Cell-free mitochondrial DNA in CSF is associated with early viral rebound, inflammation, and severity of neurocognitive deficits in HIV infection.

Cell-free mitochondiral DNA (mtDNA) is an immunogenic molecule associated with many inflammatory conditions. We evaluated the relationship between cell-free mtDNA in cerebrospinal fluid (CSF) and neurocognitive performance and inflammation during HIV infection. In a cross-sectional analysis, we evaluated the association of mtDNA levels with clinical assessments, inflammatory markers, and neurocognitive performance in 28 HIV-infected individuals. In CSF, we measured mtDNA levels by droplet digital PCR, and soluble CD14 and CD163, neurofilament light, and neopterin by ELISA. In blood and CSF, we measured soluble IP-10, MCP-1, TNF-α, and IL-6 by ELISA, and intracellular expression of IL-2, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells by flow cytometry. We also evaluated the relationship between CSF pleocytosis and mtDNA longitudinally in another set of five individuals participating in an antiretroviral treatment (ART) interruption study. Cell-free CSF mtDNA levels strongly correlated with neurocognitive performance among individuals with neurocognitive impairment (NCI) (r = 0.77, p = 0.001). CSF mtDNA also correlated with levels of IP-10 in CSF (r = 0.70, p = 0.007) and MCP-1 in blood plasma (r = 0.66, p = 0.01) in individuals with NCI. There were no significant associations between inflammatory markers and mtDNA in subjects without NCI, and levels of mtDNA did not differ between subjects with and without NCI. MtDNA levels preceded pleocytosis and HIV RNA following ART interruption. Cell-free mtDNA in CSF was strongly associated with the severity of neurocognitive dysfunction and inflammation only in individuals with NCI. Our findings suggest that within a subset of subjects cell-free CSF mtDNA is associated with inflammation and degree of NCI.

Cerebrospinal fluid markers reveal intrathecal inflammation in progressive multiple sclerosis.

OBJECTIVE: The management of complex patients with neuroimmunological diseases is hindered by an inability to reliably measure intrathecal inflammation. Currently implemented laboratory tests developed >40 years ago either are not dynamic or fail to capture low levels of central nervous system (CNS) inflammation. Therefore, we aimed to identify and validate biomarkers of CNS inflammation in 2 blinded, prospectively acquired cohorts of untreated patients with neuroimmunological diseases and embedded controls, with the ultimate goal of developing clinically useful tools. METHODS: Because biomarkers with maximum utility reflect immune phenotypes, we included an assessment of cell specificity in purified primary immune cells. Biomarkers were quantified by optimized electrochemiluminescent immunoassays. RESULTS: Among markers with cell-specific secretion, soluble CD27 is a validated biomarker of intrathecal T-cell activation, with an area under the receiver operating characteristic curve of 0.97. Comparing the quantities of cerebrospinal fluid (CSF) immune cells and their respective cell-specific soluble biomarkers (released by CSF cells as well as their counterparts in CNS tissue) provided invaluable information about stationary CNS immune responses, previously attainable via brain biopsy only. Unexpectedly, progressive and relapsing-remitting multiple sclerosis (MS) patients have comparable numbers of activated intrathecal T and B cells, which are preferentially embedded in CNS tissue in the former group. INTERPRETATION: The cell-specific biomarkers of intrathecal inflammation may improve diagnosis and management of neuroimmunological diseases and provide pharmacodynamic markers for future therapeutic developments in patients with intrathecal inflammation that is not captured by imaging, such as in progressive MS.

Flow cytometric analysis of T cell/monocyte ratio in clinically isolated syndrome identifies patients at risk of rapid disease progression.

