Evidence That Lipopolisaccharide May Contribute to the Cytokine Storm and Cellular Activation in Patients with Visceral Leishmaniasis

Background

Visceral leishmaniasis (VL) is characterized by parasite-specific immunosuppression besides an intense pro-inflammatory response. Lipopolisaccharide (LPS) has been implicated in the immune activation of T-cell deficient diseases such as HIV/AIDS and idiopathic lymphocytopenia. The source of LPS is gram-negative bacteria that enter the circulation because of immunological mucosal barrier breakdown. As gut parasitization also occurs in VL, it was hypothesized that LPS may be elevated in leishmaniasis, contributing to cell activation.

Methodology/Principal Findings

Flow cytometry analysis and immunoassays (ELISA and luminex micro-beads system) were used to quantify T-cells and soluble factors. Higher LPS and soluble CD14 levels were observed in active VL in comparison to healthy subjects, indicating that LPS was bioactive; there was a positive correlation between these molecules (r = 0.61;p<0.05). Interestingly, LPS was negatively correlated with CD4⁺ (r = −0.71;p<0.01) and CD8⁺ T-cells (r = −0.65;p<0.05). Moreover, higher levels of activation-associated molecules (HLA-DR, CD38, CD25) were seen on T lymphocytes, which were positively associated with LPS levels. Pro-inflammatory cytokines and macrophage migration inhibitory factor (MIF) were also augmented in VL patients. Consistent with the higher immune activation status, LPS levels were positively correlated with the inflammatory cytokines IL-6 (r = 0.63;p<0.05), IL-8 (r = 0.89;p<0.05), and MIF (r = 0.64;p<0.05). Also, higher plasma intestinal fatty acid binding protein (IFABP) levels were observed in VL patients, which correlated with LPS levels (r = 0.57;p<0.05).

Conclusions/Significance

Elevated levels of LPS in VL, in correlation with T-cell activation and elevated pro-inflammatory cytokines and MIF indicate that this bacterial product may contribute to the impairment in immune effector function. The cytokine storm and chronic immune hyperactivation status may contribute to the observed T-cell depletion. LPS probably originates from microbial translocation as suggested by IFABP levels and, along with Leishmania antigen-mediated immune suppression, may play a role in the immunopathogenesis of VL. These findings point to possible benefits of antimicrobial prophylaxis in conjunction with anti-Leishmania therapy.     

The influence of cognition, anxiety and psychiatric disorders over treatment adherence in uncontrolled hypertensive patients

Background

Poor adherence is estimated to cause 125 thousand deaths per year and is linked to 10% of all hospital stays in the U.S. Up to one third of elderly hypertensive patients don''t have adherence, which is responsible for high proportion of hospitalizations. Hypertension is also related to poor performance in tests that assess cognitive functions. On the other hand, poor cognitive performance is associated with low adherence to treatment.

Objective

To assess the association between cognitive function, anxiety and psychiatric disorders with adherence to drug treatment in patients with hypertension.

Methodology and Principal Findings

This a cohort studies with 56 adult patients with uncontrolled hypertension who participated of all meetings of a pharmaceutical intervention in a randomized clinical trial of pharmaceutical care. Cognitive function was measured by the Mini Mental State Examination (Mini-mental). The memory was measured by digit and word spans, tower and church shadow test, short story test and metamemory. Anxiety and psychiatric disorders were evaluated by the State Trace Anxiety Inventory and the Self-Report Questionnaire, respectively. The participants were classified as adherent or non-adherent to the drug treatment, according to the identification of plasma levels of hydrochlorothiazide. All non-adherent patients (n = 12) and 35 out 44 (79.5%) patients with adherence to treatment had at least one memory test with an altered score (P = 0.180). Participants with an unsatisfactory score in the Mini-mental had six-fold higher risk of non-adherence to treatment when compared to those with a normal score (RR = 5.8; CI 95%: 1.6–20.8; P = 0.007). The scores of anxiety and psychiatric disorders were not associated with adherence to the pharmacological treatment.

Conclusion

Cognitive deficit impairs adherence to drug therapy and should be screened as part of a program of pharmaceutical care to improve adherence to treatment.     

Detection of Microbial Translocation in HIV and SIV Infection Using the Limulus Amebocyte Lysate Assay is Masked by Serum and Plasma

Objective

Microbial translocation (MT) is thought to be a major contributor to the pathogenesis of HIV-related immune activation, and circulating lipopolysaccharide (LPS) from Gram-negative bacteria is the principle measurement of this process. However, related research has been impeded by inconsistent LPS test results.

