Neuroinflammatory TNFα Impairs Memory via Astrocyte Signaling.

The occurrence of cognitive disturbances upon CNS inflammation or infection has been correlated with increased levels of the cytokine tumor necrosis factor-α (TNFα). To date, however, no specific mechanism via which this cytokine could alter cognitive circuits has been demonstrated. Here, we show that local increase of TNFα in the hippocampal dentate gyrus activates astrocyte TNF receptor type 1 (TNFR1), which in turn triggers an astrocyte-neuron signaling cascade that results in persistent functional modification of hippocampal excitatory synapses. Astrocytic TNFR1 signaling is necessary for the hippocampal synaptic alteration and contextual learning-memory impairment observed in experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS). This process may contribute to the pathogenesis of cognitive disturbances in MS, as well as in other CNS conditions accompanied by inflammatory states or infections.

RORγt drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation.

Although the role of the T(H)1 and T(H)17 subsets of helper T cells as disease mediators in autoimmune neuroinflammation remains a subject of some debate, none of their signature cytokines are essential for disease development. Here we report that interleukin 23 (IL-23) and the transcription factor RORγt drove expression of the cytokine GM-CSF in helper T cells, whereas IL-12, interferon-γ (IFN-γ) and IL-27 acted as negative regulators. Autoreactive helper T cells specifically lacking GM-CSF failed to initiate neuroinflammation despite expression of IL-17A or IFN-γ, whereas GM-CSF secretion by Ifng(-/-)Il17a(-/-) helper T cells was sufficient to induce experimental autoimmune encephalomyelitis (EAE). During the disease effector phase, GM-CSF sustained neuroinflammation via myeloid cells that infiltrated the central nervous system. Thus, in contrast to all other known helper T cell-derived cytokines, GM-CSF serves a nonredundant function in the initiation of autoimmune inflammation regardless of helper T cell polarization.

TGFß regulates persistent neuroinflammation by controlling Th1 polarization and ROS production via monocyte-derived dendritic cells.

Intracerebral levels of Transforming Growth Factor beta (TGFß) rise rapidly during the onset of experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis (MS). We addressed the role of TGFß responsiveness in EAE by targeting the TGFß receptor in myeloid cells, determining that Tgfbr2 was specifically targeted in monocyte-derived dendritic cells (moDCs) but not in CNS resident microglia by using bone-marrow chimeric mice. TGFß responsiveness in moDCs was necessary for the remission phase since LysM(Cre) Tgfbr2(fl/fl) mice developed a chronic form of EAE characterized by severe demyelination and extensive infiltration of activated moDCs in the CNS. Tgfbr2 deficiency resulted in increased moDC IL-12 secretion that skewed T cells to produce IFN-γ, which in turn enhanced the production of moDC-derived reactive oxygen species that promote oxidative damage and demyelination. We identified SNPs in the human NOX2 (CYBB) gene that associated with the severity of MS, and significantly increased CYBB expression was recorded in PBMCs from both MS patients and from MS severity risk allele rs72619425-A carrying individuals. We thus identify a novel myeloid cell-T cell activation loop active in the CNS during chronic disease that could be therapeutically targeted. GLIA 2016;64:1925-1937.

Tissue-specific differentiation of a circulating CCR9- pDC-like common dendritic cell precursor.

The ontogenic relationship between the common dendritic cell (DC) progenitor (CDP), the committed conventional DC precursor (pre-cDC), and cDC subpopulations in lymphoid and nonlymphoid tissues has been largely unraveled. In contrast, the sequential steps of plasmacytoid DC (pDC) development are less defined, and it is unknown at which developmental stage and location final commitment to the pDC lineage occurs. Here we show that CCR9(-) pDCs from murine BM which enter the circulation and peripheral tissues have a common DC precursor function in vivo in the steady state, in contrast to CCR9(+) pDCs which are terminally differentiated. On adoptive transfer, the fate of CCR9(-) pDC-like precursors is governed by the tissues they enter. In the BM and liver, most transferred CCR9(-) pDC-like precursors differentiate into CCR9(+) pDCs, whereas in peripheral lymphoid organs, lung, and intestine, they additionally give rise to cDCs. CCR9(-) pDC-like precursors which are distinct from pre-cDCs can be generated from the CDP. Thus, CCR9(-) pDC-like cells are novel CDP-derived circulating DC precursors with pDC and cDC potential. Their final differentiation into functionally distinct pDCs and cDCs depends on tissue-specific factors allowing adaptation to local requirements under homeostatic conditions.

