Differences In Cytokine Responses Research Articles

Topical treatment with nanoliposomal Amphotericin B reduces early lesion growth but fails to induce cure in an experimental model of cutaneous leishmaniasis caused by Leishmania mexicana

Leishmania mexicana infection causes localized skin lesions that can lead to tissue damage and permanent disfigurement if not resolved. Currently, recommended treatments include intravenous administration of Amphotericin B, which is undesirable due to the associated cost and patient burden related to receiving regular injections. In this study, we evaluated the effect of topical treatment with a nanoliposomal formulation of Amphotericin B that is penetrable to the skin (SinaAmphoLeish 0.4%) in mice infected with L. mexicana by using ulcerated (BALB/c) and non-ulcerated (129SVE) models. BALB/c mice received a 4 week treatment following ulcerated lesion development, while 129SVE mice received a 10 week treatment beginning at week 5 post-infection. Although mice from both models showed comparable susceptibility to L. mexicana infection after topical treatment with SinaAmphoLeish relative to controls, 129SVE mice displayed a transient decrease in lesion sizes which eventually became similar to control mice. On other hand this treatment resulted in no reduction in the lesion sizes in BALB/c mice. 129SVE treated mice exhibited greater IFN-γ, IL-4, and IL-10 cytokine levels and higher T-cell proliferation in re-stimulated draining lymph node cells. BALB/c mice showed no differences in cytokine responses between treated and control mice. These findings indicate that topical SinaAmphoLeish treatment is not likely to be effective in the treatment of cutaneous leishmaniasis caused by L. mexicana.

Differential Gamma Interferon- and Tumor Necrosis Factor Alpha-Driven Cytokine Response Distinguishes Acute Infection of a Metatherian Host with Toxoplasma gondii and Neospora caninum.

Toxoplasma gondii and Neospora caninum (both Apicomplexa) are closely related cyst-forming coccidian parasites that differ significantly in their host ranges and ability to cause disease. Unlike eutherian mammals, Australian marsupials (metatherian mammals) have long been thought to be highly susceptible to toxoplasmosis and neosporosis because of their historical isolation from the parasites. In this study, the carnivorous fat-tailed dunnart (Sminthopsis crassicaudata) was used as a disease model to investigate the immune response and susceptibility to infection of an Australian marsupial to T. gondii and N. caninum The disease outcome was more severe in N. caninum-infected dunnarts than in T. gondii-infected dunnarts, as shown by the severity of clinical and histopathological features of disease and higher tissue parasite burdens in the tissues evaluated. Transcriptome sequencing (RNA-seq) of spleens from infected dunnarts and mitogen-stimulated dunnart splenocytes was used to define the cytokine repertoires. Changes in mRNA expression during the time course of infection were measured using quantitative reverse transcription-PCR (qRT-PCR) for key Th1 (gamma interferon [IFN-γ] and tumor necrosis factor alpha [TNF-α]), Th2 (interleukin 4 [IL-4] and IL-6), and Th17 (IL-17A) cytokines. The results show qualitative differences in cytokine responses by the fat-tailed dunnart to infection with N. caninum and T. gondii Dunnarts infected with T. gondii were capable of mounting a more effective Th1 immune response than those infected with N. caninum, indicating the role of the immune response in the outcome scenarios of parasite infection in this marsupial mammal.

Proinflammatory effects of diesel exhaust particles from moderate blend concentrations of 1st and 2nd generation biodiesel in BEAS-2B bronchial epithelial cells—The FuelHealth project

Biodiesel fuel fuels are introduced at an increasing extent as a more carbon-neutral alternative to reduce CO2-emissions, compared to conventional diesel fuel. In the present study we have investigated the impact of increasing the use of 1st generation fatty acid methyl ester (FAME) biodiesel from current 7% blend (B7) to 20% blend (B20), or by increasing the biodiesel content by adding 2nd generation hydrotreated vegetable oil (HVO) based biodiesel (SHB; Synthetic Hydrocarbon Biofuel) on toxicity of diesel exhaust particles (DEP) in an in vitro system. Human bronchial epithelial BEAS–2B cells were exposed for 4 and 20h to DEP from B7, B20 and SHB at different concentrations, and examined for effects on gene expression of interleukin 6 (IL-6), CXCL8 (IL-8), CYP1A1 and heme oxygenase-1 (HO-1). The results show that both B20 and SHB were more potent inducers of IL-6 expression compared to B7. Only B20 induced statistically significant increases in CXCL8 expression. By comparison the rank order of potency to induce CYP1A1 was SHB>B7>B20. No statistically significant difference were observed form HO-1 expression, suggesting that the differences in cytokine responses were not due to oxidative stress. The results show that even moderate increases in biodiesel blends, from 7% to 20%, may increase the proinflammatory potential of emitted DEP in BEAS–2B cells. This effect was observed for both addition of 1st generation FAME and 2nd generation HVO biodiesel.