Methods

Specimens were obtained from HIV-infected adults enrolled in the PEARLS study (ACTG A5175) and HIV-HCV co-infected participants enrolled in a study of liver disease staging using MRI elastography. Pig-tailed macaque specimens were obtained from SIV-infected and –uninfected animals. Samples were tested for LPS using the LAL assay with diazo-coupling modifications to improve sensitive detection.

Results

When exogenous LPS was added to macaque plasma, >25% inhibition of LPS detection was found in 10/10 (100%) samples at 20% plasma concentration compared to control; in contrast 5/10 (50%) samples at 2% plasma concentration (p = 0.07) and 0/10 (0%) at 0.1% plasma concentration (p = 0.004) showed >25% inhibition of LPS detection. Similarly, when LPS was added to human serum, >25% inhibition of LPS detection was found in 5/12 (42%) of samples at 2% serum concentration compared to control, while 0/12 (0%) of samples in 0.1% serum showed >25% inhibition of LPS detection (p = 0.07). Likewise, LPS detection in human sera without exogenous LPS was improved by dilution: LPS was detected in 2/12 (17%) human samples in 2% serum, ranging from 3,436–4,736 pg/mL, compared to 9/12 (75%) samples in 0.1% serum, ranging from 123 pg/mL –60,131 pg/mL (p = 0.016). In a separate validation cohort of HIV-HCV co-infected participants sampled at two different times on the same day, LPS measured in 0.2% plasma and with diazo-coupling was closely correlated between the first and second samples (R = 0.66, p<0.05).

Conclusions

Undiluted serum and plasma mask LPS detection. The extent of MT may be substantially underestimated.     

Role of secreted conjunctival mucosal cytokine and chemokine proteins in different stages of trachomatous disease

Background

Chlamydia trachomatis is responsible for trachoma, the primary cause of preventable blindness worldwide. Plans to eradicate trachoma using the World Health Organization''s SAFE program (Surgery, Antibiotics, Facial Cleanliness and Environment Improvement) have resulted in recurrence of infection and disease following cessation of treatment in many endemic countries, suggesting the need for a vaccine to control infection and trachomatous disease. Vaccine development requires, in part, knowledge of the mucosal host immune responses in both healthy and trachomatous conjuctivae—an area of research that remains insufficiently studied.

Methodology/Principal Findings

We characterized 25 secreted cytokines and chemokines from the conjunctival mucosa of individuals residing in a trachoma endemic region of Nepal using Luminex X100 multiplexing technology. Immunomodulating effects of concurrent C. trachomatis infection were also examined. We found that proinflammatory cytokines IL-1β (r = 0.259, P = 0.001) and TNFα (r = 0.168, P<0.05) were significantly associated with trachomatous disease and concurrent C. trachomatis infection compared with age and sex matched controls from the same region who did not have trachoma. In support of these findings, anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra) was negatively associated with chronic scarring trachoma (r = −0.249, P = 0.001). Additional cytokines (Th1, IL-12p40 [r = −0.212, P<0.01], and Th2, IL-4 and IL-13 [r = −0.165 and −0.189, respectively, P<0.05 for both]) were negatively associated with chronic scarring trachoma, suggesting a protective role. Conversely, a pathogenic role for the Th3/Tr1 cytokine IL-10 (r = 0.180, P<0.05) was evident with increased levels for all trachoma grades. New risk factors for chronic scarring trachoma included IL-6 and IL-15 (r = 0.259 and 0.292, respectively, P<0.005 for both) with increased levels for concurrent C. trachomatis infections (r = 0.206, P<0.05, and r = 0.304, P<0.005, respectively). Chemokine protein levels for CCL11 (Eotaxin), CXCL8 (IL-8), CXCL9 (MIG), and CCL2 (MCP-1) were elevated in chronic scarring trachoma compared with age and sex matched controls (P<0.05, for all).

Conclusions/Significance

Our quantitative detection of previously uncharacterized and partially characterized cytokines, a soluble cytokine receptor, and chemokines for each trachoma grade and associations with C. trachomatis infections provide, to date, the most comprehensive immunologic evaluation of trachoma. These findings highlight novel pathologic and protective factors involved in trachomatous disease, which will aid in designing immunomodulating therapeutics and a vaccine.     

