Increased Expression Of IFN-gamma Research Articles

Sexual dimorphism modulates metabolic and cognitive alterations under HFD nutrition and chronic stress exposure in mice. Correlation between spatial memory impairment and BDNF mRNA expression in hippocampus and spleen

AimsThe accumulated evidence suggests that lifestyle - specifically dietary habits and stress exposure - plays a detrimental role in health. The purpose of the present study was to analyze the interplay of stress, diet, and sex in metabolic and cognitive alterations. Main methodsFor this purpose, one-month-old C57Bl/6J mice were fed with a standard diet or high-fat diet (HFD). After eight weeks, one subgroup of mice from each respective diet was exposed to 20 weeks of chronic mild stress (CMS), whilst the others were left undisturbed. Key findingsAfter 28 weeks of HFD feeding, mice from both sexes were overweight, with an increase in caloric intake and abdominal and subcutaneous fat pads. Stress exposure induced a decrease in body weight, related to a decrease in caloric efficiency in both males and females. Results indicate that males are more susceptible than the females in modulating metabolic and cognitive functions under HFD and CMS. Although both sexes demonstrated HFD-induced weight gain, fat accumulation, insulin resistance, high cholesterol, only males exposed to CMS but not females have (i) impaired glucose tolerance with higher glucose level; (ii) significant prolonged latency in Barnes test, suggesting cognitive impairment; (iii) increased IFN-gamma expression in hippocampus, suggesting greater neuroinflammatory response; (iv) poorer cognitive performance related to a decrease in hippocampal and spleen BDNF mRNA expression. SignificanceThe main finding in this study is the presence of a sexual dimorphism in modulating metabolic and cognitive functions under HFD and CMS, showing males are more susceptible than females. In addition, poorer cognitive performance was related to a decrease in hippocampal BDNF mRNA expression. Interestingly, these changes were observed in the spleen as well.

IMMU-39. TIM-3 APTAMER IN COMBINATION WITH RADIOTHERAPY RESULTS IN ENHANCED SURVIVAL IN DIPG MODELS

Abstract Pediatric diffuse midline gliomas-H3-K27M-mutant are aggressive brain tumours that arise during childhood. Despite new advances in genomic knowledge and the significant number of clinical trial testing new targeted therapies, patient outcome is still insufficient. Cancer immunotherapy is opening new therapeutic options representing a hope for this orphan disease. Aptamers are single-stranded nucleic acid ligands design to achieve a remarkable affinity and specificity to their targets, comparable to antibodies. TIM-3, is a potential immune checkpoint target, typically involved in T-cell exhaustion. Recent studies showed that TIM-3 is also expressed in tumour and glial cells and it plays an important role in brain tumour responses mediated by myeloid cells. In this work, we examined the anti-tumour effect of an aptamer against TIM-3 alone or in combination with radiotherapy. Of importance, we tested TIM-3 aptamer in a murine glioma and DIPG model, where we not observed any toxicity. TIM-3 administration increased overall survival but was unable to control the disease. Of importance, TIM-3 combination with radiotherapy improved the overall survival of treated mice when compared with single treatments leading to 50% of long-term survivors. TIM-3 aptamer administration increase T-infiltration in the tumour site compared to non-treated or library control. Mechanistic studies performed on day 16 showed an increase in CD8 effector cells, a decrease in T-regulators Foxp3+ cells and an increase in IFN-gamma expression suggesting the triggering of an antitumor-immune response. Rechallenge experiments demonstrated immune memory in the long-term responders that led to reject tumour re-implantation, confirming that TIM-3 aptamer treatment in combination with RT elicits specific antitumor immunity in mouse glioma models. These results suggest that immuno-therapies approaches in combination with radiotherapy would be worth exploring in the treatment of deadly DMG-H3K27-Mutant tumours.

