Upregulation Of Cytokine mRNA Research Articles

3-Pentylcatechol, a Non-Allergenic Urushiol Derivative, Displays Anti-Helicobacter pylori Activity In Vivo.

We previously reported that 3-pentylcatechol (PC), a synthetic non-allergenic urushiol derivative, inhibited the growth of Helicobacter pylori in an in vitro assay using nutrient agar and broth. In this study, we aimed to investigate the in vivo antimicrobial activity of PC against H. pylori growing in the stomach mucous membrane. Four-week-old male C57BL/6 mice (n = 4) were orally inoculated with H. pylori Sydney Strain-1 (SS-1) for 8 weeks. Thereafter, the mice received PC (1, 5, and 15 mg/kg) and triple therapy (omeprazole, 0.7 mg/kg; metronidazole, 16.7 mg/kg; clarithromycin, 16.7 mg/kg, reference groups) once daily for 10 days. Infiltration of inflammatory cells in gastric tissue was greater in the H. pylori-infected group compared with the control group and lower in both the triple therapy- and PC-treated groups. In addition, upregulation of cytokine mRNA was reversed after infection, upon administration of triple therapy and PC. Interestingly, PC was more effective than triple therapy at all doses, even at 1/15th the dose of triple therapy. In addition, PC demonstrated synergism with triple therapy, even at low concentrations. The results suggest that PC may be more effective against H. pylori than established antibiotics.

Expression of Cytokines in Porcine Iris, Retina and Choroidal Tissues Stimulated by Microbe-associated Molecular Patterns

ABSTRACT Purpose The innate immune system is strongly implicated in the pathogenesis of uveitis. This study was designed to clarify the responses of the innate immune system in uveal tissues. Materials and Methods We utilized quantitative, real-time RT-PCR to measure mRNA of innate immune system receptors from porcine iris, choroid, and retina tissues. We used RT-PCR for cytokines to evaluate the responses of these tissues to specific ligands or extracts of whole bacteria that activate the innate immune system. We used ELISA for IL-6 on selected choroidal supernatants to confirm that the mRNA measurement correlated with protein levels. Results In each of the studied tissues, we detected the expression of important receptors belonging to the innate immune system including dectin-1, TLR4, TLR8, and NOD2. Relative mRNA expression was generally lower in the retina compared to iris or choroid. All three tissues demonstrated upregulation of cytokine mRNA in response to a range of ligands that activate the innate immune system. The measurement of IL-6 protein was consistent with results based on mRNA. Notably, the expression of mRNA for IL-23 was more pronounced than IL-12 in all three tissues after stimulation with various innate immune system ligands. Conclusions These data provide evidence of a potent innate immune response intrinsic to uveal tissues. Specific innate immune system ligands as well as bacterial extracts enhanced the production of several inflammatory cytokines. Furthermore, the observation of higher upregulation of IL-23 mRNA, compared to IL-12 in response to innate immune stimuli, suggested that a local TH17 response might be more robust than a local TH1 response in uveal tissues. Our results expand the understanding as to how the innate immune system may contribute to uveitis.

Inhibition of microglial activation in rats attenuates paraventricular nucleus inflammation in Gαi2 protein-dependent, salt-sensitive hypertension.

