical signs, bacterial counts in cerebella, brain damage, MMP-9 and cyto/chemokine levels were assessed 48 hpi. RESULTS: Analysis of bacterial load in cerebella indicated that CRO and CRO + DOX were equally effective at controlling meningococcal replication. No differences in survival were observed between mice treated with CRO (94.4%) or CRO + DOX (95.5%), (p > 0.05). Treatment with CRO + DOX significantly diminished both the number of cerebral hemorrhages (p = 0.029) and the amount of MMP-9 in the brain (p = 0.046) compared to untreated controls, but not to CRO-treated animals (p > 0.05). Levels of inflammatory markers in the brain of mice that received CRO or CRO + DOX were not significantly different (p > 0.05). Overall, there were no significant differences in the parameters assessed between the groups treated with CRO alone or CRO + DOX. CONCLUSIONS: Treatment with CRO + DOX showed similar bactericidal activity to CRO in vivo, suggesting no antagonist effect of DOX on CRO. Combined therapy significantly improved mouse survival and disease severity compared to untreated animals, but addition of DOX to CRO did not offer significant benefits over CRO monotherapy. In contrast to experimental PM, DOX has no adjunctive activity in experimental MM.

Virulence Traits of a Serogroup C Meningococcus and Isogenic cssA Mutant, Defective in Surface-Exposed Sialic Acid, in a Murine Model of Meningitis.

In serogroup C Neisseria meningitidis, the cssA (siaA) gene codes for an UDP-N-acetylglucosamine 2-epimerase that catalyzes the conversion of UDP-N-acetyl-α-d-glucosamine into N-acetyl-d-mannosamine and UDP in the first step in sialic acid biosynthesis. This enzyme is required for the biosynthesis of the (α2→9)-linked polysialic acid capsule and for lipooligosaccharide (LOS) sialylation. In this study, we have used a reference serogroup C meningococcal strain and an isogenic cssA knockout mutant to investigate the pathogenetic role of surface-exposed sialic acids in a model of meningitis based on intracisternal inoculation of BALB/c mice. Results confirmed the key role of surface-exposed sialic acids in meningococcal pathogenesis. The 50% lethal dose (LD50) of the wild-type strain 93/4286 was about four orders of magnitude lower than that of the cssA mutant. Compared to the wild-type strain, the ability of this mutant to replicate in brain and spread systemically was severely impaired. Evaluation of brain damage evidenced a significant reduction in cerebral hemorrhages in mice infected with the mutant in comparison with the levels in those challenged with the wild-type strain. Histological analysis showed the typical features of bacterial meningitis, including inflammatory cells in the subarachnoid, perivascular, and ventricular spaces especially in animals infected with the wild type. Noticeably, 80% of mice infected with the wild-type strain presented with massive bacterial localization and accompanying inflammatory infiltrate in the corpus callosum, indicating high tropism of meningococci exposing sialic acids toward this brain structure and a specific involvement of the corpus callosum in the mouse model of meningococcal meningitis.

Inhibition of DAMP signaling as an effective adjunctive treatment strategy in pneumococcal meningitis.