OC.12.4 INNATE LYMPHOID CELLS ARE INCREASED IN CELIAC DISEASE INTESTINAL MUCOSA AND HAVE A PATHOGENIC ROLE IN A MOUSE MODEL OF SMALL INTESTINE ATROPHY

Background and aim: Innate lymphoid cells (ILCs) are an emerging family of innate hematopoietic cells producing inflammatory cytokines and involved in the pathogenesis of several immune-mediated diseases. The aim of our study was to characterize the tissue distribution of ILCs in celiac disease (CD) and analyze their role in gut tissue damage. Materials and methods: ILC subpopulations were analyzed in lamina propria mononuclear cells (LPMCs), isolated from duodenal biopsies of patients with active CD, patients with inactive CD on a gluten-free diet and healthy controls (HC) and jejunal specimens of patients undergoing gastro-intestinal bypass by flow cytometry. Cytokines, transcription factors and receptors for interleukin (IL)-15, interferon (IFN)-alpha and Toll-like receptors (TLR) were assessed in ILCs either freshly isolated or following incubation of control LPMC with IL-15, IFNalpha and poly I:C, a TLR agonist, by flow cytometry. The role of ILCs in gut tissue damage was evaluated in a mouse model of poly I:C-driven small intestine atrophy. Results: The percentage of total ILCs did not differ between CD (both active and inactive) patients and HC. However, the fractions of ILCs type 1 expressing T-bet and ILCs type 3 expressing ROR-(g)t were significantly increased in active CD compared to controls, while there was no difference in term of ILCs type 2 among groups. ILCs expressed TLR3, IL-15R, little TLR2 but not IFN-alphaR and TLR4. IL-15 and poly I:C, but not IFN-alpha increased IFNgamma expression in ILCs. In vivo in mice, depletion of ILCs significantly reduced TLR3driven small intestinal damage. Conclusions: Active CD is characterized by preferential accumulation of type 1 and type 3 ILC subpopulations and these cells respond to IL-15, a cytokine over-produced in CD, by enhancing IFN-gamma production. The demonstration that ILCs express TLR3, are functionally able to respond to poly I:C with increased synthesis of inflammatory cytokines, and contribute to TLR3-driven small intestinal atrophy delineates a novel mechanism underlying the CD-associated tissue damage.

Influence of four parts of forming of Yiqi Qingwen Jidu Heji(YQQWJDHJ) to lung inflammatory cytokines of model rats infected with influenza virus FM1

To observe the influence of the 4 parts of forming of Yiqi Qingwen Jidu Heji(YQQWJDHJ) to lung inflammatory cytokines of the model rats infected with influenza virus dynamically, and to discuss the mechanism of 4 parts of forming to anti-influenza immune injury and restoration. At the different stages of infection with the model rats infected by FM1 influenza, expression in lung of TNF-alpha, IL-6, IL-1, IL-10 and IFN-gamma was detected after the intervention of 4 parts of forming using ELISA method. The expression of TNF-alpha, IL-6, IL-1 and IFN-gamma of model rats infected by FM1 were higher than the control group, the expression of IL-10 did not change. The expression of TNF-alpha was significantly reduced in 3 to 5 days after infection. By the method of relieving superficies with acrid-cold, clearing away heat and poison and replenishing Qi, the lung expression of IFN-gamma was significantly increased in the stage after infection. The method of relieving superficies with acrid-warm significantly reduced lung expression of IL-6 after infection in 1 to 3 days and on the 7th day, decreased the expression of IL-1 in 3 to 7 days, increased IFN-gamma expression on the 3rd day and the 7th day, and significantly increased the expression of IL-10 on the 1st day and in 5 to 7 days. The method of relieving superficies with acrid-cold reduced the expresssion of IL-6 after infection, and significantly increased the expression of IL-10. It could increase the expression of IL-1 after infection on the 3rd day, but reduced IL-1 expression after infection 7 days. The method of clearing away heat and poison reduced lung IL- 6 expression after infection in 3 to 7 days significantly, decreased the expression of IL-1 in 5 to 7 days, also increased the lung expression of IL-10 in 1 to 5 days significantly. The method of replenishing Qi significantly reduced the expression of IL-6 after infection on the 1st day and in 5 to 7 days, decreased the expression of IL-1 in 3 to 7 days, also significantly increased the lung IL-10 on the 5th day after infection. The method of clearing away heat and poison and replenishing Qi could be against the lung immune inflammatory damage and repair damage. The method of relieving superficies with acrid-warm demonstrated some immunity against lung injury on the 3rd day after infection and the method of relieving superficies with acrid-cold demonstrated some immunity against lung injury on the 5th days after infection.