NG2 expressed by macrophages and oligodendrocyte precursor cells is dispensable in experimental autoimmune encephalomyelitis.

Increased expression of the chondroitin proteoglycan NG2 is a prominent feature in central nervous system injury with unknown cellular source and biological relevance. Here, we describe the first detailed analysis of experimental autoimmune encephalomyelitis in NG2 knockout mice and NG2 knockout bone marrow chimeras. We show that both macrophages and oligodendrocyte progenitor cells express and secrete NG2 in response to transforming growth factor-ß. A subpopulation of macrophages expresses NG2 within leucocyte infiltrates in the central nervous system, but only oligodendrocyte progenitor cells contribute to NG2 accumulation. Notably, NG2 plays no role in experimental autoimmune encephalomyelitis initiation, progression or recuperation. In concurrence, the immune response is unaltered in NG2-deficient mice as are the extent of central nervous system damage and degree of remyelination.

Induction of inhibitory central nervous system-derived and stimulatory blood-derived dendritic cells suggests a dual role for granulocyte-macrophage colony-stimulating factor in central nervous system inflammation.

The mononuclear phagocyte system, particularly dendritic cells, plays several pivotal roles in the development of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Here, we demonstrate that functionally distinct dendritic cell subpopulations are present in the central nervous system during experimental autoimmune encephalomyelitis. At peak experimental autoimmune encephalomyelitis, the majority of dendritic cells consisted of a CD11b(+)F4/80(+) inflammatory dendritic cell subtype. Both granulocyte-macrophage colony-stimulating factor and chemokine (C-C motif) ligand 2 were previously suggested to recruit 'inflammatory' monocyte-derived dendritic cells to the central nervous system during experimental autoimmune encephalomyelitis. We show that intra-cerebral production of granulocyte-macrophage colony-stimulating factor leading to chemokine (C-C motif) ligand 2 induction and attraction of chemokine (C-C motif) receptor 2-positive precursors suffices to recruit dendritic cell populations identical to those observed in experimental autoimmune encephalomyelitis into the central nervous system of healthy mice. This does not occur with fms-like tyrosine kinase-3-ligand treatment. Both during experimental autoimmune encephalomyelitis and upon intra-cerebral granulocyte-macrophage colony-stimulating factor production, all myeloid dendritic cells, lymphoid dendritic cells and periphery-derived inflammatory dendritic cells stimulated T cell proliferation, whereas inflammatory dendritic cells that differentiated from central nervous system precursors inhibited T cell activation and pro-inflammatory cytokine production. Despite the capacity of granulocyte-macrophage colony-stimulating factor to induce central nervous system-derived inhibitory inflammatory dendritic cells, the administration of granulocyte-macrophage colony-stimulating factor into mice with experimental autoimmune encephalomyelitis resulted in exacerbated disease. Granulocyte-macrophage colony-stimulating factor thus has a dual role in the central nervous system: it directs both central nervous system-derived dendritic cells towards an inhibitory phenotype and recruits peripheral dendritic cells exhibiting pro-inflammatory functions.

Expression of the HGF receptor c-met by macrophages in experimental autoimmune encephalomyelitis.

Hepatocyte growth factor (HGF) is a pleiotropic cytokine able to evoke a wide array of cellular responses including proliferation, migration, and survival through activation of its receptor c-met. Various types of leukocytes have been described to express c-met suggesting that HGF/c-met signaling may directly influence leukocyte responses in inflammation. We have investigated the HGF/c-met pathway in experimental autoimmune encephalomyelitis (EAE), a common mouse model of multiple sclerosis (MS), in which macrophages play a dual role, contributing directly to CNS damage at disease onset but promoting recovery during remission by removing myelin debris. Here we show that during EAE both HGF and c-met are expressed in the CNS and that c-met is activated. We subsequently demonstrate that c-met is primarily expressed in inflammatory lesions by macrophages and a small number of dendritic cells (DCs) and oligodendrocyte progenitor cells (OPCs) but not by microglia or T cells. Complementary in vitro experiments show that only LPS and TNFalpha, but not IL-6, IL-10, or IL-13, are able to induce c-met expression in macrophages. In addition, using TNF signaling deficient macrophages we demonstrate that LPS and TNFalpha induce c-met through distinct pathways. Furthermore, TNFalpha- and LPS-induced c-met is functional because treatment of macrophages with recombinant HGF results in rapid phosphorylation of c-met. Interestingly, HGF/c-met signaling does not modulate cytokine expression, phagocytosis, or antigen presentation but promotes proliferation of activated macrophages. Taken together, our data indicate a pro-inflammatory role for the HGF/c-met pathway in EAE rather than a role in the initiation of repair mechanisms.