New Findings • What is the central question of this study? We hypothesized that central inflammatory processes that involve activation of microglia and astrocytes contribute to the development of Gαi2 protein‐dependent, salt‐sensitive hypertension.• What is the main finding and its importance? The main finding is that PVN‐specific inflammatory processes, driven by microglial activation, appear to be linked to the development of Gαi2 protein‐dependent, salt‐sensitive hypertension in Sprague–Dawley rats. This finding might reveal new mechanistic targets in the treatment of hypertension. The central mechanisms underlying salt‐sensitive hypertension, a significant public health issue, remain to be established. Researchers in our laboratory have reported that hypothalamic paraventricular nucleus (PVN) Gαi2 proteins mediate the sympathoinhibitory and normotensive responses to high sodium intake in salt‐resistant rats. Given the recent evidence of central inflammation in animal models of hypertension, we hypothesized that PVN inflammation contributes to Gαi2 protein‐dependent, salt‐sensitive hypertension. Male Sprague–Dawley rats received chronic intracerebroventricular infusions of a targeted Gαi2 or control scrambled oligodeoxynucleotide (ODN) and were maintained for 7 days on a normal‐salt (NS; 0.6% NaCl) or high‐salt (HS; 4% NaCl) diet; in subgroups on HS, intracerebroventricular minocycline (microglial inhibitor) was co‐infused with ODNs. Radiotelemetry was used in subgroups of rats to measure mean arterial pressure (MAP) chronically. In a separate group of rats, plasma noradrenaline, plasma renin activity, urinary angiotensinogen and mRNA levels of the PVN pro‐inflammatory cytokines TNFα, IL‐1β and IL‐6 and the anti‐inflammatory cytokine IL‐10 were assessed. In additional groups, immunohistochemistry was performed for markers of PVN and subfornical organ microglial activation and cytokine levels and PVN astrocyte activation. High salt intake evoked salt‐sensitive hypertension, increased plasma noradrenaline, PVN pro‐inflammatory cytokine mRNA upregulation, anti‐inflammatory cytokine mRNA downregulation and PVN‐specific microglial activation in rats receiving a targeted Gαi2 but not scrambled ODN. Minocycline co‐infusion significantly attenuated the increase in MAP and abolished the increase in plasma noradrenaline and inflammation in Gαi2 ODN‐infused animals on HS. Our data suggest that central Gαi2 protein prevents microglial‐mediated PVN inflammation and the development of salt‐sensitive hypertension.

Expression of inflammatory cytokines in mesenchymal stromal cells is sensitive to culture conditions and simple cell manipulations

BackgroundMesenchymal stromal cells (MSCs) can be used in several clinical applications. While MSCs are frequently cultured in fetal bovine serum for in vitro experimentation, human serum supplements are required for cells to be used in patients. Here we show how different human serum supplements and in vitro manipulations used during the cell culture impact on MSC proliferation rate and expression of inflammatory molecules. MethodsMSCs were cultured in medium supplemented with human plasma or serum combined with human platelet lysate (PL) and/or basic fibroblast growth factor (FGF2). Real time RT-PCR and western blot were used to assess expression of inflammatory cytokines. ResultsSerum with addition of FGF2 gave the fastest proliferation rate. However, serum with FGF2 also increased expression of genes encoding inflammatory cytokines. The most favorable expansion condition for chondrogenic differentiation and inhibition of cartilage matrix degrading enzymes was plasma supplemented with PL and FGF2. Detachment of cells using trypsin gave considerable upregulation of inflammatory cytokine mRNAs which lasted for up to 24 h, with concomitant increase in protein levels. Even the gentle act of changing medium led to upregulation of cytokine mRNA, caused by addition of fresh serum. DiscussionDifferent culture conditions and simple cell manipulation influence proliferation rate and expression of inflammatory genes. Supplementing culture medium with allogeneic AB serum and FGF2 during monolayer expansion supported cell expansion better than other supplements, but also induced the highest levels of inflammatory cytokines and gave inferior results for chondrogenic differentiation. The importance of the composition of the culture medium and even gentle in vitro manipulation of the cells should be taken into account in the planning of procedures using in vitro expanded MSCs.

Upregulation of cytokine mRNA in circulating leukocytes during human endotoxemia.