Statin-induced Kruppel-like factor 2 expression in human and mouse T cells reduces inflammatory and pathogenic responses

The transcription factor Krüppel-like factor 2 (KLF2) is required for the quiescent and migratory properties of naive T cells. Statins, a class of HMG-CoA reductase inhibitors, display pleiotropic immunomodulatory effects that are independent of their lipid-lowering capacity and may be beneficial as therapeutic agents for T cell-mediated inflammatory diseases. Statins upregulate KLF2 expression in endothelial cells, and this activity is associated with an antiinflammatory phenotype. We therefore hypothesized that the immunomodulatory effects of statins are due, in part, to their direct effects on T cell KLF2 gene expression. Here we report that lipophilic statin treatment of mouse and human T cells increased expression of KLF2 through a HMG-CoA/prenylation-dependent pathway. Statins also diminished T cell proliferation and IFN-gamma expression. shRNA blockade of KLF2 expression in human T cells increased IFN-gamma expression and prevented statin-induced IFN-gamma reduction. In a mouse model of myocarditis induced by heart antigen-specific CD8+ T cells, both statin treatment of the T cells and retrovirally mediated overexpression of KLF2 in the T cells had similar ameliorating effects on disease induction. We conclude that statins reduce inflammatory functions and pathogenic activity of T cells through KLF2-dependent mechanisms, and this pathway may be a potential therapeutic target for cardiovascular diseases.

Patients with Multidrug-Resistant Tuberculosis Display Impaired Th1 Responses and Enhanced Regulatory T-Cell Levels in Response to an Outbreak of Multidrug-ResistantMycobacterium tuberculosisM and Ra Strains

In Argentina, multidrug-resistant tuberculosis (MDR-TB) outbreaks emerged among hospitalized patients with AIDS in the early 1990s and thereafter disseminated to the immunocompetent community. Epidemiological, bacteriological, and genotyping data allowed the identification of certain MDR Mycobacterium tuberculosis outbreak strains, such as the so-called strain M of the Haarlem lineage and strain Ra of the Latin America and Mediterranean lineage. In the current study, we evaluated the immune responses induced by strains M and Ra in peripheral blood mononuclear cells from patients with active MDR-TB or fully drug-susceptible tuberculosis (S-TB) and in purified protein derivative-positive healthy controls (group N). Our results demonstrated that strain M was a weaker gamma interferon (IFN-gamma) inducer than H37Rv for group N. Strain M induced the highest interleukin-4 expression in CD4+ and CD8+ T cells from MDR- and S-TB patients, along with the lowest cytotoxic T-lymphocyte (CTL) activity in patients and controls. Hence, impairment of CTL activity is a hallmark of strain M and could be an evasion mechanism employed by this strain to avoid the killing of macrophages by M-specific CTL effectors. In addition, MDR-TB patients had an increased proportion of circulating regulatory T cells (Treg cells), and these cells were further expanded upon in vitro M. tuberculosis stimulation. Experimental Treg cell depletion increased IFN-gamma expression and CTL activity in TB patients, with M- and Ra-induced CTL responses remaining low in MDR-TB patients. Altogether, these results suggest that immunity to MDR strains might depend upon a balance between the individual host response and the ability of different M. tuberculosis genotypes to drive Th1 or Th2 profiles.

Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN-gamma expression in activated human T cells.

The zinc transporter ZIP8 is highly expressed in T cells derived from human subjects. T cell ZIP8 expression was markedly up-regulated upon in vitro activation. T cells collected from human subjects who had received oral zinc supplementation (15 mg/day) had higher expression of the activation marker IFN-gamma upon in vitro activation, indicating a potentiating effect of zinc on T cell activation. Similarly, in vitro zinc treatment of T cells along with activation resulted in increased IFN-gamma expression with a maximum effect at 3.1 microM. Knockdown of ZIP8 in T cells by siRNA decreased ZIP8 levels in nonactivated and activated cells and concomitantly reduced secretion of IFN-gamma and perforin, both signatures of activation. Overexpression of ZIP8 by transient transfection caused T cells to exhibit enhanced activation. Confocal microscopy established that ZIP8 is localized to the lysosome where ZIP8 abundance is increased upon activation. Loss of lysosomal labile zinc in response to activation was measured by flow cytometry using a zinc fluorophore. Zinc between 0.8 and 3.1 microM reduced CN phosphatase activity. CN was also inhibited by the CN inhibitor FK506 and ZIP8 overexpression. The results suggest that zinc at low concentrations, through inhibition of CN, sustains phosphorylation of the transcription factor CREB, yielding greater IFN-gamma expression in T cells. ZIP8, through control of zinc transport from the lysosome, may provide a secondary level of IFN-gamma regulation in T cells.