Increased bone marrow interleukin-7 (IL-7)/IL-7R levels but reduced IL-7 responsiveness in HIV-positive patients lacking CD4+ gain on antiviral therapy

Background

The bone marrow (BM) cytokine milieu might substantially affect T-lymphocyte homeostasis in HIV-positive individuals. Interleukin-7 (IL-7) is a bone marrow-derived cytokine regulating T-cell homeostasis through a CD4+-driven feedback loop. CD4+ T-lymphopenia is associated with increased free IL-7 levels and reduced IL-7R expression/function, which are only partially reverted by highly active antiretroviral therapy (HAART). We investigated the BM production, peripheral expression and signaling (pStat5+ and Bcl-2+ CD4+/CD8+ T cells) of IL-7/IL-7Rα in 30 HAART-treated HIV-positive patients who did not experience CD4+ recovery (CD4+ ≤200/µl) and who had different levels of HIV viremia; these patients included 18 immunological nonresponders (INRs; HIV-RNA≤50), 12 complete failures (CFs; HIV-RNA>1000), and 23 HIV-seronegative subjects.

Methods

We studied plasma IL-7 levels, IL-7Rα+CD4+/CD8+ T-cell proportions, IL-7Rα mRNA expression in PBMCs, spontaneous IL-7 production by BM mononuclear cells (BMMCs), and IL-7 mRNA/IL-7Rα mRNA in BMMC-derived stromal cells (SCs). We also studied T-cell responsiveness to IL-7 by measuring the proportions of pStat5+ and Bcl-2+ CD4+/CD8+ T cells.

Results

Compared to HIV-seronegative controls, CFs and INRs presented elevated plasma IL-7 levels and lower IL-7Rα CD4+/CD8+ cell-surface expression and peripheral blood production, confirming the most relevant IL-7/IL-7R disruption. Interestingly, BM investigation revealed a trend of higher spontaneous IL-7 production in INRs (p = .09 vs. CFs) with a nonsignificant trend toward higher IL-7-Rα mRNA levels in BMMC-derived stromal cells. However, upon IL-7 stimulation, the proportion of pStat5+CD4+ T cells did not increase in INRs despite higher constitutive levels (p = .06); INRs also displayed lower Bcl-2+CD8+ T-cell proportions than controls (p = .04).

Conclusions

Despite severe CD4+ T-lymphopenia and a disrupted IL-7/IL-7R profile in the periphery, INRs display elevated BM IL-7/IL-7Rα expression but impaired T-cell responsiveness to IL-7, suggesting the activity of a central compensatory pathway targeted to replenish the CD4+ compartment, which is nevertheless inappropriate to compensate the dysfunctional signaling through IL-7 receptor.     

Surgical castration of pigs affects the behavioural response to a low-dose lipopolysaccharide (LPS) challenge after weaning

The objective of the present study was to evaluate the effect of an acute stressor in the early postnatal life of pigs, surgical castration, on post-weaning behaviour, and on the behavioural, endocrine and immune responses elicited by a low-dose lipopolysaccharide (LPS) challenge after weaning. At 5-days-of-age, 64 male piglets were randomly assigned to undergo surgical castration or were left untreated (treatment). Pigs were weaned at 28 days-of-age. Behaviour post-weaning and mixing was assessed during a 1-h period, during which agonistic interactions were recorded. One day post-weaning, pigs were injected intraperitoneally with a single dose of 0 or 5 μg/kg of BW of LPS from Escherichia coli (challenge). Sickness behaviour was studied by scan sampling every 5 min for 45 min at 0, 1, 2, 3, 4, 6 and 8 h after the challenge. Blood samples were taken at 0, 2, 12 or 24 h after injection and were analysed for plasma concentrations of tumor necrosis factor-alpha (TNF-), interleukin-1beta (IL-1β), C-reactive protein (CRP), serum amyloid A (SAA) and cortisol. Results showed that non-castrated pigs were more aggressive than castrated pigs immediately after weaning (P < 0.05). Administration of LPS provoked behaviours characteristic of sickness including a reduction in general activity, as well as decreased eating and exploratory behaviours (P < 0.05). These altered behaviours occurred predominantly 3-h post injection (P < 0.05). Significant treatment by challenge interactions showed that castration reduced the occurrence of sickness behaviours induced by LPS, such as depressed general activity (P < 0.01), anorexia (P < 0.01) and reduced exploratory behaviours (P < 0.05). LPS administration increased TNF- levels (P < 0.05), with peak concentrations 2 h after injection (P < 0.01). CRP levels of LPS-treated pigs were higher than saline-treated animals at 12 h (P < 0.05). LPS administration tended to increase plasma SAA levels (P < 0.1), but did not increase cortisol levels (P > 0.1). However, castration did not affect the response of pro-inflammatory cytokines, acute phase proteins and cortisol to the challenge. These results show that surgical castration reduces aggressiveness at weaning and affects specific sickness behaviours but not the endocrine and immune responses elicited by low-dose endotoxin challenge in weaned pigs.     