Endotoxemia induces pronounced changes in leukocyte count and enhances the release of many cytokines. However, the molecular regulation of this cytokine release is poorly characterized in humans. The time course of mRNA expression of 24 cytokines in circulating leukocytes was studied in a well-standardized model of human endotoxemia (2ng/kg). Real-time polymerase chain reaction (RT-PCR) was used to quantify the lipopolysaccharide (LPS)-inducible mRNA levels of leukocytes from 16 healthy volunteers in a randomized, placebo-controlled trial. Baseline mRNA levels of interleukins including IL-1α, IL-3, IL-5, IL-6, IL-12p40, IL-13, IL-15, IL-17, granulocyte colony-stimulating factor (G-CSF) and granulocyte monocyte CSF (GM-CSF) were below detectable levels in normal blood of the healthy participants. After 2h, LPS infusion increased median mRNA levels of IL-1α by >1100-fold and IL-1β and IL-8 by 33-fold and 46-fold, respectively. In contrast, levels of tumor necrosis factor (TNF-α) and IL-10 mRNA increased by only 7-fold, whereas changes in mRNA expression of other cytokines showed either a more than two fold increase or were undetectable. In vitro incubation of whole blood with 50pg/mL LPS for 2h enhanced transcription levels of IL-1α mRNA by >10,000-fold, IL-6 and IL-12p40 by >1000-fold, IL-1β by 400-fold, TNF-α by 100-fold, IL-8, IL-18, interferon γ (IFN-γ) and G-CSF by >10-25-fold, and IL-10, IL-12p35, TNF-β, and IL-13 by 10-25-fold. Only half of the 24 evaluated cytokines were expressed at the mRNA level in circulating leukocytes under basal conditions and after an LPS challenge. Only IL-1α, IL-1β, IL-10, IL-8, and TNF-α were upregulated in the circulating leukocytes, whereas several other cytokines (including IL-6 and G-CSF), were expressed on the mRNA level following in vitro incubation of blood with LPS. In addition, IL-1α and IL-1β might be potential diagnostic targets in inflammatory diseases.

Abstract TP110: Polymerase δ-Interacting Protein 2 Regulates Astrocyte Activation in Ischemic Stroke

Introduction: Polymerase δ-Interacting Protein 2 (Poldip2) is a binding partner of Nox4 NADPH oxidase and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Nox4 and CEACAM1 have been implicated in stroke, but with different outcomes: both contribute to neurodegeneration but only CEACAM1 contributes to blood brain barrier (BBB) dysfunction. However, the underlying mechanisms are still obscure. Hypothesis: Poldip2+/- mice may be protected from the consequences of transient middle cerebral artery occlusion (tMCAO). Methods: tMCAO was induced in wild type (WT) and Poldip2+/- mice. The volume of the ischemic lesion was measured in TTC-stained sections. BBB breakdown was evaluated by Evans blue dye extravasation. Poldip2 protein expression was evaluated by immunofluorescence and western blotting. RT-PCR was used to measure mRNA levels of cytokines, MMPs and TIMPs. Astrocytes were transfected with Poldip2 siRNA in culture and mRNA levels of cytokines were evaluated. Results: Poldip2+/- and WT mice displayed comparable infarct sizes following tMCAO (n=6). A decrease in Evans blue dye extravasation was observed in Poldip2+/- mice (25±3 vs 6±2uM/g) (n=7). Upregulation of cytokine mRNA following tMCAO was also attenuated in Poldip2+/- mice (n=5): MCP-1(253±34 vs 83±23AU), IL-6 (134±38 vs 38±10AU), TNFα (39±12 vs 12±3AU), MMP-2 (19±3 vs 10±1AU), MMP-9 (253±12 vs 13±3AU) and TIMP-1 (286±110 vs 61±15AU). Poldip2 protein expression increased in the ischemic brain of WT mice after tMCAO (69±4 vs 20±5AU) and was predominantly located in astrocytes (n=4). Poldip2 protein expression was also increased in astrocytes following oxygen and glucose deprivation (79±15 vs 27±5AU) (n=6), and Poldip2 siRNA prevented cytokine induction under these conditions. Conclusions: In conclusion, Poldip2 contributes to stroke-induced BBB breakdown via its ability to inhibit a pro- inflammatory response in perivascular astrocytes.