Repression of interferon-γ expression in T cells by Prospero-related Homeobox protein

Interferon-gamma (IFN-gamma) is a major proinflammatory effector and regulatory cytokine produced by activated T cells and NK cells. IFN-gamma has been shown to play pivotal roles in fundamental immunological processes such as inflammatory reactions, cell-mediated immunity and autoimmunity. A variety of human disorders have now been linked to irregular IFN-gamma expression. In order to achieve proper IFN-gamma-mediated immunological effects, IFN-gamma expression in T cells is subject to both positive and negative regulation. In this study, we report for the first time the negative regulation of IFN-gamma expression by Prospero-related Homeobox (Prox1). In Jurkat T cells and primary human CD4+ T cells, Prox1 expression decreases quickly upon T cell activation, concurrent with a dramatic increase in IFN-gamma expression. Reporter analysis and chromatin immunoprecipitation (ChIP) revealed that Prox1 associates with and inhibits the transcription activity of IFN-,gammapromoter in activated Jurkat T cells. Co-immunoprecipitation and GST pull-down assay demonstrated a direct binding between Prox1 and the nuclear receptor peroxisome proliferator-activated receptor gamma (PPPARgamma, which is also an IFN-gamma repressor in T cells. By introducing deletions and mutations into Prox1, we show that the repression of IFN-gamma promoter by Prox1 is largely dependent upon the physical interaction between Prox1 and PPPARgamma Furthermore, PPPARgammaantagonist treatment removes Prox1 from IFN-gamma promoter and attenuates repression of IFN-gamma expression by Prox1. These findings establish Prox1 as a new negative regulator of IFN-gamma expression in T cells and will aid in the understanding of IFN-gamma transcription regulation mechanisms.

Cytokine expression and synovial pathology in the initiation and spontaneous resolution phases of adjuvant arthritis: interleukin-17 expression is upregulated in early disease.

The aim of this study was to understand the immune processes controlling the initiation and spontaneous resolution of adjuvant arthritis (AA). We investigated synovial T-cell recruitment and mRNA expression of IL-17 and other important disease related cytokines, IFN-gamma, IL-2, IL-4, TNF and TGF-beta in inguinal lymph node (ILN) and synovial membrane (SM). Arthritis severity was assessed by a numerical rating score and rats were sacrificed every 3--4 days postadjuvant induction. Further assessment involved quantitative radiology and histology of the ankle joints on each day, and the ILN and SM were removed for RNA extraction. Cytokine mRNA expression was measured using RT-PCR and densitometry. Paraffin sections of rat ankle joints were stained for T-cells (CD3) by immunohistochemistry. In the ILN, there was an increase in IL-17, TNF and IFN-gamma expression in the early stages of disease, with a secondary sustained increase in IFN-gamma expression. In the SM, there was expression of T-cell cytokines in early arthritis (day 13), and prolonged TNF and TGF-beta expression, which reflected disease progression. IL-4 mRNA expression increased in the later stages of AA. Synovial T-cell numbers transiently increased at day 6, and remained high from days 13--28. Increased pro-inflammatory cytokine expression, including IL-17, in the ILN reflects the initiating events in the early stage of disease. IL-17 may therefore play an important role in the pathogenesis of AA. The increase in IL-4 (an anti-inflammatory cytokine) in the SM in the later stages of AA suggests that IL-4 is involved in the spontaneous resolution of AA. The initial increase in IFN-gamma in the ILN may reflect a pro-inflammatory response, while the prolonged secondary increase may indicate activation of regulatory T-cells.

Cytokine expression in peripheral blood lymphocytes before and after aspirin desensitization in aspirin-exacerbated respiratory disease