Whole Blood Cytokine Response to Local Traffic-Related Particulate Matter in Peruvian Children With and Without Asthma.

This study sought to investigate if acute phase immune responses of whole blood from Peruvian children with controlled and uncontrolled asthma differed from children without asthma, following exposure to traffic-related particulate matter (TRPM). TRPM, including particulate matter from diesel combustion, has been shown to stimulate acute airway inflammation in individuals with and without asthma. For this study, a whole blood assay (WBA) was used to test peripheral whole blood samples from 27 children with asthma, and 12 without asthma. Participant blood samples were stimulated, ex vivo, for 24-h with an aqueous extract of TRPM that was collected near study area highways in Lima, Peru. All participant blood samples were tested against the same TRPM extract, in addition to purified bacterial endotoxin and pyrogen-free water, which served as positive and negative WBA controls, respectively. The innate and adaptive cytokine responses were evaluated in cell-free supernatants of the whole blood incubations. Comparatively similar levels were recorded for nine out of the 10 cytokines measured [e.g., – Interleukin (IL)-1β, IL-6, IL-10], regardless of study participant asthma status. However, IL-8 levels in TRPM-stimulated blood from children with uncontrolled asthma were diminished, compared to subjects without asthma (633 pg/ml vs. 1,023 pg/ml, respectively; p < 0.01); IL-8 responses for subjects with controlled asthma were also reduced, but to a lesser degree (799 pg/ml vs. 1,023 pg/ml, respectively; p = 0.10). These relationships were present before, and after, adjusting for age, sex, obesity/overweight status, C-reactive protein levels, and residential proximity to the study area’s major roadway. For tests conducted with endotoxin, there were no discernible differences in cytokine response between groups, for all cytokines measured. The WBA testing conducted for this study highlighted the capacity of the TRPM extract to potently elicit the release of IL-8 from the human whole blood system. Although the small sample size of the study limits the capacity to draw definitive conclusions, the IL-8 responses suggest that that asthma control may be associated with the regulation of a key mediator in neutrophil chemotaxis, at a systemic level, following exposure to PM derived from traffic-related sources.

Editorial: Infection and Inflammation: Potential Triggers of Sudden Infant Deaths.

The Editorial on the Research Topic Infection and Inflammation: Potential Triggers of Sudden Infant Deaths Among infants in industrialized countries, sudden infant death syndrome (SIDS) is still the major cause of death between 1 month and 1 year of age. SIDS is a diagnosis of exclusion, a subset of sudden unexpected deaths in infancy (SUDI), those deaths that remain unexplained following a thorough postmortem, assessment of the medical history, and investigation of the scene of death. The risk factors for SUDI/SIDS identified in epidemiological studies parallel risk factors associated with susceptibility to infection in children, and various studies have identified microorganisms or their products in significant proportions of these deaths (Blackwell et al.). Even in a country, such as Hungary, with a historically low incidence of SUDI/SIDS, there is evidence that infection and inflammation contribute to these deaths (Toro et al.). This collection provides insights into a variety of approaches for the investigation of how genetic, environmental, and developmental risk factors could dysregulate or potentiate the effects of inflammatory responses to “mild” infections that are often reported prior to death of the infant. Exploration of the role of infectious agents in these deaths has been limited by two factors. The evidence does not fit Koch’s postulates: no single organism has been isolated from all cases of SUDI/SIDS. A variety of organisms, both viruses and bacteria with various virulence factors, has been identified in different studies (Blackwell et al.; Bettelheim and Goldwater; Goldwater). In addition, there is no animal model that accurately reflects all the genetic, developmental, and environmental risk factors associated with SUDI/SIDS. This problem and examples of the models developed are reviewed by Blood-Siegfried. The common thread in the investigation of infection does not appear to be a specific infectious agent but the infant’s inflammatory response to the infection. Therefore, it is important to develop models to assess risk factors that can influence levels of inflammatory mediators, which can affect the major physiological mechanisms proposed to explain SUDI/SIDS – anaphylaxis, poor arousal, hypoxia and apnea, shock, cardiac arrhythmias, hyperthermia, and hypoglycemia. The differences in the incidence of SIDS among ethnic groups and the excess of male infants indicated that genetic factors might be involved in triggering these deaths. Potential genetic contributions to these deaths and the case for whole exome sequencing are assessed here by Morris. Genetic variations in the immune/inflammatory responses in relation to SUDI are reviewed by Ferrante and Opdal. The potential contribution of two cytokines and polymorphisms in their genes, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), is explored by Moscovis et al. and Moscovis et al. in relation to these responses to infectious agents and interactions with environmental risk factors such as cigarette smoke. The differences in cytokine responses of male and female fetuses identified by Burns et al. indicate that females might be better able to control inflammatory responses in utero. Two important risk factors for SUDI/SIDS are low birth weight and premature birth. These were explored in relation to two environmental factors that could affect fetal development during pregnancy, exposure to cigarette smoke, and evidence of inflammation. Evidence of inflammation was inversely correlated to gestational age. Exposure to cigarette smoke was also significantly associated with low birth weight and age at delivery. The effects of both evidence of inflammation and exposure to cigarette smoke were more pronounced for male infants (Pringle et al.). In the absence of a widely accepted model for these infant deaths, their investigation requires the cooperation of a variety of disciplines to attempt to identify why these children have died suddenly. Current evidence indicates that inflammatory responses are dysregulated by combinations of infectious agents and cigarette smoke. Developmental stage of the infant and hormone levels might also affect these responses (Blackwell et al.). The approaches and techniques developed to examine SUDI/SIDS need to be applied to stillbirths, which have been suggested to be part of the spectrum of infant deaths associated with SIDS (1). While the risk factors are similar, investigations of stillbirths need to take into consideration the responses to infectious agents of both the mother and the infant (Blackwell). New techniques emerging for studies of infection and inflammation will complement the standard studies carried in routine forensic examinations: assessment of microbiome for both viral and bacterial pathogens; whole genome sequencing of DNA from infants; and screening for a range of inflammatory mediators. These techniques are not usually available for diagnosis of infant deaths. Progress in this area will require cooperation between forensic medicine and pathology services and research groups with the expertise to complement investigations carried out by standard protocols.