TLR15 Is Unique to Avian and Reptilian Lineages and Recognizes a Yeast-Derived Agonist

The TLRs represent a family of pattern recognition receptors critical in the induction of vertebrate immune responses. Between 10 and 13 different TLR genes can be identified in each vertebrate species, with many represented as orthologous genes in different species. The agonist specificity of orthologous TLR is also highly conserved. In contrast, TLR15 can only be identified in avian and reptilian genomes, suggesting that this receptor arose ~320 million years ago after divergence of the bird/reptile and mammalian lineages. Transfection of a constitutively active form of chicken TLR15 led to NF-κB activation in HEK293 cells and induced cytokine mRNA upregulation in chicken cell lines. Full-length TLR15 mediated NF-κB induction in response to lysates from yeast, but not those derived from viral or bacterial pathogens, or a panel of well-characterized TLR agonists. TLR15 responses were induced by whole-cell lysates derived from Candida albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, but not zymosan preparations from S. cerevisiae. The ability of yeast lysate to activate TLR15-dependent NF-κB pathways (in transfection assays) or stimulate IL-1β mRNA upregulation in chicken macrophages was abrogated by heat inactivation or pre-exposure of the lysate to PMSF. Identification of yeast as an agonist source for TLR15 provides a functional framework for consideration of this TLR within the context of pattern recognition receptor evolution and may impact on the development of novel adjuvants.

Excessive innate immune response and mutant D222G/N in severe A (H1N1) pandemic influenza

Explore the role of viral factors and immune response in patients with severe pandemic pdmH1N1 illness without significant co-morbidity. Seven patients with pdmH1N1 influenza, bilateral chest X-rays infiltrates, requiring mechanical ventilator support were consecutively recruited. Seven age- and gender-matched healthy individuals served as controls. Four patients were viremic, two with the mutant D222G/N pdmH1N1.Microarray analyses of peripheral blood leukocytes suggested a marked granulocytes activation, but no up-regulation of inflammatory cytokine mRNA. Patients with severe pdmH1NI had a marked systemic complement activation, and in contrast to the lack of cytokine mRNA up-regulation in blood leukocytes, plasma levels of a broad range of inflammatory mediators, including IP-10, and mediators involved in pulmonary remodelling were markedly elevated. Patients with mutant virus had particularly high IP-10 levels, and the most pronounced complement activation. In severe pdmH1N1, viremia was common and the D222G/N mutant was found in half of the viremic patients. Host immune response was characterized by strong activation of the innate immune system, including complement and granulocytes activation, increased serum levels of inflammation and pulmonary remodelling markers, possibly contributing to the observed tissue damage. However, few patients were included and further studies are needed to characterize the immune response in severe pdmH1N1 infection.

The relationship of mucosal bacteria to duodenal histopathology, cytokine mRNA, and clinical disease activity in cats with inflammatory bowel disease

Feline inflammatory bowel disease (IBD) is the term applied to a group of poorly understood enteropathies that are considered a consequence of uncontrolled intestinal inflammation in response to a combination of elusive environmental, enteric microbial, and immunoregulatory factors in genetically susceptible cats. The present study sought to examine the relationship of mucosal bacteria to intestinal inflammation and clinical disease activity in cats with inflammatory bowel disease. Duodenal biopsies were collected from 27 cats: 17 undergoing diagnostic investigation of signs of gastrointestinal disease, and 10 healthy controls. Subjective duodenal histopathology ranged from normal (10), through mild (6), moderate (8), and severe (3) IBD. The number and spatial distribution of mucosal bacteria was determined by fluorescence in situ hybridization with probes to 16S rDNA. Mucosal inflammation was evaluated by objective histopathology and cytokine profiles of duodenal biopsies. The number of mucosa-associated Enterobacteriaceae was higher in cats with signs of gastrointestinal disease than healthy cats ( P < 0.001). Total numbers of mucosal bacteria were strongly associated with changes in mucosal architecture ( P < 0.001) and the density of cellular infiltrates, particularly macrophages ( P < 0.002) and CD3 +lymphocytes ( P < 0.05). The number of Enterobacteriaceae, E. coli, and Clostridium spp. correlated with abnormalities in mucosal architecture (principally atrophy and fusion), upregulation of cytokine mRNA (particularly IL-1, -8 and -12), and the number of clinical signs exhibited by the affected cats. These data establish that the density and composition of the mucosal flora is related to the presence and severity of intestinal inflammation in cats and suggest that mucosal bacteria are involved in the etiopathogenesis of feline IBD.