BACKGROUND: Pneumococcal meningitis remains a potentially lethal and debilitating disease, mainly due to brain damage from sustained inflammation. The release of danger-associated molecular patterns (DAMPs), like myeloid-related protein 14 (MRP14) and high mobility group box 1 protein (HMGB1), plays a major role in persistence of inflammation. In this study, we evaluated if paquinimod, an MRP14-inhibitor, and an anti-HMGB1 antibody can improve clinical outcome as adjunctive therapeutics in pneumococcal meningitis. METHODS: We tested the adjuvant administration of paquinimod and the anti-HMGB1 antibody in our pneumococcal meningitis mouse model assessing clinical (clinical score, open-field-test, temperature) and pathophysiological parameters (intracranial pressure, white blood cell count in CSF, bleeding area) as well as bacterial titers in blood and brain 24 h after administration and 48 h after infection. Furthermore, we explored the interactions of these two agents with dexamethasone, the standard adjuvant treatment in pneumococcal meningitis (PM), and daptomycin, a non-bacteriolytic antibiotic preventing pathogen-associated molecular pattern (PAMP) release. RESULTS: Adjunctive inhibition of MRP14 or HMGB1 reduced mortality in mice with PM. This effect was lost when the two anti-DAMP agents were given simultaneously, possibly due to excessive immunosuppression. Combining anti-PAMP (daptomycin) and anti-DAMP treatments did not produce synergistic results; instead, the anti-DAMP treatment alone was sufficient and superior. The combination of anti-HMGB1 with dexamethasone did not diminish the effect of the former. CONCLUSIONS: DAMP inhibition possesses good potential as an adjuvant treatment approach in PM, as it improves clinical outcome and can be given together with the standard adjuvant dexamethasone without drug effect loss in experimental PM.

NK-cell dysfunction in human renal carcinoma reveals diacylglycerol kinase as key regulator and target for therapeutic intervention.

The relevance of NK cells in tumor control is well established in mouse models and human hematologic malignancies; however, their contribution to the control of human solid tumors remains disputed due to problems with in situ detection and reports of functional inactivity in the tumor milieu. In this study, we established a reliable in situ detection method for NK cells. Moreover, we performed analysis to elucidate mechanisms that impair NK-cell function in the tumor milieu and thereby identify therapeutic targets that allow recovery of NK-cell functionality. It was observed that NK cells from clear cell renal cell carcinoma (ccRCC), compared to NK cells from nontumor kidney and peripheral blood lymphocytes (PBLs), displayed conjoint phenotypic alterations and dysfunction induced by the tumor milieu, which were associated mechanistically with high levels of signaling attenuator diacylglycerol kinase (DGK)-α and blunted mitogen-activated protein kinase pathway activation (ERK1/2, Jun kinase). Reinstating NK-cell functionality was possible by DGK inhibition or brief IL-2 culture, interventions that de-repressed the ERK pathway. The extent of alteration and magnitude of recovery could be linked to NK-cell frequency within ccRCC-infiltrating lymphocytes, possibly explaining the observed survival benefit of patients with NK(high) tumors. In conclusion, DGK-mediated dampening of the ERK pathway ensuing in NK-cell dysfunction was identified as an important escape mechanism in ccRCC. DGK and the ERK pathway thus emerge as promising therapeutic targets to restore suppressed NK-cell activity for the improvement of antitumor immunity.

High DGK-α and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention.

CD8(+) tumor-infiltrating T cells (CD8-TILs) are found in many types of tumors including human renal cell carcinoma. However, tumor rejection rarely occurs, suggesting limited functional activity in the tumor microenvironment. In this study, we document that CD8-TILs are unresponsive to CD3 stimulation, showing neither lytic activity, nor lytic granule exocytosis, nor IFN-γ production. Mechanistically, no deficits in TCR proximal signaling molecules (lymphocyte-specific protein tyrosine kinase, phospholipase Cγ) were identified. In contrast, distal TCR signaling was suppressed, as T cells of TILs showed strongly reduced steady-state phosphorylation of the MAPK ERK and were unable to increase phosphorylation of ERK and JNK as well as AKT and AKT client proteins (IκB, GSK3) after stimulation. These deficits were tumor-specific as they were not observed in CD8(+) T cells infiltrating non-tumor kidney areas (CD8(+) non-tumor kidney-infiltrating lymphocytes; CD8-NILs). Diacylglycerol kinase-α (DGK-α) was more highly expressed in CD8-TILs compared with that in CD8-NILs, and its inhibition improved ERK phosphorylation and lytic granule exocytosis. Cultivation of TILs in low-dose IL-2 reduced DGK-α protein levels, increased steady-state phosphorylation of ERK, improved stimulation-induced phosphorylation of ERK and AKT, and allowed more CD8-TILs to degranulate and to produce IFN-γ. Additionally, the protein level of the AKT client molecule p27kip, an inhibitory cell cycle protein, was reduced, whereas cyclin E, which promotes G1-S phase transition, was increased. These results indicate that the tumor-inflicted deficits of TILs are reversible. DGK-α inhibition and provision of IL-2 signals could be strategies to recruit the natural CD8(+) T cells to the anti-tumor response and may help prevent inactivation of adoptively transferred T cells thereby improving therapeutic efficacy.