BACKGROUND: Multiple sclerosis is a chronic inflammatory central nervous system disease diagnosed by clinical presentation and characteristic magnetic resonance imaging findings. The role of cerebrospinal fluid (CSF) analysis has been emphasized in particular in the context of differential diagnosis in patients with a first episode suggestive of multiple sclerosis. OBJECTIVE: We investigated here the potential additional value of analysis of CSF cellularity by fluorescence activated cell sorting (FACS) in the setting of a routine diagnostic work-up in our inpatient clinic. METHODS: CSF cells from back-up samples from patients with suspected chronic inflammatory central nervous system disorder were analyzed by FACS and correlated with clinical data, magnetic resonance imaging findings and oligoclonal band status. RESULTS: We found distinct changes of T cell/monocyte (CD4/CD14) and B cell/monocyte (CD20/CD14) ratios between clinically isolated syndrome (CIS)/multiple sclerosis and other neurologic diseases or other inflammatory neurologic diseases. In particular, patients with a rapid transition from CIS to multiple sclerosis had an elevated CD4/CD14 ratio. A subgroup analysis showed diagnostic value of CD4/CD8 ratio in the differential diagnosis of CIS/multiple sclerosis to neurosarcoidosis. CONCLUSION: The diagnostic and prognostic accuracy of autoimmune neuroinflammatory diseases can be improved by FACS analysis of CSF cells.

Soluble CD14 in cerebrospinal fluid is associated with markers of inflammation and axonal damage in untreated HIV-infected patients: a retrospective cross-sectional study.

BACKGROUND: HIV-associated cognitive impairment has declined since the introduction of combination antiretroviral treatment (cART). However, milder forms of cognitive impairment persist. Inflammation in the cerebrospinal fluid (CSF) has been associated with cognitive impairment, and CSF neurofilament light chain protein (NFL) and CSF neopterin concentrations are increased in those patients. Microbial translocation in HIV infection has been suggested to contribute to chronic inflammation, and lipopolysaccharide (LPS) and soluble CD14 (sCD14) are markers of microbial translocation and the resulting monocyte activation, respectively. We hypothesised that microbial translocation contributes to inflammation and axonal damage in the central nervous system (CNS) in untreated HIV infection. METHODS: We analyzed paired samples of plasma and CSF from 62 HIV-infected, untreated patients without cognitive symptoms from Sahlgrenska University Hospital, Gothenburg, Sweden. Measurements of neopterin and NFL in CSF were available from previous studies. Plasma and CSF sCD14 was measured using ELISA (R&D, Minneapolis, MN), and plasma and CSF LPS was measured using LAL colorimetric assay (Lonza, Walkersville, MD, USA). Univariate and multivariate regression analyses were performed. RESULTS: LPS in plasma was associated with plasma sCD14 (r = 0.31, P = 0.015), and plasma sCD14 was associated with CSF sCD14 (r = 0.32, P = 0.012). Furthermore, CSF sCD14 was associated with NFL (r = 0.32, P = 0.031) and neopterin (r = 0.32, P = 0.012) in CSF. LPS was not detectable in CSF. In a multivariate regression model CSF sCD14 remained associated with NFL and neopterin after adjusting for age, CD4+ cell count, and HIV RNA in CSF. CONCLUSIONS: In a group of untreated, HIV-infected patients LPS was associated with sCD14 in plasma, and plasma sCD14 was associated CSF sCD14. CSF sCD14 were associated with markers of CNS inflammation and axonal damage. This suggest that microbial translocation might be a driver of systemic and CNS inflammation. However, LPS was not detectable in the CSF, and since sCD14 is a marker of monocyte activation sCD14 may be increased due to other causes than microbial translocation. Further studies regarding cognitive impairment and biomarkers are warranted to fully understand causality.

Diagnostic accuracy of presepsin (sCD14-ST) for prediction of bacterial infection in cerebrospinal fluid samples from children with suspected bacterial meningitis or ventriculitis.