Aspirin intolerance is the hallmark of aspirin-exacerbated respiratory disease (AERD). Overproduction of cysteinyl-leukotrienes (Cys-LTs) has been implicated as major mediators of AERD; however, the LT receptor antagonist montelukast is only partially effective in inhibiting aspirin responses. Several studies have documented the importance of cytokine production by T lymphocytes in asthma. Peripheral blood lymphocyte (PBL) cytokine expression and its relation to aspirin desensitization in aspirin-sensitive patients with asthma have not been studied. This study was performed to examine PBL cytokine expression in aspirin-sensitive patients who have asthma before and after aspirin desensitization. A 42-year-old white woman with a history of severe asthma, nasal polyps, aspirin sensitivity, and chronic sinusitis was treated with aspirin desensitization. Blood was taken before and after aspirin desensitization, and PBL cytokine expression was studied by flow cytometry. Aspirin desensitization differentially affects interferon (IFN) gamma expression. This effect results in an increase in IFN-gamma expression by CD4(+) lymphocytes and a decrease in IFN-gamma expression by CD8(+) lymphocytes. Aspirin desensitization in an aspirin-sensitive patient with asthma resulted in an increase in IFN-gamma expression by CD4(+) lymphocytes and a decrease in IFN-gamma expression by CD8(+) lymphocytes, the significance of which needs additional investigation.

Increased Interleukin‐10 in Helicobacter pylori Infection Could Be Involved in the Mechanism Protecting From Allergy

A protective effect of Helicobacter pylori infection against allergic diseases has been reported. The increasing incidence of childhood allergy in developed countries may be a result of reduced stimulation of the immune system by early chronic infections, with the protective effect of gastrointestinal microbes being mediated by regulatory T lymphocytes and production of interleukin (IL)-10. To elucidate a possible mechanism involved in protecting against the development of atopy, we measured expression of IL-10 in gastric mucosa of children with H pylori gastritis. Gastric biopsies were performed during endoscopy in 48 children (median age, 9 years), 32 of whom had H pylori gastritis and 16 of whom served as controls. Interferon-gamma (IFN-gamma), interleukin-1beta (IL-1beta), and IL-10 were measured in tissue homogenate by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). The amounts of IFN-gamma, IL-1beta, and IL-10 transcripts were quantified via competitive RT-PCR with use of dilution series of specific competitors. Expression of IFN-gamma and IL-10 were significantly higher in H pylori-infected children. No direct correlation with age was found, but a further increase in IL-10 expression was found in H pylori-infected children older than 4 years, whereas in control subjects, IL-10 expression tended to be lower in older children. IL-1beta expression was similar in infected children and control subjects. In H pylori-infected children, the prevalence of allergy was significantly higher in children with lower cytokine expression in gastric mucosa. In children, H pylori-induced inflammatory response is associated with development of cell-mediated immunity of T-helper 1 type, as demonstrated by increased IFN-gamma expression. The significantly increased expression of gastric IL-10 in H pylori-infected children and its further increase in older children suggest that this chronic infection may influence IL-10 production even beyond the age of 4 years. H pylori may be one of the infections with the potential to modulate immune responses.

Local Complement C3 Expression is Upregulated in Humoral and Cellular Rejection of Renal Allografts

Evidence on the role of the complement system in transplantation pathology has been accelerated by the discovery of C4d as an in situ marker of antibody-mediated rejection. However, a local or systemic source of complement expression during acute rejection is under discussion. Thus, we quantitatively analyzed local RNA expression of complement component C3 as a pivotal molecule in active humoral and cellular rejection of renal allografts. After laser microdissection, real-time RT-PCR was performed for C3 using RNA extracted from tubuli and glomeruli of 68 paraffin-embedded renal allograft biopsies. Protocol and indication biopsies with signs of humoral and/or cellular rejection were investigated. Quantitative expression analysis of cytokines (IFN gamma, MCP-1, IL2, IL8) potentially influencing local C3 expression was performed. We observed a significant increase in median expression level of C3 mRNA in tubuli of C4d-positive indication biopsies, and in tubuli from indication biopsies with signs of T-cell-mediated cellular rejection. Highest expression levels were found in C4d-positive indication biopsies with signs of cellular rejection. Biopsies with upregulated C3 showed increased IFN gamma expression, suggesting allograft-infiltrating T-cells as potential stimulus for local C3 expression. Therefore, locally synthesized complement component C3 contributes to both humoral and cellular rejection, with tubular epithelial cells being a major source.