Performance and clinical outcomes in telestroke remain robust during the COVID-pandemic: insight into the NEVAS network.

BACKGROUND: The COVID-19 pandemic had significant impact on global healthcare, including stroke management. Telemedical stroke networks have emerged with positive results for patient outcome in rural areas without stroke expertise. However, telestroke faced enormous on-site challenges during the pandemic. So far, data on performance and clinical outcomes in telestroke settings during the COVID-pandemic are scarce. METHODS: We retrospectively analyzed data from stroke patients treated in four spoke hospitals of the Bavarian telestroke network NEVAS in 2020-2021 and 2019 as reference year and compared the 3 years for various parameters. Primary outcome was functional outcome according to the modified Rankin scale (mRS). Secondary outcome parameters included time intervals, periprocedural intracranial hemorrhage rates, and mortality. RESULTS: In 2019-2021, 2820 patients were treated for acute ischemic stroke with an admission decrease of 10% during the pandemic. Of those, 241 received only IVT and 204 were transferred to our center for MT. Door-to-imaging, door-to-needle, and symptom-onset-to-groin times remained comparable in the 3 years. Complication rates remained at a low level. Good clinical outcome rates (mRS 0-2) at discharge remained stable for all stroke patients (82-84%) and for those treated with IVT (64-77%). Good clinical outcome rates at 3 month follow-up for MT patients declined in 2020 (23% vs. 35% in 2019) but recovered again in 2021 (42%). Mortality rates did not increase for all patient groups analyzed. CONCLUSIONS: Stroke care remained robust during the COVID-pandemic within our network, indicating that well-established telestroke networks can overcome unexpected critical challenges such as a pandemic, guaranteeing best practice stroke care in rural areas.

Basilar artery occlusion: drip-and-ship versus direct-to-center for mechanical thrombectomy within the Neurovascular Network of Southwest Bavaria (NEVAS).

BACKGROUND: Recent clinical trials revealed a substantial clinical benefit for mechanical thrombectomy (MT) in patients with basilar artery occlusion (BAO). While urban areas are sufficiently covered with comprehensive stroke centers and MT expertise, rural areas lack such resources. Structured telemedical stroke networks offer rural hospitals instant consultation by stroke experts, enabling swift administration of intravenous thrombolysis (IVT) on-site and transportation for MT. For BAO patients, data on performance and clinical outcomes in telemedical stroke networks are lacking. METHODS: We retrospectively analyzed data from patients with acute BAO eligible for MT: those treated directly in our comprehensive stroke center (direct-to-center/DC) and those treated in rural hospitals that were telemedically consulted by the Neurovascular Network of Southwest Bavaria (NEVAS) and transferred to our center for MT (drip-and-ship, DS). Key time intervals, stroke management performance and functional outcome after 90 days were compared. RESULTS: Baseline characteristics, including premorbid status and stroke severity, were comparable. Time from symptom onset to IVT was identical in both groups (118 min). There was a delay of 180 min until recanalization in DS patients, mainly due to patient transport for MT. Procedural treatment time intervals, success of recanalization and complications were comparable. Clinical outcome at 3 months follow-up of DS patients was not inferior to DC patients. CONCLUSION: We show for the first time that patients with BAO in rural areas benefit from a structured telemedicine network such as NEVAS, regarding both on-site processing and drip-and-ship for MT. Clinical outcomes are comparable among DS and DC patients.