The influence of atopy and asthma on immune responses in inner‐city adults

Asthma in the inner-city population is usually atopic in nature, and is associated with significant morbidity and mortality. However, the underlying immune abnormalities that underlie asthma in urban adults have not been well defined. We investigated the influence of atopy and asthma on cytokine responses of inner-city adult women to define immune abnormalities associated with asthma and atopy. Blood samples were collected from 509 of 606 inner-city women enrolled in the Urban Environment and Childhood Asthma (URECA) study. We tested for associations between atopy and asthma status and cytokine responses in peripheral blood mononuclear cells incubated ex vivo with a panel of innate and adaptive immune stimulants. Atopic subjects had heightened Th2 cytokine responses (IL-4, IL-5, IL-13) to cockroach and dust mite antigens, tetanus toxoid, and phytohemagglutinin (P < 0.05 for all). Differences in cytokine responses were greatest in response to stimulation with cockroach and dust mite. In a multivariate analysis, atopy was broadly related to increased Th2-like responses to all antigens and PHA, while asthma was only weakly related to mitogen-induced IL-4 and IL-5 responses. There were few asthma or allergy-related differences in responses to innate stimuli, including IFN-α and IFN-γ responses. In this inner-city adult female population, atopy is associated with enhanced Th2 responses to allergens and other stimuli, and there was little or no additional signal attributable to asthma. In particular, these data indicate that altered systemic interferon and innate immune responses are not associated with allergies and/or asthma in inner-city women.

Cytokine Response after Stimulation with Key Commensal Bacteria Differ in Post-Infectious Irritable Bowel Syndrome (PI-IBS) Patients Compared to Healthy Controls.

BackgroundMicrobial dysbiosis and prolonged immune activation resulting in low-grade inflammation and intestinal barrier dysfunction have been suggested to be underlying causes of post-infectious irritable bowel syndrome (PI-IBS). The aim of this study was to evaluate the difference in cytokine response between mucosal specimens of PI-IBS patients and healthy controls (HC) after ex vivo stimulation with key anaerobic bacteria.MethodsColonic biopsies from 11 PI-IBS patients and 10 HC were stimulated ex vivo with the commensal bacteria Bacteroides ovatus, Ruminococcus gnavus, Akkermansia muciniphila, Subdoligranulum variabile and Eubacterium limosum, respectively. The cytokine release (IL-1β, IL-2, IL-8, IL-10, IL-13, IL-17, TNF-α and IFN-γ) in stimulation supernatants was analyzed using the LUMINEX assay. Comparison of cytokine release between PI-IBS patients and healthy controls was performed taking both unstimulated and bacterially stimulated mucosal specimens into account.Key ResultsIL-13 release from mucosal specimens without bacterial stimulation was significantly lower in PI-IBS patients compared to HC (p < 0.05). After stimulation with Subdoligranulum variabile, IL-1β release from PI-IBS patients was significantly increased compared to HC (p < 0.05). Stimulation with Eubacterium limosum resulted in a significantly decreased IL-10 release in HC compared to PI-IBS patients (p < 0.05) and a tendency to decreased IL-13 release in HC compared to PI-IBS patients (p = 0.07).Conclusions & InferencesPI-IBS patients differ from HC with regard to cytokine release ex vivo after stimulation with selected commensal bacteria. Hence, our results support that the pathogenesis of PI-IBS comprises an altered immune response against commensal gut microbes.