Innate and Acquired Immune System in Patients Developing Interferon-α-Related Autoimmune Thyroiditis: A Prospective Study

In this prospective study, we investigated whether the development of interferon (IFN)-alpha-related autoimmune thyroiditis (IFN-AT) was correlated with the sequential changes of cytokine pattern induced by IFNalpha in the peripheral lymphocytes. We enrolled 18 hepatitis C virus (HCV)-positive patients who developed IFN-AT, eight patients with euthyroidism [IFN-AT(Eu)] and 10 with thyroid dysfunction [IFN-AT(Dy)]. Twenty HCV-positive patients without IFN-AT acted as control group (Co-HCV+). Intracellular expression of IFNgamma and IL-4 was evaluated by multicolor flow-cytometry analysis in peripheral lymphocytes in vitro stimulated by phorbol-12-myristate-13-acetate (PMA) (25 ng/ml) and ionomycin (1 mug/ml) in presence of monensin (5 microm). At the appearance of thyroid disease, both IFN-AT(Eu) and IFN-AT(Dy) patients showed a significant increase of IFNgamma expression in CD3+CD56+ and CD3-CD56+ cells but not in CD4+ and CD8+ cells. At this time point, IFN-AT(Eu) but not IFN-AT(Dy) patients also showed an increase of IL-4 expression in CD3+CD56+ cells and CD4+ cells. Six months later, IFN-AT(Eu) patients maintained high expression of IL-4 in CD4+ and CD3+CD56+ cells without any further increase in IFNgamma expression. By contrast, IFN-AT(Dy) patients showed an increase of IFNgamma expression in CD4+ and CD8+ cells, with a concomitant decrease of IL-4 expression in CD4+ cells. Type 2 immune response is activated early and specifically in patients with IFN-AT who remain euthyroid throughout the follow-up. Predominant in patients developing thyroid dysfunction, by contrast, is the type 1 immune response that seems to occur earlier in innate than acquired immune system.

Augmented fumonisin B1 toxicity in co-cultures: evidence for crosstalk between macrophages and non-parenchymatous liver epithelial cells involving proinflammatory cytokines.

Fumonisin B1, a common mycotoxin produced by Fusarium verticillioides found in corn, causes several fatal animal diseases. Liver and kidney are target organs of fumonisin B1 in laboratory animals, but primary rodent hepatocytes and liver slices were resistant to fumonisin B1-induced cytotoxic effects. We have shown that fumonisin B1 induces expression of tumor necrosis factor (TNF)alpha, interferon (IFN)gamma, and interleukine (IL) 12, in mouse liver. In various models of acute liver injury, a positive amplification loop involving TNFalpha, IFNgamma, and IL-12 has been implied that involves Kupffer cells (macrophages), hepatic lymphocytes and non-parenchymatous liver epithelial cells (NPECs). In the current study, cellular interactions in fumonisin B1-induced toxicity were investigated, using co-cultures of murine macrophages (J774A.1) and NPECs (NMuLi). Treatment of the co-cultures with fumonisin B1-produced cytotoxicity, whereas either J774A.1 or NMuLi cultures alone showed no response to the mycotoxin. Accumulation of sphinganine occurred to the similar extent in individual cultures as well as co-cultures. Expression of TNFalpha and IL-12 was increased in co-cultures but not in individual cultures. Transfer of conditioned medium from fumonisin B1-treated J774A.1 cells to NMuLi cultures produced an increase in IFNgamma expression in NMuLi cells. Results indicated that macrophages and liver epithelial cells interact in response to fumonisin B1 and potentiate the cytokines expression, which may have implications in making hepatocytes responsive to cytotoxicity of fumonisin B1.