Increased sleep fragmentation leads to impaired off-line consolidation of motor memories in humans

A growing literature supports a role for sleep after training in long-term memory consolidation and enhancement. Consequently, interrupted sleep should result in cognitive deficits. Recent evidence from an animal study indeed showed that optimal memory consolidation during sleep requires a certain amount of uninterrupted sleep.Sleep continuity is disrupted in various medical disorders. We compared performance on a motor sequence learning task (MST) in relatively young subjects with obstructive sleep apnea (n = 16; apnea-hypopnea index 17.1±2.6/h [SEM]) to a carefully matched control group (n = 15, apnea-hypopnea index 3.7±0.4/h, p<0.001. Apart from AHI, oxygen nadir and arousal index, there were no significant differences between groups in total sleep time, sleep efficiency and sleep architecture as well as subjective measures of sleepiness based on standard questionnaires. In addition performance on the psychomotor vigilance task (reaction time and lapses), which is highly sensitive to sleep deprivation showed no differences as well as initial learning performance during the training phase. However there was a significant difference in the primary outcome of immediate overnight improvement on the MST between the two groups (controls = 14.7±4%, patients = 1.1±3.6%; P = 0.023) as well as plateau performance (controls = 24.0±5.3%, patients = 10.1±2.0%; P = 0.017) and this difference was predicted by the arousal index (p = 0.02) rather than oxygen saturation (nadir and time below 90% saturation. Taken together, this outcome provides evidence that there is a clear minimum requirement of sleep continuity in humans to ensure optimal sleep dependent memory processes. It also provides important new information about the cognitive impact of obstructive sleep apnea and challenges its current definitions.     

Expensive egos: narcissistic males have higher cortisol

Background

Narcissism is characterized by grandiosity, low empathy, and entitlement. There has been limited research regarding the hormonal correlates of narcissism, despite the potential health implications. This study examined the role of participant narcissism and sex on basal cortisol concentrations in an undergraduate population.

Methods and Findings

Participants were 106 undergraduate students (79 females, 27 males, mean age 20.1 years) from one Midwestern and one Southwestern American university. Narcissism was assessed using the Narcissistic Personality Inventory, and basal cortisol concentrations were collected from saliva samples in a laboratory setting. Regression analyses examined the effect of narcissism and sex on cortisol (log). There were no sex differences in basal cortisol, F(1,97) = .20, p = .65, and narcissism scores, F(1,97) = .00, p = .99. Stepwise linear regression models of sex and narcissism and their interaction predicting cortisol concentrations showed no main effects when including covariates, but a significant interaction, β = .27, p = .04. Narcissism was not related to cortisol in females, but significantly predicted cortisol in males. Examining the effect of unhealthy versus healthy narcissism on cortisol found that unhealthy narcissism was marginally related to cortisol in females, β = .27, p = .06, but significantly predicted higher basal cortisol in males, β = .72, p = .01, even when controlling for potential confounds. No relationship was found between sex, narcissism, or their interaction on self-reported stress.

Conclusions

Our findings suggest that the HPA axis is chronically activated in males with unhealthy narcissism. This constant activation of the HPA axis may have important health implications.     

Impact of genetic variation in SORCS1 on memory retention

Objective

We previously reported that genetic variants in SORCS1 increase the risk of AD, that over-expression of SorCS1 reduces γ-secretase activity and Aβ levels, and that SorCS1 suppression increases γ-secretase processing of APP and Aβ levels. We now explored the effect of variation in SORCS1 on memory.

Methods

We explored associations between SORCS1-SNPs and memory retention in the NIA-LOAD case control dataset (162 cases,670 controls) and a cohort of Caribbean Hispanics (549 cases,544 controls) using single marker and haplotype analyses.

Results

Three SNPs in intron 1, were associated with memory retention in the NIA-LOAD dataset or the Caribbean Hispanic dataset (rs10884402(A allele:β = −0.15,p = 0.008), rs7078098(C allele:β = 0.18,p = 0.007) and rs950809(C allele:β = 0.17,p = 0.008)) and all three SNPs were significant in a meta-analysis of both datasets (0.002ConclusionsVariation in intron 1 in SORCS1 is associated with memory changes in AD.     