Site-specific anti-tumor immunity: differences in DC function, TGF-beta production and numbers of intratumoral Foxp3+ Treg.

Gliomas localized within the CNS are generally not rejected by the immune system despite being immunogenic. This failure of the immune system has been associated both with glioma-derived immunosuppressive molecules and the immune-privileged state of the CNS. However, the relative contribution of tumor location to the glioma-mediated immunosuppression, as well as the immune mechanisms involved in the failure of glioma rejection are not fully defined. We report here that syngeneic GL261 gliomas growing either intracranially or subcutaneously in mice are infiltrated by DC and T cells. However, only subcutaneous gliomas elicit an effective anti-tumor immune response. In contrast to DC infiltrating subcutaneously grown GL261 gliomas, tumor-infiltrating DC from intracranial gliomas do not activate antigen-dependent T-cell proliferation in vitro. In addition, brain-localized GL261 gliomas are characterized by significantly higher numbers of Foxp3(+) Treg and higher levels of TGF-beta1 mRNA and protein expression when compared with GL261 gliomas in the skin. Our data show that gliomas in the CNS, but not in the skin, give rise to TGF-beta production and accumulation of both Treg and functionally impaired DC. Thus, not the tumor itself, but its location dictates the efficiency of the anti-tumor immune response.

Surveillance of invasive Aedes mosquitoes along Swiss traffic axes reveals different dispersal modes for Aedes albopictus and Ae. japonicus.

Over the past three decades, Europe has witnessed an increased spread of invasive aedine mosquito species, most notably Aedes albopictus, a key vector of chikungunya, dengue and Zika virus. While its distribution in southern Europe is well documented, its dispersal modes across the Alps remain poorly investigated, preventing a projection of future scenarios beyond its current range in order to target mosquito control. To monitor the presence and frequency of invasive Aedes mosquitoes across and beyond the Alps we set oviposition and BG-Sentinel traps at potential points of entry with a focus on motorway service areas across Switzerland. We placed the traps from June to September and controlled them for the presence of mosquitoes every other week between 2013 and 2018. Over the six years of surveillance we identified three invasive Aedes species, including Ae. albopictus, Ae. japonicus and Ae. koreicus. Based on the frequency and distribution patterns we conclude that Ae. albopictus and Ae. koreicus are being passively spread primarily along the European route E35 from Italy to Germany, crossing the Alps, while Ae. japonicus has been expanding its range from northern Switzerland across the country most likely through active dispersal.

Higher body mass index (BMI) is associated with reduced glucocorticoid inhibition of inflammatory cytokine production following acute psychosocial stress in men.