Children with temporary external ventricular drains (EVD) are prone to nosocomial infections. Diagnosis of bacterial meningitis and ventriculitis in these children is challenging due to frequent blood contamination of cerebrospinal fluid (CSF) and the presence of chemical ventriculitis. The aim of this study was to compare diagnostic accuracy of presepsin (sCD14-ST), a novel biomarker of bacterial infection in CSF, to predict bacterial infection in comparison to the accuracy of established biomarkers like those demonstrated in biochemical analysis of CSF. We conducted a prospective study with 18 children with suspected bacterial meningitis or ventriculitis who had 66 episodes of disease. CSF samples were taken from external ventricular drainage. We measured presepsin in CSF, as well as CSF leukocyte count, glucose, and proteins. CSF was also taken to prove bacterial infection with culture methods or with 16S rRNA gene broad-range PCR (SepsiTest; Molzym, Germany). Infection was clinically confirmed in 57 (86%) episodes of suspected meningitis or ventriculitis. Chemical ventriculitis was diagnosed in 9 (14%) episodes of suspected meningitis or ventriculitis. Diagnostic accuracies presented as area under the curve (AUC) for sCD14-ST, leukocytes, and proteins measured in CSF were 0.877 (95% confidence interval [CI], 0.793 to 0.961), 0.798 (95% CI, 0.677 to 0.920), and 0.857 (95% CI, 0.749 to 0.964), respectively. With CSF culture, we detected bacteria in 17 samples, compared to 37 detected with broad-range PCR. It was found that presepsin was present at a significantly higher level in children with clinically proven ventriculitis than in those without meningitis or ventriculitis. Diagnostic accuracies of presepsin were superior to those of leukocytes or proteins in CSF. Presepsin-guided 16S rRNA gene PCR could be used in everyday clinical practice to improve etiological diagnosis of meningitis and ventriculitis and to prescribe more appropriate antibiotics.

Monocyte and microglial activation in patients with mood-stabilized bipolar disorder.

BACKGROUND: Bipolar disorder is associated with medical comorbidities that have been linked to systemic inflammatory mechanisms. There is, however, limited evidence supporting a role of neuroinflammation in bipolar disorder. Here we tested whether microglial activation and associated tissue remodelling processes are related to bipolar disorder by analyzing markers in cerebrospinal fluid (CSF) and serum from patients and healthy controls. METHODS: Serum was sampled from euthymic patients with bipolar disorder and healthy controls, and CSF was sampled from a large subset of these individuals. The levels of monocyte chemoattractant protein-1 (MCP-1), YKL-40, soluble cluster of differentiation 14 (sCD14), tissue inhibitor of metalloproteinases-1 (TIMP-1) and tissue inhibitor of metalloproteinases-2 (TIMP-2), were measured, and we adjusted comparisons between patients and controls for confounding factors. RESULTS: We obtained serum samples from 221 patients and 112 controls and CSF samples from 125 patients and 87 controls. We found increased CSF levels of MCP-1 and YKL-40 and increased serum levels of sCD14 and YKL-40 in patients compared with controls; these differences remained after controlling for confounding factors, such as age, sex, smoking, blood-CSF barrier function, acute-phase proteins and body mass index. The CSF levels of MCP-1 and YKL-40 correlated with the serum levels, whereas the differences between patients and controls in CSF levels of MCP-1 and YKL-40 were independent of serum levels. LIMITATIONS: The cross-sectional study design precludes conclusions about causality. CONCLUSION: Our results suggest that both neuroinflammatory and systemic inflammatory processes are involved in the pathophysiology of bipolar disorder. Importantly, markers of immunological processes in the brain were independent of peripheral immunological activity.

Very low levels of 25-hydroxyvitamin D are not associated with immunologic changes or clinical outcome in South African patients with HIV-associated cryptococcal meningitis.

BACKGROUND: Vitamin D deficiency is associated with impaired immune responses and increased susceptibility to a number of intracellular pathogens in individuals infected with human immunodeficiency virus (HIV). It is not known whether such an association exists with Cryptococcus neoformans. METHODS: Levels of 25-hydroxyvitamin D (25[OH]D) were measured in 150 patients with cryptococcal meningitis (CM) and 150 HIV-infected controls in Cape Town, South Africa, and associations between vitamin D deficiency and CM were examined. The 25-hydroxyvitamin D levels and cryptococcal notifications were analyzed for evidence of reciprocal seasonality. Associations between 25(OH)D levels and disease severity, immune responses, and microbiological clearance were investigated in the patients with CM. RESULTS: Vitamin D deficiency (plasma 25[OH]D ≤50 nmol/L) was present in 74% of patients. Vitamin D deficiency was not associated with CM (adjusted odds ratio, 0.93 [95% confidence interval, .6-1.6]; P = .796). Levels of 25(OH)D showed marked seasonality, but no reciprocal seasonality was seen in CM notifications. No significant associations were found between 25(OH)D levels and fungal burden or levels of tumor necrosis factor α, interferon γ, interleukin 6, soluble CD14, or neopterin in cerebrospinal fluid. Rates of fungal clearance did not vary according to vitamin D status. CONCLUSIONS: Vitamin D deficiency does not predispose to the development of CM, or lead to impaired immune responses or microbiological clearance in HIV-infected patients with CM.