Peripheral Immune Response to Marine Corps Martial Arts Training

This study measured changes in the profile of circulating immune cells and stress proteins following an acute bout of U.S. Marine Corps Martial Arts training. Seven male Marines participated in this study. Blood samples were collected prior to the start of training (baseline) and during recovery at 0-, 15-, and 60-min post. Blood measures include cortisol, catecholamines, CBC (with differential), lymphocyte subsets, T helper cell cytokines GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-13, IL-17a and TNF-α, IgG, IgM, and serum creatine kinase. Data was analyzed using rm-ANOVA with Bonferroni corrections (p<0.05). Total catecholamines were significantly elevated at 0-min and remained elevated at 60-min. Total white blood cell counts and neutrophils were significantly elevated at 0- and 15-min returning to baseline values by 60-min. IgG was significantly elevated at 0- and returned to baseline at 15-min. CD3+ and CD4+ cell counts both had non-significant elevations at 0-min but were significantly lower at 15- and 60-min compared to baseline. Total lymphocytes and CD19+ cells were significantly lower at 60-min than baseline. There were no significant differences in cytokine responses. We conclude the Marine Corps Martial Arts Program is able to induce a significant alteration in the distribution of lymphocyte subsets in the peripheral circulation following a training bout but does not shift the balance of Th1/Th2 cytokines or induce a systemic inflammatory response. Supported by a grant through the Office of Naval Research.

Male adolescent rats display blunted cytokine responses in the CNS after acute ethanol or lipopolysaccharide exposure

Alcohol induces widespread changes in cytokine expression, with recent data from our laboratory having demonstrated that, during acute ethanol intoxication, adult rats exhibit consistent increases in interleukin (IL)-6 mRNA expression in several brain regions, while showing reductions in IL-1 and TNFα expression. Given evidence indicating that adolescence may be an ontogenetic period in which some neuroimmune processes and cells may not yet have fully matured, the purpose of the current experiments was to examine potential age differences in the central cytokine response of adolescent (P31–33days of age) and adult (69–71days of age) rats to either an acute immune (lipopolysaccharide; LPS) or non-immune challenge (ethanol). In Experiment 1, male Sprague–Dawley rats were given an intraperitoneal (i.p.) injection of either sterile saline, LPS (250μg/kg), or ethanol (4-g/kg), and then trunk blood and brain tissue were collected 3h later for measurement of blood ethanol concentrations (BECs), plasma endotoxin, and central mRNA expression of several immune-related gene targets. In Experiment 2, the response to intragastrically (i.g.) administered ethanol was examined and compared to animals given tap water (i.g.). Results showed that LPS stimulated robust increases in expression of IL-1, IL-6, TNFα, and IκBα in the hippocampus, PVN, and amygdala, and that these increases were generally less pronounced in adolescents relative to adults. Following an i.p. ethanol challenge, IL-6 and IκBα expression was significantly increased in both ages in the PVN and amygdala, and adults exhibited even greater increases in IκBα than adolescents. I.g. administration of ethanol also increased IL-6 and IκBα expression in all three brain regions, with hippocampal IL-6 elevated even more so in adults compared to adolescents. Furthermore, assessment of plasma endotoxin concentrations revealed (i) whereas robust increases in plasma endotoxin were observed in adults injected with LPS, no corresponding elevations were seen in adolescents after LPS; and (ii) neither adolescents nor adults demonstrated increases in plasma endotoxin concentrations following i.p. or i.g. ethanol administration. Analysis of BECs indicated that, for both routes of exposure, adolescents exhibited lower BECs than adults. Taken together, these data suggest that categorically different mechanisms are involved in the central cytokine response to antigen exposure versus ethanol administration. Furthermore, these findings confirm once again that acute ethanol intoxication is a potent activator of brain cytokines, and calls for future studies to identify the mechanisms underlying age-related differences in the cytokine response observed during ethanol intoxication.