BACKGROUND: Body mass index (BMI) and mental stress seem to exert part of their cardiovascular risk by eliciting inflammation. However, the adverse effects of stress on inflammatory activity with BMI are not fully understood. We investigated whether higher BMI is associated with reduced glucocorticoid inhibition of inflammatory cytokine production following stress in men while controlling for age and blood pressure. We measured glucocorticoid inhibition of lipopolysaccharide (LPS)-stimulated release of the proinflammatory cytokine tumor necrosis factor (TNF)-alpha. METHODS: Forty-two men (age range 21-65 years; BMI range 21-34 kg/m(2)) underwent the Trier Social Stress Test (combination of mock job interview and mental arithmetic task). Whole blood samples were taken immediately before and after stress, and during recovery up to 60 min post-stress. Glucocorticoid sensitivity of LPS-stimulated TNF-alpha expression was assessed in vitro with and without coincubating increasing doses of dexamethasone. Moreover, salivary cortisol was measured during the experiment and on a normal day for assessment of baseline circadian cortisol. RESULTS: Higher BMI was associated with lower glucocorticoid sensitivity of monocyte TNF-alpha production after stress (main effect of BMI: p<0.001) and with more pronounced decreases of glucocorticoid sensitivity following stress (interaction of stress-by-BMI: p=0.002). Neither LPS-stimulated TNF-alpha release nor baseline glucocorticoid sensitivity were associated with BMI. Similarly, BMI was not associated with salivary cortisol, either in reaction to stress or in circadian cortisol secretion. CONCLUSIONS: Our data suggest that with increasing BMI, glucocorticoids are less able to inhibit TNF-alpha production following stress. This might suggest a new mechanism linking BMI with elevated risk for adverse cardiovascular outcomes following stress.

Chronic social stress induces peripheral and central immune activation, blunted mesolimbic dopamine function, and reduced reward-directed behaviour in mice.

Psychosocial stress is a major risk factor for depression, stress leads to peripheral and central immune activation, immune activation is associated with blunted dopamine (DA) neural function, DA function underlies reward interest, and reduced reward interest is a core symptom of depression. These states might be inter-independent in a complex causal pathway. Whilst animal-model evidence exists for some specific steps in the pathway, there is currently no animal model in which it has been demonstrated that social stress leads to each of these immune, neural and behavioural states. Such a model would provide important existential evidence for the complex pathway and would enable the study of causality and mediating mechanisms at specific steps in the pathway. Therefore, in the present mouse study we investigated for effects of 15-day resident-intruder chronic social stress (CSS) on each of these states. Relative to controls, CSS mice exhibited higher spleen levels of granulocytes, inflammatory monocytes and T helper 17 cells; plasma levels of inducible nitric oxide synthase; and liver expression of genes encoding kynurenine pathway enzymes. CSS led in the ventral tegmental area to higher levels of kynurenine and the microglia markers Iba1 and Cd11b and higher binding activity of DA D1 receptor; and in the nucleus accumbens (NAcc) to higher kynurenine, lower DA turnover and lower c-fos expression. Pharmacological challenge with DA reuptake inhibitor identified attenuation of DA stimulatory effects on locomotor activity and NAcc c-fos expression in CSS mice. In behavioural tests of operant responding for sucrose reward validated as sensitive assays for NAcc DA function, CSS mice exhibited less reward-directed behaviour. Therefore, this mouse study demonstrates that a chronic social stressor leads to changes in each of the immune, neural and behavioural states proposed to mediate between stress and disruption of DA-dependent reward processing. The model can now be applied to investigate causality and, if demonstrated, underlying mechanisms in specific steps of this immune-neural-behavioural pathway, and thereby to identify potential therapeutic targets.

Insecticide susceptibility of Aedes albopictus and Ae. aegypti from Brazil and the Swiss-Italian border region.

BACKGROUND: Aedes aegypti and Ae. albopictus are two highly invasive mosquito species, both vectors of several viruses, including dengue, chikungunya and Zika. While Ae. aegypti is the primary vector in the tropics and sub-tropics, Ae. albopictus is increasingly under the public health watch as it has been implicated in arbovirus-transmission in more temperate regions, including continental Europe. Vector control using insecticides is the pillar of most control programmes; hence development of insecticide resistance is of great concern. As part of a Brazilian-Swiss Joint Research Programme we set out to assess whether there are any signs of existing or incipient insecticide resistance primarily against the larvicide Bacillus thuringiensis svar. israelensis (Bti), but also against currently applied and potentially alternative insecticides in our areas, Recife (Brazil) and the Swiss-Italian border region. METHODS: Following World Health Organization guidelines, dose-response curves for a range of insecticides were established for both colonized and field caught Ae. aegypti and Ae. albopictus. The larvicides included Bti, two of its toxins, Cry11Aa and Cry4Ba, Lysinibacillus sphaericus, Vectomax CG®, a formulated combination of Bti and L. sphaericus, and diflubenzuron. In addition to the larvicides, the Swiss-Italian Ae. albopictus populations were also tested against five adulticides (bendiocarb, dichlorodiphenyltrichloroethane, malathion, permethrin and λ-cyhalothrin). RESULTS: Showing a similar dose-response, all mosquito populations were fully susceptible to the larvicides tested and, in particular, to Bti which is currently used both in Brazil and Switzerland. In addition, there were no signs of incipient resistance against Bti as larvae were equally susceptible to the individual toxins, Cry11Aa and Cry4Ba. The field-caught Swiss-Italian populations were susceptible to the adulticides tested but DDT mortality rates showed signs of reduced susceptibility. CONCLUSIONS: The insecticides currently used for mosquito control in Switzerland and Brazil are still effective against the target populations. The present study provides an important reference as relatively few insecticide susceptibility surveys have been carried out with Ae. albopictus.