Co-infection by Semliki forest virus and malarial parasite modulates viral multiplication, pathogenesis and cytokines in mice

Environmental, technological and societal factors continue to have a dramatic effect on infectious diseases worldwide and are considered to be facilitating the emergence of several infectious diseases at a time. Co-infection with different species of viral and malaria infections are currently emerging problems of dual infection in the developing as well as developed countries. Understanding of interactions between the host, malaria and virus infection is of current concern and we have initiated studies to delineate the mechanisms involved during the progression of Semliki forest virus (SFV) and Plasmodium yoelii (P. yoelii) infection in mice. Enhanced virus multiplication and up-regulation of cytokine mRNA level in P. yoelii and SFV co-infected mice were observed on day 4 post-infection compared to respective controls. Collectively, our observations indicate that malaria infection may influence virus multiplication, pathogenesis and up-regulation of cytokine mRNA during co-infection in mice.

A role for the Mycoplasma pneumoniae adhesin P1 in interleukin (IL)-4 synthesis and release from rodent mast cells

Mycoplasma pneumoniae is a respiratory tract pathogen associated with exacerbations in patients with chronic asthma, yet relatively little is known about the potential role of this organism in asthma pathogenesis. Our previous studies demonstrated that RBL-2H3 mast cells co-cultured with M. pneumoniae released preformed inflammatory mediators, synthesized multiple cytokine mRNA species, and released IL-4 protein. In this study, we sought to determine the mechanism by which M. pneumoniae activates mast cell cytokine production. Cytokine mRNA upregulation and IL-4 protein production in RBL cells were induced almost exclusively by plastic-adherent M. pneumoniae cultures (MpA). Organisms grown under non-adherent conditions (MpN) were unable to induce cytokine responses efficiently. Western blots demonstrated that MpA was enriched for P1, the major M. pneumoniae adhesin, compared to MpN. M. pneumoniae-induced IL-4 release from RBL cells was inhibited >85% by anti-P1 monoclonal antibodies. Additionally, a P1-deficient strain of the bacteria was unable to efficiently induce IL-4 release. Desialation of RBL cell surface glycoproteins by neuraminidase treatment eliminated IL-4 release. We conclude that P1 plays an important role in M. pneumoniae-induced cytokine responses in RBL mast cells and that direct contact between the organism and sialated residues on the RBL surface mediates this activation.

IL-12 family members: differential kinetics of their TLR4-mediated induction by Salmonella Enteritidis and the impact of IL-10 in bone marrow-derived macrophages