Modeling cortisol dynamics in the neuro-endocrine axis distinguishes normal, depression, and post-traumatic stress disorder (PTSD) in humans

Cortisol, secreted in the adrenal cortex in response to stress, is an informative biomarker that distinguishes anxiety disorders such as major depression and post-traumatic stress disorder (PTSD) from normal subjects. Yehuda et al. proposed a hypothesis that, in humans, the hypersensitive hypothalamus-pituitary-adrenal (HPA) axis is responsible for the occurrence of differing levels of cortisol in anxiety disorders. Specifically, PTSD subjects have lower cortisol levels during the late subjective night in comparison to normal subjects, and this was assumed to occur due to strong negative feedback loops in the HPA axis. In the present work, to address this hypothesis, we modeled the cortisol dynamics using nonlinear ordinary differential equations and estimated the kinetic parameters of the model to fit the experimental data of three categories, namely, normal, depressed, and PTSD human subjects. We concatenated the subjects (n = 3) in each category and created a model subject (n = 1) without considering the patient-to-patient variability in each case. The parameters of the model for the three categories were simultaneously obtained through global optimization. Bifurcation analysis carried out with the optimized parameters exhibited two supercritical Hopf points and, for the choice of parameters, the oscillations were found to be circadian in nature. The fitted kinetic parameters indicate that PTSD subjects have a strong negative feedback loop and, as a result, the predicted oscillating cortisol levels are extremely low at the nadir in contrast to normal subjects, albeit within the endocrinologic range. We also simulated the phenotypes for each of the categories and, as observed in the clinical data of PTSD patients, the simulated cortisol levels are consistently low at the nadir, and correspondingly the negative feedback was found to be extremely strong. These results from the model support the hypothesis that high stress intensity and strong negative feedback loop may cause hypersensitive neuro-endocrine axis that results in hypocortisolemia in PTSD.     

Association between IgM anti-herpes simplex virus and plasma amyloid-beta levels

Objective

Herpes simplex virus (HSV) reactivation has been identified as a possible risk factor for Alzheimer''s disease (AD) and plasma amyloid-beta (Aβ) levels might be considered as possible biomarkers of the risk of AD. The aim of our study was to investigate the association between anti-HSV antibodies and plasma Aβ levels.

Methods

The study sample consisted of 1222 subjects (73.9 y in mean) from the Three-City cohort. IgM and IgG anti-HSV antibodies were quantified using an ELISA kit, and plasma levels of Aβ_1–40 and Aβ_1–42 were measured using an xMAP-based assay technology. Cross-sectional analyses of the associations between anti-HSV antibodies and plasma Aβ levels were performed by multi-linear regression.

Results

After adjustment for study center, age, sex, education, and apolipoprotein E-e4 polymorphism, plasma Aβ_1–42 and Aβ_1–40 levels were specifically inversely associated with anti-HSV IgM levels (β = −20.7, P = 0.001 and β = −92.4, P = 0.007, respectively). In a sub-sample with information on CLU- and CR1-linked SNPs genotyping (n = 754), additional adjustment for CR1 or CLU markers did not modify these associations (adjustment for CR1 rs6656401, β = −25.6, P = 0.002 for Aβ_1–42 and β = −132.7, P = 0.002 for Aβ_1–40; adjustment for CLU rs2279590, β = −25.6, P = 0.002 for Aβ_1–42 and β = −134.8, P = 0.002 for Aβ_1–40). No association between the plasma Aβ_1–42-to-Aβ_1–40 ratio and anti-HSV IgM or IgG were evidenced.

Conclusion

High anti-HSV IgM levels, markers of HSV reactivation, are associated with lower plasma Aβ_1–40 and Aβ_1–42 levels, which suggest a possible involvement of the virus in the alterations of the APP processing and potentially in the pathogenesis of AD in human.     

Interleukin and growth factor levels in subretinal fluid in rhegmatogenous retinal detachment: a case-control study

Background

Rhegmatogenous retinal detachment (RRD) is a major cause of visual loss in developed countries. Proliferative vitreoretinopathy (PVR), an eye-sight threatening complication of RRD surgery, resembles a wound-healing process with inflammation, scar tissue formation, and membrane contraction. This study was performed to determine the possible involvement of a wide range of cytokines in the future development of PVR, and to identify predictors of PVR and visual outcome.

Methodology

A multiplex immunoassay was used for the simultaneous detection of 29 different cytokines in subretinal fluid samples from patients with primary RRD. Of 306 samples that were collected and stored in our BioBank between 2001 and 2008, 21 samples from patients who developed postoperative PVR were compared with 54 age-, sex-, and storage-time–matched RRD control patients who had an uncomplicated postoperative course during the overall follow-up period.