Surveillance and Control of Aedes albopictus in the Swiss-Italian Border Region: Differences in Egg Densities between Intervention and Non-intervention Areas.

BACKGROUND: Aedes albopictus, the Asian tiger mosquito, originates from the tropical and subtropical regions of Southeast Asia. Over the recent decades it has been passively spread across the globe, primarily through the used tyre trade and passive transportation along major traffic routes. A. albopictus is a proven vector for many arboviruses, most notably chikungunya and dengue, with recent outbreaks also in continental Europe. In southern Switzerland, in the Canton of Ticino A. albopictus was spotted for the first time in 2003. Since then the local authorities have implemented a control programme based on larval source reduction. Despite these efforts, mosquito densities have increased over the last decade, casting doubts on the effectiveness of such larval control programmes. METHODOLOGY/PRINCIPAL FINDINGS: The Italian communities just across the Swiss-Italian border lack a control programme. This motivated us to compare the intervention and the non-intervention areas side by side in an attempt to find evidence for, or against, the effectiveness of larval A. albopictus control. Using ovitraps and a randomised sampling scheme, we examined the seasonal and spatial abundance of A. albopictus in sylvatic and urban environments across the Swiss-Italian border in 2012 and 2013. In the urban environments of the non-intervention area, egg densities were 2.26 times higher as compared to the intervention area. In the sylvatic environments, as compared to the urban environments, egg densities were 36% in the intervention area and 18% in the non-intervention area. CONCLUSIONS/SIGNIFICANCE: Though alternative explanations are also valid, the results support the hypothesis that the Ticino intervention programme does have an impact. At the same time the data also suggest that current larval interventions fall short in gaining full control over the mosquito, calling for the evaluation of additional, or alternative, approaches. Ideally, these should also consider inclusion of the neighbouring Italian communities in the surveillance and control efforts.

First report of the invasive mosquito species Aedes koreicus in the Swiss-Italian border region.

BACKGROUND: In 2012 and 2013, an entomological survey of Aedes albopictus, the Asian tiger mosquito, was carried out in the border region of southern Switzerland and northern Italy, using ovitraps. In July 2013, besides A. albopictus already known to the region several unusual eggs were recovered. FINDINGS: A total of 548 seemingly different eggs were found within three communities: Chiasso (Switzerland), and Como and Brunate (Italy). Proteomic diagnostics based on matrix-assisted laser desorption/ionization mass-spectrometry (MALDI-TOF MS) and morphological identification of one reared adult revealed the presence of at least 18 A. (Finlaya) koreicus (Edwards, 1917) specimens. A. koreicus is a species native to Southeast Asia and is competent to transmit Japanese encephalitis and potentially other arboviruses, as well as the dog heartworm Dirofilaria immitis. While new to Switzerland, this invasive species has previously been reported from Belgium, north-eastern Italy and European Russia. CONCLUSIONS: This is the first report of the introduction of this exotic mosquito species into Switzerland and Lombardy, Italy, suggesting the range of A. koreicus is expanding in Central Europe. As A. koreicus is competent to vector pathogens its establishment imposes a risk to public and veterinary health. From a technical point of view, the presence of A. koreicus alongside A. albopictus requires careful analysis and reliable diagnostics. As a diagnostic tool the use of the recently developed MALDI-TOF MS approach has proofed to be a very useful approach, particularly since hatching rates of A. koreicus seem to be low, making identification by classic morphology difficult, if not impossible.

Reduced ß-amyloid pathology in an APP transgenic mouse model of Alzheimer's disease lacking functional B and T cells.