The members of the IL-12 family of heterodimeric cytokines play a pivotal role in initiation and regulation of cell-mediated immunity. Best known is IL-12p70, which promotes an immune response towards T(h)1 bias. Other members of this family (IL-23, IL-27) are less well characterized in terms of induction and function. Using either heat-killed or viable Salmonella Enteritidis or LPS as a stimulus, the kinetics of mRNA production of each member of the IL-12 family (p19, p28, p35, p40, Ebstein-Barr-Virus-induced gene 3 (EBI-3)) were determined in BMDMPhi originating from wild-type, Toll-like receptor (TLR)2- and/or TLR4-deficient mice. It was found that following either type of stimulation, a characteristic mRNA expression pattern was observed for each cytokine subunit. Whereas p19 was induced early and transiently, p40 and p35 were up-regulated later and then continuously, but the secretion of IL-23 and IL-12p70 was significantly reduced by IL-10. The up-regulation of p28 mRNA occurred also delayed and declined afterwards, whereas the initial high-level expression of EBI-3 remained almost unchanged in BMDMPhi. Furthermore, a splice variant of the EBI-3 mRNA was discovered. In this context, the cytokine mRNA up-regulation by whole Salmonella Enteritidis is mediated chiefly by TLR4, but depends on additional pattern recognition receptors other than TLR2 expressed by macrophages.

Tissue- and time-dependent upregulation of cytokine mRNA in a murine model for the multiple organ dysfunction syndrome.

We sought to quantitate the course of specific cytokine mRNA expression in tissues that exhibit increasing histopathological changes in time in an animal model for the multiple organ dysfunction syndrome (MODS). The development of treatment protocols for MODS requires elucidation of the mechanisms and mediators involved. To devise logical interventions, it is necessary to collect data on cytokine expression at tissue level during the development of MODS. Ninety-four C57BL/6 mice were given an intraperitoneal injection of 40 microg of lipopolysaccharide (LPS), followed by zymosan at a dose of 0.8 mg/g body weight 6 days later (day 0). Six additional animals did not receive zymosan and acted as controls. At several time points after zymosan injection, 6 randomly assigned, zymosan-treated animals were killed, and their livers, lungs, spleens, and kidneys were collected. mRNA expression of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, macrophage migration inhibiting factor, IL-12, interferon-gamma, and IL-10 was measured using a real-time reverse transcription-polymerase chain reaction assay. The injection of zymosan induced an acute peritonitis, followed by an apparent recovery. From approximately day 6 onwards, animals started to display MODS-like symptoms. During the peritonitis phase, up-regulation of cytokine mRNA was limited. During the period of apparent recovery, cytokine mRNA expression strongly increased, mostly reaching its maximum at day 9 when deterioration of the clinical condition had already set in. The up-regulation of tumor necrosis factor-alpha mRNA was most pronounced, especially in the lungs and liver. Interventions should preferentially be targeted against multiple cytokines and, at least in this model, there may be a treatment window well after the initial challenge.

Hemozoin-inducible proinflammatory events in vivo: potential role in malaria infection.

During malaria infection, high levels of proinflammatory molecules (e.g., cytokines, chemokines) correlate with disease severity. Even if their role as activators of the host immune response has been studied, the direct contribution of hemozoin (HZ), a parasite metabolite, to such a strong induction is not fully understood. Previous in vitro studies demonstrated that both Plasmodium falciparum HZ and synthetic HZ (sHZ), beta-hematin, induce macrophage/monocyte chemokine and proinflammatory cytokine secretion. In the present study, we investigated the proinflammatory properties of sHZ in vivo. To this end, increasing doses of sHZ were injected either i.v. or into an air pouch generated on the dorsum of BALB/c mice over a 24-h period. Our results showed that sHZ is a strong modulator of leukocyte recruitment and more specifically of neutrophil and monocyte populations. In addition, evaluation of chemokine and cytokine mRNA and protein expression revealed that sHZ induces the expression of chemokines, macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL2, and monocyte chemoattractant protein-1/CCL2; chemokine receptors, CCR1, CCR2, CCR5, CXCR2, and CXCR4; cytokines, IL-1beta and IL-6; and myeloid-related proteins, S100A8, S100A9, and S100A8/A9, in the air pouch exudates. Of interest, chemokine and cytokine mRNA up-regulation were also detected in the liver of i.v. sHZ-injected mice. In conclusion, our study demonstrates that sHZ is a potent proinflammatory agent in vivo, which could contribute to the immunopathology related to malaria.