The glial marker YKL-40 is decreased in synucleinopathies.

BACKGROUND: Microglia are resident immunosurveillant cells in the central nervous system, and astrocytes are important for blood flow, plasticity, and neurotransmitter regulation. The aim of this study was to investigate whether astrocyte and microglial activation, estimated through markers in cerebrospinal fluid and serum, differed between synucleinopathies, tauopathies, and controls. METHODS: We analyzed the glial activation markers YKL-40 and soluble CD14 in serum and cerebrospinal fluid from 37 controls, 50 patients with Parkinson's disease (PD), and 79 P+ patients (those with progressive supranuclear palsy, corticobasal degeneration, and multiple system atrophy). RESULTS: Cerebrospinal fluid levels of YKL-40 were decreased significantly in patients who had PD compared with controls (P < 0.05), patients who had multiple system atrophy (P < 0.01), and patients who had tauopathies (P < 0.0001). In addition, cerebrospinal fluid levels of YKL-40 were significantly lower in patients who had synucleinopathies than in those who had tauopathies (P < 0.0001). CONCLUSIONS: The decreased cerebrospinal fluid levels of YKL-40 suggest that glial activation is reduced in the brains of patients who have Parkinson's disease and synucleinopathies compared with patients who have tauopathies and controls.

Role of soluble CD14 in cerebrospinal fluid as a regulator of glial functions.

Proteomic analysis of cerebrospinal fluid (CSF) samples derived from patients with Alzheimer's disease (AD) or Parkinson's disease (PD) was performed. On the basis of liquid chromatography-tandem mass spectrometry, two-dimensional gel electrophoresis analysis, and Western blot validation, it was found that the level of soluble form of monocyte differentiation antigen CD14 precursor was elevated in CSF from AD or PD patients compared with normal subjects. The soluble CD14 protein and mRNA expression was detected in microglia cells, indicating that microglia may be a cellular source of soluble CD14 in CSF. Next, the role of soluble CD14 in the regulation of glial functions was investigated. Soluble CD14 inhibited lipopolysaccharide (LPS)- or LPS/interferon-gamma-induced nitric oxide production and cell death of microglia and astrocytes. Soluble CD14 suppressed glial neurotoxicity in a coculture of glia/neuroblastoma. In addition, soluble CD14 moderately enhanced phagocytic activity of microglia. These results suggest that microglia-derived soluble CD14 is a candidate CSF biomarker for AD and PD, and the soluble CD14 may inhibit glial activation by interfering with LPS effects.

YKL-40 is a CSF biomarker of intrathecal inflammation in secondary progressive multiple sclerosis.

YKL-40 (CHI3L1) is a glycoprotein predominantly produced by reactive astrocytes in chronic active MS lesions, which are common in secondary progressive MS. In this study, YKL-40 was investigated in different stages of MS and in relation to MRI findings. YKL-40 levels in CSF samples from two independent patient cohorts of MS patients were determined with ELISA. CSF YKL-40 was increased in patients with active relapsing-remitting MS and correlated with the number of gadolinium enhancing lesions. Patients with secondary progressive MS had similar high levels of YKL-40, whereas not active relapsing-remitting MS patients had YKL-40 levels comparable to healthy controls.

(1→3)-ß-D-Glucan Levels Correlate With Neurocognitive Functioning in HIV-Infected Persons on Suppressive Antiretroviral Therapy: A Cohort Study.