INTRODUCTION: In Alzheimer's disease, accumulation and pathological aggregation of amyloid ß-peptide is accompanied by the induction of complex immune responses, which have been attributed both beneficial and detrimental properties. Such responses implicate various cell types of the innate and adaptive arm of the immunesystem, both inside the central nervous system, and in the periphery. To investigate the role of the adaptive immune system in brain ß-amyloidosis, PSAPP transgenic mice, an established mouse model of Alzheimer's disease, were crossbred with the recombination activating gene-2 knockout (Rag2 ko) mice lacking functional B and T cells. In a second experimental paradigm, aged PSAPP mice were reconstituted with bone marrow cells from either Rag2 ko or wildtype control mice. RESULTS: Analyses from both experimental approaches revealed reduced ß-amyloid pathology and decreased brain amyloid ß-peptide levels in PSAPP mice lacking functional adaptive immune cells. The decrease in brain ß-amyloid pathology was associated with enhanced microgliosis and increased phagocytosis of amyloid ß-peptide aggregates. CONCLUSION: The results of this study demonstrate an impact of the adaptive immunity on cerebral ß-amyloid pathology in vivo and suggest an influence on microglia-mediated amyloid ß-peptide clearance as a possible underlying mechanism.

Lymph node stromal cells acquire peptide-MHCII complexes from dendritic cells and induce antigen-specific CD4⁺ T cell tolerance.

Dendritic cells (DCs), and more recently lymph node stromal cells (LNSCs), have been described to tolerize self-reactive CD8(+) T cells in LNs. Although LNSCs express MHCII, it is unknown whether they can also impact CD4(+) T cell functions. We show that the promoter IV (pIV) of class II transactivator (CIITA), the master regulator of MHCII expression, controls endogenous MHCII expression by LNSCs. Unexpectedly, LNSCs also acquire peptide-MHCII complexes from DCs and induce CD4(+) T cell dysfunction by presenting transferred complexes to naive CD4(+) T cells and preventing their proliferation and survival. Our data reveals a novel, alternative mechanism where LN-resident stromal cells tolerize CD4(+) T cells through the presentation of self-antigens via transferred peptide-MHCII complexes of DC origin.

MHC class II-restricted antigen presentation by plasmacytoid dendritic cells inhibits T cell-mediated autoimmunity.

Although plasmacytoid dendritic cells (pDCs) express major histocompatibility complex class II (MHCII) molecules, and can capture, process, and present antigens (Ags), direct demonstrations that they function as professional Ag-presenting cells (APCs) in vivo during ongoing immune responses remain lacking. We demonstrate that mice exhibiting a selective abrogation of MHCII expression by pDCs develop exacerbated experimental autoimmune encephalomyelitis (EAE) as a consequence of enhanced priming of encephalitogenic CD4(+) T cell responses in secondary lymphoid tissues. After EAE induction, pDCs are recruited to lymph nodes and establish MHCII-dependent myelin-Ag-specific contacts with CD4(+) T cells. These interactions promote the selective expansion of myelin-Ag-specific natural regulatory T cells that dampen the autoimmune T cell response. pDCs thus function as APCs during the course of EAE and confer a natural protection against autoimmune disease development that is mediated directly by their ability to present of Ags to CD4(+) T cells in vivo.

N-glycoprotein profiling of lung adenocarcinoma pleural effusions by shotgun proteomics.

BACKGROUND: Malignant pleural effusion of advanced lung adenocarcinoma may be a valid source for detection of biomarkers, such as N-glycosylated proteins (N-GP), because tumor cells grow during weeks in this liquid. The authors aimed for creation of N-GP effusion profiles from routine cytology specimens to detect relevant biomarkers. METHODS: Hundred microliters of malignant pleural effusions of 5 patients with lung adenocarcinoma and 5 nonmalignant controls were used for triplicate N-GP capture by solid-phase extraction. After trypsin digest and PNGase F release, a liquid chromatography separation connected online to a tandem mass spectrometer was performed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). RESULTS: In the total of 10 samples, 170 and 278 nonredundant proteins were detected with probabilities of >or=.9 and >or=.5, respectively. The specificity for the N-glycomotif was 88% at P >or= .9. Penetration into the moderate to low protein concentration range (microg-ng/mL) occurred, and several proteins associated with tumor progression or metastasis were identified, including CA-125, CD44, CD166, lysosome-associated membrane glycoprotein 2 (LAMP-2), multimerin 2, and periostin. MS identifications were correlated with the corresponding immunoreactivity in either effusion fluid or tumor tissue. CONCLUSIONS: In conclusion, reduction of sample complexity by N-GP capturing allows detection of proteins in the mug to ng/mL range. Pleural effusion is a useful source for biomarker research in lung cancer.