Macrophage-neutrophil interaction: a paradigm for chronic inflammation revisited.

Macrophages have been described as 'factories' of pro-inflammatory cytokines. Several years ago the present investigators reported that binding of inactive myeloperoxidase (iMPO) to the macrophage-mannose receptor resulted in the induction of TNF and other cytokines. Also, if endothelial cells were incubated with iMPO, but not enzymatically active myeloperoxidase (MPO), upregulation of cytokine mRNA and cytokines was observed. Taken in their entirety, the data suggest a dichotomy of function for myeloperoxidase; that is, enzymatically active MPO functions primarily in cell killing through the 'cytotoxic triad' and iMPO functions as an immunoregulatory molecule through the induction of numerous cytokines. These studies underscore a previously unrecognized interaction among neutrophils, endothelial cells and macrophages, resulting in the induction of TNF and perpetuation of inflammation. The inflammation induced could be relevant in a number of diseases in which neutrophils play a prominent role. The importance of this interaction in the pathogenesis of rheumatoid arthritis is currently under investigation.

Cytokine profiles in seronegative volunteers immunized with a recombinant canarypox and gp120 prime-boost HIV-1 vaccine. NIAID AIDS Vaccine Evaluation Group.

To study memory T cell proliferative responses and cytokine profiles induced in HIV-1 seronegative volunteers immunized with a live recombinant canarypox vector expressing HIV-1 antigens (ALVAC-HIV) and boosted with a recombinant gp120 subunit vaccine. HIV-specific T cell proliferative responses and cytokines were measured 2 weeks after vaccination. Cytokines secreted by T helper 1 cells (Th1) [interleukin (IL)-2 and interferon-gamma (IFN-gamma)] and T helper 2 (Th2) cells (IL-4, IL-5, IL-6, and IL-10) were assessed both at the mRNA and the protein level. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with HIV antigens. Subsequently, T cell proliferation was measured in a standard lymphoproliferation assay; secreted cytokines were measured using an enzyme-linked immunosorbent assay and upregulation of cytokine mRNA was measured using reverse transcriptase polymerase chain reaction. All individuals who had received ALVAC-HIV followed by the protein vaccine exhibited HIV-1-specific T cell proliferative responses. Moreover, the PBMC of all prime-boost vaccinated individuals produced detectable IFN-gamma and IL-10 in response to stimulation with HIV-1 envelope glycoprotein antigens; 83% also had detectable levels of IL-2 and IL-6, 71% had detectable levels of IL-4, and 86% had detectable levels of IL-5. These data indicate that this vaccination regimen was inducing both Th1- and Th2-type responses to HIV-1 envelope antigens. This prime-boost vaccination approach elicited T cell help for the generation of cytotoxic T lymphocyte responses as well as help for antibody production and so promises to generate a broad HIV-1-specific immune response.

Listeria monocytogenes and Serratia marcescens infections as models for Th1/Th2 immunity in laboratory cats