Elevated expression OF IFN-gamma in the HIV-1 infected brain

We determined the extent of expression of three cytokines (IFN-gamma, IL-4, and TNF-alpha ) in brain tissue infected with human immunodeficiency virus-1 (HIV-1). The selections were IFN-gamma as a Th1 cytokine, IL- 4 as a Th2 cytokine, and TNF-alpha as a pro-inflammatory cytokine (and because of its prior implication in brain tissue damage due to HIV-1 infection). Based on current models for pathogenesis of HIV-1-associated dementia (HAD), in the periphery, Th1 cytokines are considered to be salutary, whereas Th2 cytokines are regarded as deleterious. However, we hypothesized that in the CNS these roles are reversed. Post-mortem temporal lobe tissue specimens from 16 HIV-1-seropositive patients and 11 HIV-1-seronegative controls were stained for IFN-gamma, IL-4, and TNF-alpha utilizing immunohistochemistry and alkaline phosphatase. HIV-1 infection causes alterations of brain cytokine expression that include increased IFN-gamma expression for HIV-1-seropositive vs. HIV-1-seronegative individuals. There was increased expression of IFN-gamma for HIV-1-seropositive individuals with or without HAD, with or without the broader category of neuropsychiatric impairment (NPI), and with or without opportunistic infections (OIs) compared to HIV-1-seronegatives. A significant inverse correlation between IFN-gamma vs. IL-4 in HIV-1-seropositives with HAD and in seronegative individuals was observed. There was an inverse correlation in seropositives between IFN-gamma vs. TNF-alpha, a positive trend with HAD, significant without HAD, significant with NPI and significant without OIs. Between IL-4 vs. TNF-alpha there was a correlation (trend) in seropositives, a trend with NPI, significant without NPI, and a trend without OI. Due to HIV-1 infection of the brain and neurological disease there is a prominent increased expression of IFN-gamma, an inverse expression of IFN-gamma vs. TNF-alpha, and TNF-alpha vs. IL-4. The inverse correlation between increased IFN-gamma and decreased IL-4 expression is consistent with the stimulation of activated macrophages, and T cells, greater toxicity in the HIV-1-infected brain, and is supportive of the significance of IFN-gamma in HIV-1-infected patients.

Kinetics of TNF-alpha and IFN-gamma mRNA expression in islets and spleen of NOD mice.

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop diabetes similar to the human disease. Cytokines produced by islet-infiltrating mononuclear cells may be directly cytotoxic and can be involved in islet destruction coordinated by CD4+ and CD8+ cells. We utilized a semiquantitative RT-PCR assay to analyze in vitro the mRNA expression of TNF-alpha and IFN-gamma cytokine genes in isolated islets (N = 100) and spleen cells (5 x 10(5) cells) from female NOD mice during the development of diabetes and from female CBA-j mice as a related control strain that does not develop diabetes. Cytokine mRNAs were measured at 2, 4, 8, 14 and 28 weeks of age from the onset of insulitis to the development of overt diabetes. An increase in IFN-gamma expression in islets was observed for females aged 28 weeks (149 +/- 29 arbitrary units (AU), P<0.05, Student t-test) with advanced destructive insulitis when compared with CBA-j mice, while TNF-alpha was expressed in both NOD and CBA-j female islets at the same level at all ages studied. In contrast, TNF-alpha in spleen was expressed at higher levels in NOD females at 14 weeks (99 +/- 8 AU, P<0.05) and 28 weeks (144 +/- 17 AU, P<0.05) of age when compared to CBA-j mice. The data suggest that IFN-gamma and TNF-alpha expression in pancreatic islets of female NOD mice is associated with beta cell destruction and overt diabetes.

Glucan phosphate potentiates endotoxin-induced interferon-γ expression in immunocompetent mice, but attenuates induction of endotoxin tolerance

Glucan phosphate has been shown to enhance antimicrobial immunity in a variety of experimental models. However, the mechanisms by which glucans enhance resistance to infection remain largely unknown. Interferon-gamma (IFN-gamma) is a key regulator of both innate and acquired immunity. Suppression of IFN-gamma production is a prominent feature of the altered immune response that follows major trauma or sepsis. The present studies were designed to determine the effect of glucan phosphate on IFN-gamma expression in normal mice and endotoxin [lipopolysaccharide (LPS)]-tolerant mice. The model of LPS tolerance was used because it results in patterns of cytokine expression similar to those commonly observed following severe trauma or sepsis. Glucan treatment potentiated LPS-induced IFN-gamma expression in control mice. The induction of LPS tolerance resulted in marked suppression of LPS-induced IFN-gamma production. However, co-administration of glucan with LPS, during the tolerance induction phase, attenuated the LPS-tolerant response. Interleukin-12 (IL-12) and IL-18 are important mediators of LPS-induced IFN-gamma production. LPS-induced IL-12 p40 mRNA expression was increased in the spleens of glucan-treated mice compared with controls. Induction of LPS tolerance caused marked suppression of IL-12 production, a response that was attenuated by glucan treatment. IL-18 was constitutively expressed in both control and LPS-tolerant mice, and LPS-induced serum levels of IL-18 were increased in mice treated with glucan. T cells isolated from glucan-treated mice exhibited increased IFN-gamma expression in response to IL-12 and IL-18, as well as increased expression of the IL-12 and IL-18 receptors. The ability of glucan to potentiate IFN-gamma expression in control mice provides a potential mechanism by which glucan enhances antimicrobial immunity. The ability of glucan to attenuate suppressed IFN-gamma expression in LPS-tolerant mice denotes its potential benefit for the treatment of trauma and sepsis-induced immunosuppression.