Variations in anticipatory cognitive stress appraisal and differential proinflammatory cytokine expression in response to acute stress.

OBJECTIVE: Anticipatory cognitive appraisal can affect the stress-induced release of stress hormones and stress hormones can modulate monocyte cytokine expression. We investigated whether anticipatory cognitive appraisal processes would predict changes in monocyte cytokine expression following psychosocial stress in relation to stress hormone release. METHODS: Forty-four men (mean age 43+/-2 years; mean arterial blood pressure (MAP) 102+/-2 mmHg; mean body mass index (BMI) 26+/-.4kg/m(2)) completed the Primary Appraisal Secondary Appraisal (PASA) scale before undergoing the Trier Social Stress Test (combination of mock job interview and mental arithmetic task). Lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 expression by monocytes was assessed in vitro immediately before and after stress, and during recovery up to 60min post-stress. Moreover, we repeatedly measured salivary cortisol as well as plasma epinephrine and norepinephrine levels. RESULTS: Stress hormones increased and cytokines decreased following stress (all p<0.05). Correlation analyses showed that a higher PASA "stress index" was associated with higher expression (area under the curve, AUC) of total LPS-stimulated TNF-alpha (r=.33, p=.03) and IL-6 (r=.32, p=.33) between rest and 60min post-stress. While controlling for age, BMI, and MAP, higher values in the primary PASA scale "control expectancy" predicted lower TNF-alpha expression following stress (ss=-.42, p=0.003). Higher "control expectancy" (ss=-.32, p=0.031) and lower "challenge" (ss=.30, p=0.046) predicted lower IL-6 expression. None of the stress hormones predicted expression of any cytokine. CONCLUSIONS: We found that anticipatory cognitive stress appraisal modulates monocyte inflammatory activity following stress suggesting that interventions aiming at improving coping skills might modify the monocyte cytokine response.

Modulation of therapeutic antibody effector functions by glycosylation engineering: influence of Golgi enzyme localization domain and co-expression of heterologous beta1, 4-N-acetylglucosaminyltransferase III and Golgi alpha-mannosidase II.

The effector functions elicited by IgG antibodies strongly depend on the carbohydrate moiety linked to the Fc region of the protein. Therefore several approaches have been developed to rationally manipulate these glycans and improve the biological functions of the antibody. Overexpression of recombinant beta1,4-N-acetylglucosaminyltransferase III (GnT-III) in production cell lines leads to antibodies enriched in bisected oligosaccharides. Moreover, GnT-III overexpression leads to increases in non-fucosylated and hybrid oligosaccharides. Such antibody glycovariants have increased antibody-dependent cellular cytotoxicity (ADCC). To explore a further variable besides overexpression of GnT-III, we exchanged the localization domain of GnT-III with that of other Golgi-resident enzymes. Our results indicate that chimeric GnT-III can compete even more efficiently against the endogenous core alpha1,6-fucosyltransferase (alpha1,6-FucT) and Golgi alpha-mannosidase II (ManII) leading to higher proportions of bisected non-fucosylated hybrid glycans ("Glyco-1" antibody). The co-expression of GnT-III and ManII led to a similar degree of non-fucosylation as that obtained for Glyco-1, but the majority of the oligosaccharides linked to this antibody ("Glyco-2") are of the complex type. These glycovariants feature strongly increased ADCC activity compared to the unmodified antibody, while Glyco-1 (hybrid-rich) features reduced complement-dependent cytotoxicity (CDC) compared to Glyco-2 or unmodified antibody. We show that apart from GnT-III overexpression, engineering of GnT-III localization is a versatile tool to modulate the biological activities of antibodies relevant for their therapeutic application.