Polymorphisms in cytokine genes IL6, TNF, IL10, IL17A and IFNG influence susceptibility to complicated skin and skin structure infections.

Complicated skin and skin structure infections (cSSSIs) are caused by Gram-positive and Gram-negative, aerobic and anaerobic pathogens, with a polymicrobial aetiology being frequent. Recognition of invading pathogens by the immune system results in the production of pro- and anti-inflammatory cytokines, which are extremely important for intercellular communication and control of infection. This study assessed whether genetic variation in genes encoding cytokines influences the susceptibility to cSSSIs. For the association study, 318 patients with cSSSI and 328 healthy controls were genotyped for single nucleotide polymorphisms (SNPs) in cytokine genes IL1A, IL1B, IL1RN, TNF, IL10, IL17A, IL17F and IFNG. For immunological validation, peripheral blood mononuclear cells (PBMCs) from 74 healthy individuals, genotyped for SNPs of interest, were stimulated with Staphylococcus aureus or Escherichia coli and corresponding cytokine levels were determined by enzyme-linked immunosorbent assay (ELISA). Polymorphisms IL6 rs1800797, TNF rs1800629, IL10 rs1800871, IL17A rs8193036 and IFNG rs2069705 influenced susceptibility to cSSSIs. No differences in cytokine responses, stratified for genotype, were detected after PBMC stimulation. No association with cSSSIs was observed for polymorphisms IL1A rs17561 and rs1800587, IL1B rs16944 and rs1143627, IL1RN rs4251961, TNF rs361525, IL10 rs1800896, IL17A rs2275913 and IL17F rs763780. In conclusion, polymorphisms in IL6, TNF, IL10, IL17A and IFNG are associated with susceptibility to cSSSIs.

The risk for behavioural deficits is determined by the maternal immune response to prenatal immune challenge in a neurodevelopmental model

Schizophrenia is a highly disabling psychiatric disorder with a proposed neurodevelopmental basis. One mechanism through which genetic and environmental risk factors might act is by triggering persistent brain inflammation, as evidenced by long-lasting neuro-immunological disturbances in patients. Our goal was to investigate whether microglia activation is a neurobiological correlate to the altered behaviour in the maternal immune activation (MIA) model, a well-validated animal model with relevance to schizophrenia. A recent observation in the MIA model is the differential maternal body weight response to the immune stimulus, correlated with a different behavioural outcome in the offspring. Although it is generally assumed that the differences in maternal weight response reflect differences in cytokine response, this has not been investigated so far. Our aim was to investigate whether (i) the maternal weight response to MIA reflects differences in the maternal cytokine response, (ii) the differential behavioural phenotype of the offspring extends to depressive symptoms such as anhedonia and (iii) there are changes in chronic microglia activation dependent on the behavioural phenotype. Based on a dose-response study, MIA was induced in pregnant rats by injecting 4mg/kg Poly I:C at gestational day 15. Serum samples were collected to assess the amount of TNF-α in the maternal blood following MIA. MIA offspring were divided into weight loss (WL; n=14) and weight gain (WG; n=10) groups, depending on the maternal body weight response to Poly I:C. Adult offspring were behaviourally phenotyped for prepulse inhibition, locomotor activity with and without amphetamine and MK-801 challenge, and sucrose preference. Finally, microglia activation was scored on CD11b- and Iba1-immunohistochemically stained sections. Pregnant dams that lost weight following MIA showed increased levels of TNF-α compared to controls, unlike dams that gained weight following MIA. Poly I:C WL offspring showed the most severe behavioural outcome. Poly I:C WG offspring, on the other hand, did not show clear behavioural deficits. Most interestingly a reduced sucrose preference indicative of anhedonia was found in Poly I:C WL but not Poly I:C WG offspring compared to controls. Finally, there were no significant differences in microglia activation scores between any of the investigated groups. The individual maternal immune response to MIA is an important determinant of the behavioural outcome in offspring, including negative symptoms such as anhedonia. We failed to find any significant difference in the level of microglia activation between Poly I:C WL, Poly I:C WG and control offspring.

Eosinophils Are Important for Protection, Immunoregulation and Pathology during Infection with Nematode Microfilariae

Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO) and major basic protein-1 (MBP-1), during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL), we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity.

Establishment of cell lines from both myeloma bone marrow and plasmacytoma: SNU_MM1393_BM and SNU_MM1393_SC from a single patient.