Findings

Levels of IL-1α, IL-2, IL-3, IL-6, VEGF, and ICAM-1 were significantly higher (P<0.05) in patients who developed postoperative PVR after reattachment surgery than in patients with an uncomplicated postoperative course, whereas levels of IL-1β, IL-4, IL-5, IL-7, IL-9, IL-10, IL-11, IL-12p70, IL-13, IL-15, IL-17, IL-18, IL-21, IL-22, IL-23, IL-25, IL-33, TNF-α, IFN-γ, IGF-1, bFGF, HGF, and NGF were not (P>0.05). Multivariate logistic regression analysis revealed that IL-3 (P = 0.001), IL-6 (P = 0.047), ICAM-1 (P = 0.010), and preoperative visual acuity (P = 0.026) were independent predictors of postoperative PVR. Linear regression analysis showed that ICAM-1 (P = 0.005) and preoperative logMAR visual acuity (P = 0.001) were predictive of final visual outcome after primary RRD repair.

Conclusions/Significance

Our findings indicate that after RRD onset an exaggerated response of certain cytokines may predispose to PVR. Sampling at a time close to the onset of primary RRD may thus provide clues as to which biological events may initiate the development of PVR and, most importantly, may provide a means for therapeutic control.     

The accumulation and prognosis value of tumor infiltrating IL-17 producing cells in esophageal squamous cell carcinoma

Background

The role of IL-17 producing cells in tumors is controversial. In the present study, we investigated the prognostic value of measuring tumor-infiltrating IL-17 producing cell levels in human esophageal squamous cell carcinoma (ESCC).

Methodology/Principal Findings

Immunohistochemical staining was performed to investigate the levels of IL-17+ tumor infiltrating lymphocytes (TILs), as well as CD8+ cytotoxic T lymphocytes (CTLs) and CD57+ natural killer (NK) cells from 181 ESCC patients. The prognostic value of measuring the densities of IL-17+TILs and the correlation with CTLs and NK was evaluated. IL-17 producing cells were detected in esophageal squamous cell carcinoma tissues. The IL-17 producing cells were major CD4 positive, but Foxp3 negative. The median level of IL-17+TILs was 3.90 cells/high power microscopic field (HPF). The density of IL-17 producing cells correlated negatively with T stage (P = 0.042). The higher densities of tumor infiltrating IL-17+ lymphocytes were associated with better overall survival (P = 0.031). Furthermore, we found that there were positive correlations between levels of IL-17 producing cells and the densities of CD8+cells, as well as CD57+cells (r = 0.198, P = 0.008 for CD8+ cells and r = 0.261, P<0.001 for CD57+ cells, respectively). The prognosis analysis also showed that the higher levels of CD8+ CTLs and CD57+ NK cells correlated with better overall survival of ESCC patients.

Conclusions

Our study suggests that tumor infiltrating IL-17 producing cells in ESCC patients may have protective roles in the tumor microenvironment and may be treated as a prognostic marker for ESCC patients.     

Increased Responsiveness of Peripheral Blood Mononuclear Cells to In Vitro TLR 2, 4 and 7 Ligand Stimulation in Chronic Pain Patients

Glial activation via Toll-like receptor (TLR) signaling has been shown in animals to play an important role in the initiation and establishment of chronic pain. However, our ability to assess this central immune reactivity in clinical pain populations is currently lacking. Peripheral blood mononuclear cells (PBMCs) are an accessible source of TLR expressing cells that may mirror similarities in TLR responsiveness of the central nervous system. The aim of this study was to characterize the IL-1β response to various TLR agonists in isolated PBMCs from chronic pain sufferers (on and not on opioids) and pain-free controls. Venous blood was collected from 11 chronic pain sufferers on opioids (≥ 20 mg of morphine / day), 8 chronic pain sufferers not on opioids and 11 pain-free controls. PBMCs were isolated and stimulated in vitro with a TLR2 (Pam3CSK4), TLR4 (LPS) or TLR7 (imiquimod) agonist. IL-1β released into the supernatant was measured with ELISA. Significantly increased IL-1β expression was found in PBMCs from chronic pain sufferers (on and not on opioids) compared with pain-free controls for TLR2 (F _{(6, 277)} = 15, P<0.0001), TLR4 (F _{(8, 263)} = 3, P = 0.002) and TLR7 (F _(2,201) = 5, P = 0.005) agonists. These data demonstrate that PBMCs from chronic pain sufferers were more responsive to TLR agonists compared with controls, suggesting peripheral cells may have the potential to become a source of biomarkers for chronic pain.     