Nitric oxide synthesis during acute SIVmac251 infection of macaques

During HIV1 infection, nitric oxide (NO) could significantly contribute to immune dysregulation by its multiple effects on the modulation of the host immune response. The in vivo regulation of NO production is attributable to several nitric oxide synthases, one of which is a cytokine-inducible enzyme (iNOS). In vitro experiments suggest that iNOS expression in macrophages may be directly modulated by HIV infection. Acute infection of macaques with a pathogenic strain of the simian immunodeficiency virus (SIV) represents a relevant animal model for the in vivo study of the relationships between iNOS expression and lentiviral replication. Indeed, acute infection in this model is characterized by high rates of viral replication associated with early cytokine dysregulations, in the absence of opportunistic infection. In our experiment, two cynomolgus macaques were inoculated intravenously with a pathogenic isolate of SIVmac251, and iNOS gene expression was investigated ex vivo during acute infection in mononuclear cells obtained from bronchoalveolar lavage (BALMCs). An enhancement of this gene expression was observed as early as the second week of infection, at the time of peak of systemic viraemia, and increased until day 31 p.i. This overexpression was concomitant with a marked linear increase in IFN gamma expression in BALMCs. At the time of systemic viral load peak, the production of NO in plasma of these two monkeys was evidenced by the detection of large amounts of nitrate.

Immunomodulatory effects of IL-12 on allergic lung inflammation depend on timing of doses.

We investigated the effects of IL-12 on a murine model of allergic lung inflammation. Administration of IL-12 was timed to interfere with either allergic sensitization (early dosage) or the hypersensitivity inflammatory response in the lung (late dosage), or both (early and late dosages). Comparisons of IL-12- and PBS-treated animals within each treatment group revealed several noticeable effects of IL-12. Early dosage, and the combination of early and late dosages, strikingly decreased ragweed-specific serum IgE, tracheal ring reactivity to acetylcholine, and BAL eosinophilia following allergen challenge. In contrast, late dosage had no effect on IgE levels and only a minimal effect on tracheal ring reactivity, but had a modest effect on recruitment of eosinophils. Early dosage down-regulated IL-5 and IL-10, but did not alter IL-4 or IFN-gamma expression. Late dosage down-regulated IL-5, up-regulated IL-10 and IFN-gamma, but did not change IL-4 expression. The combination of early and late dosage down-regulated IL-4, IL-5, and IL-10 expression, but increased IFN-gamma expression and production in the BAL cells and fluids. Taken together, these results indicate that IL-12 has potent immunomodulatory effects on allergic lung inflammation that depend on the timing of IL-12 administration relative to allergic sensitization and allergen challenge.

CD4+ T cell associated cytokine gene expression during experimental infection with Listeria monocytogenes: the mRNA phenotype of granuloma formation.

In murine listeriosis, elimination of bacteria and immunity to re-infection critically depend on Thy-1+CD4- cells, while cell-mediated inflammatory phenomena like delayed-type hypersensitivity and granuloma formation are mediated by CD4+ T cells. In an attempt to correlate T cell phenotype and function with a particular set of cytokines produced in vivo, we examined the cytokine gene expression profile associated with the presence or absence of CD4+ and/or CD8+ cells in the livers of mice during experimental infection with Listeria monocytogenes. T cell subset depletion was achieved by i.p. administration of saturating amounts of the appropriate mAbs, and mRNA detection was carried out using a qualitative and semi-quantitative polymerase chain reaction-based mRNA amplification protocol. In both primary and secondary infection, the presence of CD4+ cells was a prerequisite for granuloma formation, and was found to be closely associated with mRNA expression for IL-2, IL-3 and IL-4, a 5-fold increase in expression of tumor necrosis factor (TNF)-alpha and granulocyte macrophage colony stimulating factor, and a 25-fold increase in expression of IFN-gamma and TNF-beta mRNAs, suggesting a role for these cytokines in granuloma formation. In striking contrast, depletion of CD8+ cells did not result in reduced mRNA expression for any one of the cytokines studied, implying that CD8+ T cell mediated cure and prevention of listeriosis may operate via qualitatively distinct mechanisms.