Purpose. We tried to establish clinically relevant human myeloma cell lines that can contribute to the understanding of multiple myeloma (MM). Materials and Methods. Mononuclear cells obtained from MM patient's bone marrow were injected via tail vein in an NRG/SCID mouse. Fourteen weeks after the injection, tumor developed at subcutis of the mouse. The engraftment of MM cells into mouse bone marrow (BM) was also observed. We separated and cultured cells from subcutis and BM. Results. After the separation and culture of cells from subcutis and BM, we established two cell lines originating from a single patient (SNU_MM1393_BM and SNU_MM1393_SC). Karyotype of the two newly established MM cell lines showed tetraploidy which is different from the karyotype of the patient (diploidy) indicating clonal evolution. In contrast to SNU_MM1393_BM, cell proliferation of SNU_MM1393_SC was IL-6 independent. SNU_MM1393_BM and SNU_MM1393_SC showed high degree of resistance against bortezomib compared to U266 cell line. SNU_MM1393_BM had the greater lethality compared to SNU_MM1393_SC. Conclusion. Two cell lines harboring different site tropisms established from a single patient showed differences in cytokine response and lethality. Our newly established cell lines could be used as a tool to understand the biology of multiple myeloma.

189: Differences in cytokine responses during infection with high and low pathogenic Ebola viruses

To date five Ebolavirus species have been identified, differing in pathogenicity. The highly pathogenic Zaire ebolavirus (EBOV) causes a hemorrhagic disease in humans with case fatality rates up to 90%. A hallmark of EBOV infection is a dysregulated immune response, including high levels of cytokines. In contrast, Reston ebolavirus (RESTV) seems to be nonpathogenic in humans, as only asymptomatic cases of RESTV infection in humans have been reported. The mechanisms of protection against RESTV in humans are not yet understood. As cytokines might play a role in the pathogenesis of EBOV infection, we analyzed the cytokine response in EBOV- and RESTV-infected human macrophages and dendritic cells. These cells not only orchestrate innate and adaptive immune responses but are also early targets of Ebolaviruses. We observed degradation of I κ Bα and nuclear translocation of p65 in EBOV-infected macrophages, indicating activation of NFκB signaling. qRT-PCR and Luminex assays revealed induction of selected cytokines in EBOV-infected macrophages. Most analyzed cytokines and chemokines were upregulated in EBOV infection at 6 hpi or later, whereas others, including IL10, did not show increased secretion levels. In contrast, p65 was not translocated into the nucleus in RESTV-infected macrophages and no significant changes in cytokine mRNA levels or secreted protein levels were observed. To investigate if this effect is due to inhibition of pro-inflammatory signaling pathways, RESTV-infected macrophages were treated with LPS at 1 dpi and cytokine production was determined. Similar levels of pro-inflammatory cytokines, including IL6 and TNFα, were detected in RESTV-infected and LPS-treated macrophages compared to non-infected LPS-treated cells, suggesting that RESTV is not able to inhibit LPS-mediated cytokine induction. Our data support the hypothesis that high pathogenicity correlates with high cytokine levels in Ebolavirus infection. Currently we are investigating which cellular signaling pathways are involved in the observed differential cytokine induction in EBOV versus RESTV infection.

CCR2 Deficiency, Dysregulation of Notch Signaling, and Spontaneous Pulmonary Arterial Hypertension

In pulmonary arterial hypertension (PAH), there is overexpression of the chemokine, C-C chemokine ligand type 2 (CCL2), and infiltration of myeloid cells into the pulmonary vasculature. Inhibition of CCL2 in animals decreases PAH, suggesting that the CCL2 receptor (CCR2) plays a role in PAH development. To test this hypothesis, we exposed wild-type (WT) and CCR2-deficient (Ccr2(-/-)) mice to chronic hypobaric hypoxia to induce PAH. After hypoxic stress, Ccr2(-/-) mice displayed a more severe PAH phenotype, as demonstrated by increased right ventricular (RV) systolic pressures, RV hypertrophy, and tachycardia relative to WT mice. However, these mice also exhibited increased RV systolic pressures and increased pulmonary artery muscularization under normoxic conditions. Moreover, Ccr2(-/-) mice displayed decreased pulmonary vascular branching at 3 weeks of age and increased vascular muscularization at birth, suggesting that an abnormality in pulmonary vascular development leads to spontaneous PAH in these animals. No significant differences in cytokine responses were observed between WT and Ccr2(-/-) mice during either normoxia or hypoxia. However, Ccr2(-/-) mice displayed increased Notch-3 signaling and dysregulated Notch ligand expression, suggesting a possible cause for their abnormal pulmonary vascular development. Our findings imply that CCR2 does not directly contribute to the development of PAH, but does play a previously unrecognized role in pulmonary vasculature development and remodeling wherein the absence of CCR2 results in spontaneous PAH, most likely via dysregulation of Notch signaling. Our results demonstrate that CCR2 has impacts beyond leukocyte recruitment, and is required for the proper expression of Notch signaling molecules.