The role of circulating serotonin in the development of chronic obstructive pulmonary disease

Background

Cigarette smoking is a major risk factor in the development of age-related chronic obstructive pulmonary disease (COPD). The serotonin transporter (SERT) gene polymorphism has been reported to be associated with COPD, and the degree of cigarette smoking has been shown to be a significant mediator in this relationship. The interrelation between circulating serotonin (5-hydroxytyptamine, 5-HT), cigarette smoking and COPD is however largely unknown. The current study aimed at investigating the mediation effects of plasma 5-HT on cigarette smoking-induced COPD and the relation between plasma 5-HT levels and age.

Methods

The association between plasma 5-HT, age and COPD was analyzed in a total of 62 COPD patients (ever-smokers) and 117 control subjects (healthy non-smokers and ever-smokers). Plasma 5-HT levels were measured by enzyme-linked immuno assay (EIA).

Results

The elevated plasma 5-HT levels were significantly associated with increased odds for COPD (OR = 1.221, 95% CI = 1.123 to 1.319, p<0.0001). The effect remained significant after being adjusted for age and pack-years smoked (OR = 1.271, 95% CI = 1.134 to 1.408, p = 0.0003). Furthermore, plasma 5-HT was found to mediate the relation between pack-years smoked and COPD. A positive correlation (r = 0.303, p = 0.017) was found between plasma 5-HT levels and age in COPD, but not in the control subjects (r = −0.149, p = 0.108).

Conclusion

Our results suggest that cigarette smoke-induced COPD is partially mediated by the plasma levels of 5-HT, and that these become elevated with increased age in COPD. The elevated plasma 5-HT levels in COPD might contribute to the pathogenesis of this disease.     

Interleukin-8 is activated in patients with chronic liver diseases and associated with hepatic macrophage accumulation in human liver fibrosis

Background

Interleukin-8 (IL-8, CXCL8) is a potent chemoattractant for neutrophils and contributes to acute liver inflammation. Much less is known about IL-8 in chronic liver diseases (CLD), but elevated levels were reported from alcoholic and hepatitis C-related CLD. We investigated the regulation of IL-8, its receptors CXCR1 and CXCR2 and possible IL-8 responding cells in CLD patients.

Methodology

Serum IL-8 levels were measured in CLD patients (n = 200) and healthy controls (n = 141). Intrahepatic IL-8, CXCR1 and CXCR2 gene expression was quantified from liver samples (n = 41), alongside immunohistochemical neutrophil (MPO) and macrophage (CD68) stainings. CXCR1 and CXCR2 expression was analyzed on purified monocytes from patients (n = 111) and controls (n = 31). In vitro analyses explored IL-8 secretion by different leukocyte subsets.

Principal Findings

IL-8 serum levels were significantly increased in CLD patients, especially in end-stage cirrhosis. Interestingly, patients with cholestatic diseases exhibited highest IL-8 serum concentrations. IL-8 correlated with liver function, inflammatory cytokines and non-invasive fibrosis markers. Intrahepatically, IL-8 and CXCR1 expression were strongly up-regulated. However, intrahepatic IL-8 could only be associated to neutrophil infiltration in patients with primary biliary cirrhosis (PBC). In non-cholestatic cirrhosis, increased IL-8 and CXCR1 levels were associated with hepatic macrophage accumulation. In line, CXCR1, but not CXCR2 or CXCR3, expression was increased on circulating monocytes from cirrhotic patients. Moreover, monocyte-derived macrophages from CLD patients, especially the non-classical CD16⁺ subtype, displayed enhanced IL-8 secretion in vitro.

Conclusions

IL-8 is strongly activated in CLD, thus likely contributing to hepatic inflammation. Our study suggests a novel role of IL-8 for recruitment and activation of hepatic macrophages via CXCR1 in human liver cirrhosis.     

Plasma cytokine profiles in subjects with high-functioning autism spectrum disorders

Background

Accumulating evidence suggests that dysregulation of the immune system is involved in the pathophysiology of autism spectrum disorders (ASD). The aim of the study was to explore immunological markers in peripheral plasma samples from non-medicated subjects with high-functioning ASD.