Microbial translocation from the gut is associated with immune dysfunction, persistent inflammation, and likely plays a role in the pathogenesis of neurocognitive dysfunction during HIV infection. (1→3)-ß-D-Glucan (BDG) is a component of most fungal cell walls and might be a useful indicator of gut mucosal barrier impairment. The objective of this study was to evaluate whether higher blood BDG levels correlate with impaired neurocognitive functioning in a cohort of HIV-infected adults with suppressed levels of HIV RNA in blood plasma. In this cross-sectional cohort study, we measured levels of BDG in blood plasma and cerebrospinal fluid (CSF) supernatant samples in a cohort of adults with acute/early HIV infection, who initiated antiretroviral therapy (ART) during the earliest phase of infection and achieved suppressed levels of HIV RNA in blood plasma (<50 copies/mL) thereafter. We compared BDG with established biomarkers of microbial translocation, immune activation, and cognitive dysfunction (evaluated by global deficit score). We found that higher blood BDG levels were significantly related to higher global deficit scores, reflecting worse neurocognitive performance (Spearman r = 0.47; P = 0.042) among HIV-infected adults with suppressed viral loads who initiated ART early in infection. Two CSF samples presented elevated BDG levels. Interestingly, these 2 samples originated from the 2 subjects with the highest global deficit scores of the cohort. BDG may be a promising independent biomarker associated with neurocognitive functioning in virologically suppressed HIV-infected individuals.

Cerebrospinal fluid levels of soluble CD14 in inflammatory and non-inflammatory diseases of the CNS: upregulation during bacterial infections and viral meningitis.

The CD14 antigen, an important cell surface molecule of monocytic cells, is involved in cellular activation: it binds lipopolysaccharide and other cellular lipid structures. Brain macrophages play a pivotal role during inflammatory reactions of the CNS parenchyma, ventricles and meninges. A soluble form of CD14 (sCD14) was measured in paired cerebrospinal fluid (CSF) and serum samples from 91 patients with different neurological diseases. Mean levels of circulating sCD14 in CSF in a control group of 22 patients with neurologic complaints but no neurological deficit on clinical examination were 0.19 +/- 0.06 (mean +/- SD) mg/l. The CSF/blood ratios of sCD14 was 49 +/- 16 x 10(-3), while those of albumin were 4.4 +/- 1.4 x 10(-3). These extremely high CSF/blood ratios of the sCD14 molecule compared to albumin indicate a local cerebral production. No significant changes in CSF sCD14 levels were found in patients with non-inflammatory neurological diseases (NID). In contrast, CSF sCD14 levels were markedly elevated during acute meningitis, but there was no direct correlation between sCD14 and monocyte count in the CSF. Thus, sCD14 could not originate in the CSF compartment from monocytes alone. The highest values for sCD14 were found in CSF during infections with various pathogens such as Staphylococcus aureus or Listeria monocytogenes. While sCD14 serum levels dramatically increased during acute bacterial meningitis, sCD14 ratios did not correlate with albumin ratios during the course of disease. Therefore, increased CSF sCD14 may originate from cerebral production by activated or infiltrated macrophages rather than passive diffusion from the blood, while elevated sCD14 serum levels resulted from enhanced local production. Increased CSF and serum sCD14 values were also observed in meningitis caused by viral infection. As in bacterial meningitis, sCD14 in CSF specimens did not correlate with the function of the blood/CSF barrier. Repeated lumbar punctures revealed a normalization of CSF sCD14 levels during clinical recovery. These results provide the first evidence for local production of sCD14 within the CNS. Our findings further indicate that sCD14 in CSF is a reliable marker for activation of macrophages within the CNS during inflammatory processes.

Monocyte activation markers in cerebrospinal fluid associated with impaired neurocognitive testing in advanced HIV infection.