Five species of bacteria known to be naturally-occurring pathogens of cats were screened for their ability to grow in feline macrophages in vitro, and to induce antibodies and delayed type hypersensitivity (DTH) responses in vivo. Two of these organisms, L. monocytogenes and S. marcescens, were selected for further study based on clear-cut differences in their in vitro and in vivo behavior. Listeria was macrophage tropic, induced DTH, and evoked poor antibody responses post-recovery, whereas Serratia remained extracellular, did not induce a DTH reaction, and produced high titer of antibodies. Young specific pathogen free cats were then inoculated subcutaneously into the drainage areas of the right and left popliteal and auricular lymph nodes with either L. monocytogenes or S. marcescens. Each of the four lymph nodes were then removed in sequence over a two week period, weighed, cultured for viable bacteria, and RNA extracted for Th1/Th2 cytokine mRNA quantitation. Antibody responses and delayed type hypersensitivity responses were also measured. Identical to pilot studies, cats infected with Serratia developed very high levels of antibody compared to Listeria infected cats but no DTH, while Listeria infected cats produced negligible or low titers of antibodies and strong DTH. Immunity to Listeria occurred around 168 h post infection as evidenced by the disappearance of living bacteria from the nodes, while immunity to Serratia took over 264 h. Pronounced lymph node hyperplasia occurred in both infections, but persisted longer for Serratia. Enlargement of Serratia infected nodes was associated with marked follicular, primary and secondary germinal center and medullary hyperplasia. Germinal center formation in Listeria stimulated nodes was much less intense and dense accumulations of macrophages dissected between follicles downward from the subcapsular sinuses. Although functional and histologic studies showed a clear-cut cell-mediated vs. humoral response in the respective Listeria and Serratia infections, preferential cytokine mRNA upregulation was observed for only two of the five major Th1/Th2 cytokines measured. Interferon- γ, a Th1 cytokine, was much more elevated in the Listeria stimulated nodes, but TNF- α (also a Th1 cytokine) was more elevated in Serratia infected nodes. Interleukin-12, an important Th1 cytokine, was elevated to equal levels in both infections as were the Th2 cytokines IL-4 and IL-10.

Cytokines and adhesion molecules in allergic rhinitis.

This review summarizes our current knowledge of nasal allergic inflammation based on studies of cytokines, chemokines, and adhesion molecules in allergic rhinitis. The article also includes some aspects of viral rhinitis. Due to artificial or natural allergen exposure, an increase in the number of eosinophils and basophils, mast cells, IgE-positive cells, macrophages, monocyte-like cells, Langerhans cells, and activated T-cells can be observed within the mucosa and on the mucosal surface. Mediators are known to be released in response to allergens, but do not seem to be adequate to initiate the cell recruitment. After antigen challenge, the release of proinflammatory and regulatory cytokines could be demonstrated, and TH2-type cytokine mRNA upregulation in allergic mucosa has been shown. Proinflammatory cytokines initiate an adhesion cascade and activate T-cells that create an "atopic" cytokine environment within the tissue, which also may be linked to the long-term selective recruitment of eosinophils. However, the acute selective migration of eosinophils after allergen challenge is not fully understood, nor is the role of chemokines in allergic and viral rhinitis. Allergic rhinitis clearly represents an inflammatory reaction.

Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines.

We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.

Up-regulation of cytokine mRNA, adhesion molecule proteins, and MHC class II proteins in salivary glands of TGF-beta1 knockout mice: MHC class II is a factor in the pathogenesis of TGF-beta1 knockout mice.

Mice homozygous for a disrupted TGF-beta1 allele develop multiple lymphoproliferative disorders similar to those seen in the pseudolymphoma of Sjögren's syndrome. At 2 wk of age, these TGF-beta1 mutant mice begin to develop wasting syndrome and die at around 4 to 5 wk of age. We studied salivary glands from symptomatic mutant mice >14 days of age. Reverse transcriptase-PCR analysis showed up-regulation of proinflammatory cytokine genes such as IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, and IFN-gamma in these mutant mice. Enhanced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1), and MHC class II as well as CD4-positive T lymphocyte infiltration was detected by immunostaining. To elucidate the role of MHC class II, salivary glands from TGF-beta1/MHC class II double knockout mice were used to investigate the expression of adhesion molecules and MHC class II. In spite of the existence of basal intercellular adhesion molecule-1 expression on vessels, there was neither MHC class II expression, enhanced vascular cell adhesion molecule-1 expression, nor lymphocytic infiltration in the salivary glands. These results suggest that MHC class II plays a significant role in the pathogenesis of TGF-beta1 mutant mice. Although the mechanism that initiates multiple inflammatory diseases in these mice remains unclear, the context reported here would provide insight into the immunopathology of Sjögren's syndrome.