Methodology/Principal Findings

A multiplex assay for cytokines and chemokines was applied to plasma samples from male subjects with high-functioning ASD (n = 28) and matched controls (n = 28). Among a total of 48 analytes examined, the plasma concentrations of IL-1β, IL-1RA, IL-5, IL-8, IL-12(p70), IL-13, IL-17 and GRO-α were significantly higher in subjects with ASD compared with the corresponding values of matched controls after correction for multiple comparisons.

Conclusion/Significance

The results suggest that abnormal immune responses as assessed by multiplex analysis of cytokines may serve as one of the biological trait markers for ASD.     

Inflammation’s Association with Metabolic Profiles before and after a Twelve-Week Clinical Trial in Drug-Na?ve Patients with Bipolar II Disorder

Inflammation is thought to be involved in the pathophysiology of bipolar disorder (BP) and metabolic syndrome. Prior studies evaluated the association between metabolic profiles and cytokines only during certain mood states instead of their changes during treatment. We enrolled drug-naïve patients with BP-II and investigated the correlation between changes in mood symptoms and metabolic indices with changes in plasma cytokine levels after 12 weeks of pharmacological treatment. Drug-naïve patients (n = 117) diagnosed with BP-II according to DSM-IV criteria were recruited. Metabolic profiles (cholesterol, triglyceride, HbA1C, fasting serum glucose, body mass index (BMI) and plasma cytokines (TNF-α, CRP, IL-6, and TGF-β) were measured at baseline and 2, 8, and 12 weeks post-treatment. To adjust within-subject dependence over repeated assessments, multiple linear regressions with generalized estimating equation methods were used. Seventy-six (65.0%) patients completed the intervention. Changes in plasma CRP were significantly associated with changes in BMI (P = 1.7E-7) and triglyceride (P = 0.005) levels. Changes in plasma TGF-β1 were significantly associated with changes in BMI (P = 8.2E-6), cholesterol (P = 0.004), and triglyceride (P = 0.006) levels. However, changes in plasma TNF-α and IL-6 were not associated with changes in any of the metabolic indices. Changes in Hamilton Depression Rating Scale scores were significantly associated with changes in IL-6 (P = 0.003) levels; changes in Young Mania Rating Scale scores were significantly associated with changes in CRP (P = 0.006) and TNF-α (P = 0.039) levels. Plasma CRP and TGF-β1 levels were positively correlated with several metabolic indices in BP-II after 12 weeks of pharmacological intervention. We also hypothesize that clinical symptoms are correlated with certain cytokines. These new findings might be important evidence that inflammation is the pathophysiology of clinical symptoms and metabolic disturbance in BP-II.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01188148","term_id":"NCT01188148"}}NCT01188148.     

Plasma cell-free DNA levels are elevated in acute Puumala hantavirus infection

Introduction

Puumala hantavirus (PUUV) causes a hemorrhagic fever with renal syndrome called nephropathia epidemica (NE). The aim of the present study was to evaluate plasma cell-free DNA (cf-DNA) levels and urinary cf-DNA excretion in acute NE as well as their associations with the severity of the disease.

Methods

Total plasma cf-DNA was quantified directly in plasma of 61 patients and urine of 20 patients with acute NE. We also carried out a qualitative high-sensitivity lab-on-a-chip DNA assay in 20 patients to elucidate the appearance of cf-DNA in plasma and urine.

Results

The maximum plasma cf-DNA values taken during acute NE were significantly higher than the control values taken after the hospitalization period (median 1.33 µg/ml, range 0.94–3.29 µg/ml vs. median 0.77 µg/ml, range 0.55–0.99 µg/ml, P<0.001). The maximum plasma cf-DNA levels correlated positively with maximum blood leukocyte count (r = 0.388, P = 0.002) and the length of hospital stay (r = 0.376, P = 0.003), and inversely with minimum blood platelet count (r = −0.297, P = 0.020). Qualitative analysis of plasma cf-DNA revealed that in most of the patients cf-DNA displayed a low-molecular weight appearance, corresponding to the size of apoptotic DNA (150–200 bp). The visually graded maximum cf-DNA band intensity correlated positively with the maximum quantity of total plasma cf-DNA (r = 0.513, P = 0.021). Maximum urinary excretion of cf-DNA in turn was not markedly increased during the acute phase of NE and did not correlate with any of the variables reflecting severity of the disease or with the maximum plasma cf-DNA level.

Conclusions

The plasma levels of cf-DNA are elevated during acute PUUV infection and correlate with the apoptotic cf-DNA-band intensity. The plasma cf-DNA concentration correlates with some variables reflecting the severity of the disease. The urinary excretion of cf-DNA does not reflect the degree of inflammation in the kidney.