BACKGROUND: Activated monocytes/macrophages play a role in severe forms of HIV-associated neurocognitive disorders (HAND), but little is known about the mechanisms driving milder forms that are prevalent despite combination antiretroviral therapy (cART). To examine relationships of monocyte activation markers to HAND of varying severity, we compared plasma and cerebrospinal fluid (CSF) biomarker levels with neurocognitive test scores in HIV+ subjects. METHODS: Plasma and CSF soluble CD14 (sCD14), CCL2, and interleukin (IL) 6 were measured by enzyme-linked immunosorbent assay in 67 HIV+ subjects with nadir CD4 <300, and CSF inflammatory biomarkers were measured by multiplex assay in 14 subjects on suppressive cART. RESULTS: Eighty-two percent were on cART, with 31% having undetectable plasma viral load (VL). CSF sCD14 was increased in subjects with impaired neurocognitive testing (P = 0.02), correlated inversely with global T scores in subjects with detectable but not undetectable plasma VL (P = 0.02), and yielded higher area under the receiver operating characteristic curve values for predicting impaired T scores (0.659) than plasma or CSF VL and current or nadir CD4 counts in single-marker and multivariate models. CSF sCD14, IL-6, IL-8, CCL2, CCL3, CXCL10, and interferon (IFN) gamma were increased in subjects on suppressive cART regardless of cognitive status and predicted patient class in unsupervised analyses, with IL-8, CCL2, and IFNγ explaining most of the variance. CONCLUSIONS: CSF sCD14 is associated with impaired neurocognitive testing in patients with HIV on nonsuppressive cART, suggesting potential utility as a biomarker to monitor HAND progression. CSF sCD14, IL-6, IL-8, CCL2, CCL3, CXCL10, and IFNγ remain elevated in patients on suppressive cART regardless of cognitive status, implying ongoing intrathecal inflammation even in the absence of clinical manifestations.

Soluble CD14 levels in the serum, synovial fluid, and cerebrospinal fluid of patients with various stages of Lyme disease.

Levels of circulating soluble CD14 (sCD14) in patients with various stages of Lyme disease (LD) were examined. Patients with early or untreated late LD had significantly higher levels of sCD14 than did healthy controls (P=.0001 and .0007, respectively); levels returned to normal within 3 months after antibiotic therapy. Patients with persistent posttreatment symptoms of LD had sCD14 levels equivalent to those of healthy controls. Differences in the serum sCD14 levels in patients with various stages of LD are likely to be directly correlated with differences in bacterial burden, suggesting that posttreatment symptoms may not require continued presence of the organism. sCD14 levels in the cerebrospinal fluid (CSF) of patients with any stage of LD were no different from those of control subjects. Levels of synovial fluid sCD14 from patients with Borrelia burgdorferi in their joints were elevated, compared with levels in normal serum, and may play a role in the pathogenesis of arthritis.

Leukocyte trafficking in free-flowing cerebrospinal fluid of normal rhesus macaques (Macaca mulatta).

Cellular components in free-flowing cerebrospinal fluid (CSF) of normal rhesus macaques were characterized. Microscopic counting enumerated the total number of leukocytes, percentage of polymorphonuclear cells (PMN), leukocytes with nonspecific esterase (NSE), and those reducing nitro blue tetrazolium (NBT). Flow cytometric analysis further identified CD4, CD8, CD14, and CD20 positive leukocytes. These experiments established reliable techniques for evaluating cellular components in CSF from rhesus macaques and documented the difference in the CD4/CD8 ratio between peripheral blood (PB) and CSF compartments under normal physiological conditions.

The origin and function of soluble CD14 in experimental bacterial meningitis.

Murine experimental meningitis models induced by either Escherichia coli LPS, live Streptococcus pneumoniae, or Listeria monocytogenes were used to study the origin and potential function of soluble CD14 (sCD14) in the brain during bacterial meningitis. Whereas intracerebral infection caused only a minor and/or transient increase of sCD14 levels in the serum, dramatically elevated concentrations of sCD14 were detected in the cerebrospinal fluid. Reverse-transcriptase PCR and FACS analysis of the leukocytes invading the subarachnoid compartment revealed an active amplification of CD14 transcription and concomitant surface expression. These findings were confirmed by in situ hybridization and immunohistochemical analysis. In contrast, parenchymal astrocytes and microglial cells were shown not to significantly contribute to the elevated levels of sCD14. Simultaneous intracerebral inoculation of rsCD14 and S. pneumoniae resulted in a markedly increased local cytokine response. Taken together, these data provide the first evidence that sCD14 can act as an inflammatory co-ligand in vivo. Thus, during bacterial meningitis, sCD14 is massively released by intrathecal leukocytes, and the sCD14 found in the cerebrospinal fluid can play an important role in the pathogenesis of this disease.