Associations between polymorphisms in the antiviral TRIM genes and measles vaccine immunity

The role of polymorphisms within the antiviral tripartite motif (TRIM) genes in measles vaccine adaptive immune responses was examined. A limited association was found between TRIM5 (rs7122620) and TRIM25 (rs205499) gene polymorphisms and measles-specific antibody levels. However, many associations were found between TRIM gene SNPs and variations in cellular responses (IFN-γ Elispot and secreted cytokines IL-2, IL-6, IL-10, IFN-γ, and TNF-α). TRIM22 rs2291841 was significantly associated with an increased IFN-γ Elispot response (35 vs. 102 SFC per 2×10(5)PBMC, p=0.009, q=0.71) in Caucasians. A non-synonymous TRIM25 rs205498 (in LD with other SNPs, r(2)≥0.56), as well as the TRIM25 AAAGGAAAGGAGT haplotype, was associated with a decreased IFN-γ Elispot response (t-statistic -2.32, p=0.02) in African-Americans. We also identified polymorphisms in the TRIM5, TRIM22, and TRIM25 genes that were associated with significant differences in cytokine responses. Additional studies are necessary to replicate our findings and to examine the functional consequences of these associations.

Early immune outcome of retroperitoneal laparoscopic radical nephrectomy for localized renal cell carcinoma: a prospective, randomized study

Objectives: We evaluated differences in cytokine responses and T-lymphocyte subsets following retroperitoneal laparoscopic andconventional open radical nephrectomies for localized renal cellcarcinoma (RCC).Methods: A total of 62 patients with T1N0M0 staged RCC were randomized to either retro-laparoscopic (n = 31) or open (n = 31) radical nephrectomy. Plasma levels of interleukin-1β (IL-1β), IL-6, andtumour necrosis factor-alpha (TNF-α) were measured separately by enzyme linked immunosorbent assay (ELISA) preoperatively and on postoperative days 1 and 5. Levels of CD3+, CD4+ and CD8+ as well as the CD4+:CD8+ ratio were acquired by flow cytometry at the same time points.Results: Levels of IL-1β, IL-6 and TNF-α increased significantly compared to preoperative values in both groups (p &lt; 0.05) on postoperative day 1, and all the parameters in the open group were significantly higher than those in the retro-laparoscopy group (p &lt; 0.05). On postoperative day 1, the levels of CD3+ and CD4+ and the CD4+:CD8+ ratio decreased markedly compared to preoperative values for both groups (p &lt; 0.05). Elevations of the CD4+:CD8+ ratio in the retro-laparoscopy group (p &lt; 0.05) and the CD8+ level in the open group (p &lt; 0.05) were observed when compared with the other group. On postoperative day 5, the levels of CD3+ and CD4+ and the CD4+:CD8+ ratio in the retro-laparoscopy group, as well as the level of CD8+ in the open group, returned to about preoperative levels (p &lt; 0.05). Follow-up ranged from 4 to14 months postoperatively in all 62 patients with a 100% cancer specific survival rate in both groups.Conclusions: Retroperitoneal laparoscopic radical nephrectomy is associated with the milder cytokine responses caused by trauma and inflammation and the better preserved distribution ofT-lymphocytes.

P004 Determination of the cytokines and chemokines level in the serum and csf of JE and non-JE viral encephalitis cases

Introduction Acute encephalitis syndrome cases with multiple aetiologies are occurring every year in eastern Uttar Pradesh. Japanese encephalitis is the leading cause of the viral encephalitis with known aetiology, however majority of them are unknown. During viral infection release of cytokines and chemokines by host immune cells play an important role in inhibition of virus. The difference in cytokine response may be one of the mechanisms accounting for different severities among different viral infections of the central nervous system. The present study determines the cytokines and chemokines level in the serum and CSF of JE and non-JE viral encephalitis cases and compare its significance between the group. Methods The study was conducted on 45 AES patients. Out of 45, twelve were JE patients as determined by JE IgM capture ELISA in CSF. Rest 33 were AES cases with unknown etiology. CSF and serum samples from the patients were collected and immediately stored at −80 °C. The levels of cytokines TNF- α , IFN- γ , IL-10 and chemokines IP-10, and RANTES in the CSF and serum samples were analyzed by use of the Duo ELISA kit (R&D Systems) as per the manufacturers’ instructions. Differences of cytokine level between groups (JE and non-JE) were examined by Mann–Whitney U test. P Results Serum and CSF levels of the TNF- α and RANTES in both groups were high but had no significant difference in the rise of the cytokines level ( P > 0.05). The serum and CSF level of IFN- γ , IL-10 and IP-10 were significantly higher in Non JE patients than in JE patients ( P α level was higher than in serum. RANTES level was higher in both the serum and CSF of the JE and non-JE group but had no significant difference. Conclusion Higher level of TNF- α in CSF of JE patient shows the increase in severity of neuronal damage in the JE cases. Increased level of RANTES in both the group reflects the activation and recruitment of the immune cells in response to viral infections. Significant increase in the IFN-Y and IP-10 level in the non-JE group shows the interferon mediated protective immune response in the non-JE encephalitis cases. Significantly increased level of IL-10 in non-JE group could be related with the Th-2 mediated protective immune response which may contribute in lessen the severity and enhance the survival of non-JE encephalitis patients. The present study helps in understanding the differential immune-response by the host immune cells against the JE and non-JE encephalitis causing viruses.

Increased Levels of Circulating Cytokines with HIV-Related Immunosuppression

Cytokines may contribute to the severity of CD4 cell depletion with human immunodeficiency virus (HIV) infection, but quantitative relationships are not well defined. Serum and plasma from 181 HIV-infected individuals were tested with Millipore 30-plex Luminex cytokine assays. Within-individual correlations among cytokines were summarized by two-dimensional hierarchical cluster analysis. Associations with age, sex, race, CD4 count, and HIV viral load were determined with linear regression models. Tests for statistical significance were corrected for multiple comparisons, using a false discovery rate of 0.1. African-Americans had significantly higher levels than whites of six cytokines (IL-2, IL-5, IL-7, IL-15, fractalkine, and IFN-γ), and lower levels of MCP-1. Females had higher fractalkine levels than males. Age was not associated with levels of any cytokine. Six cytokines, including the T-helper (Th) type 1 cytokine IL-15, the Th2 cytokines IL-1ra and IL-10, the chemokines fractalkine and MCP-1, and the growth factor G-CSF were each inversely associated with CD4 count; no cytokine was directly associated with CD4 count. Fractalkine was directly associated with HIV viral load, adjusted for CD4 count. Cytokines clustered by primary function (e.g., Th1, Th2, proinflammatory, chemokines, or growth factors) whereas individuals clustered according to cytokine levels (generally high, intermediate, or low) had significantly different CD4 counts [medians (interquartile range) of 60 (17-162), 131 (62-321), and 155 (44-467), respectively; p<0.0001]. CD4 deficiency is associated with generalized increases in cytokines of various functions. Racial differences in cytokine response to HIV infection could contribute to disparities in disease progression.

Comparison of the response to experimentally induced short-term inflammation in the temporomandibular and metacarpophalangeal joints of horses

To investigate the relationship between inflammatory responses of the temporomandibular joint (TMJ) and the metacarpophalangeal (MCP) joint in clinically normal horses. 7 mature horses. In each horse, 1 TMJ and 1 MCP joint were injected with lipopolysaccharide (LPS; 0.0025 μg). The contralateral TMJ and MCP joint were injected with saline (0.9% NaCl) solution. Synovial fluid samples were collected from all 4 joints over 24 hours after injection. Concentrations of interleukin-6, tumor necrosis factor-α, transforming growth factor-β, and total protein were measured via immunoassay. Horses were assessed for clinical signs of joint inflammation at each time point. Concentrations of interleukin-6 were not significantly different between LPS-injected MCP joints and TMJs at any time point. Transforming growth factor-β concentrations were significantly increased in MCP joints, compared with concentrations in TMJs, at 12 and 24 hours after injection. Tumor necrosis factor-α concentrations were significantly higher in LPS-injected TMJs than in LPS-injected MCP joints at 1 and 6 hours after injection. Total protein concentration did not differ significantly between LPS-injected MCP joints and TMJs. Injection of LPS induced clinical inflammation at all time points; additionally, 2 MCP joints (but no TMJs) had an inflammatory response to injection of saline solution. The inflammatory response to LPS appeared to be attenuated more quickly in TMJs than in MCP joints of horses. The difference in response suggested that a lack of clinical osteoarthritis in the TMJ of horses could be attributable to a difference in cytokine response.