Rabies virus P protein is a cofactor of RNA polymerase. We investigated other potential roles of P (CVS strain) by searching for cellular partners using two-hybrid screening. We isolated a cDNA encoding the signal transducer and activator of transcription 1 (STAT1) that is a critical component of interferon type I (IFN-alpha/beta) and type II (IFN-gamma) signaling. We confirmed this interaction by glutathione S-transferase-pull-down assay. Deletion mutant analysis indicated that the carboxy-terminal part of P interacted with a region containing the DNA-binding domain and the coiled-coil domain of STAT1. The expression of P protein inhibits IFN-alpha- and IFN-gamma-induced transcriptional responses, thus impairing the IFN-induced antiviral state. Mechanistic studies indicate that P protein does not induce STAT1 degradation and does not interfere with STAT1 phosphorylation but prevents IFN-induced STAT1 nuclear accumulation. These results indicate that rabies P protein overcomes the antiviral response of the infected cells.

Ubiquitination, endocytosis, and lysosomal degradation of the IFNAR1 (interferon alpha receptor 1) subunit of the type I interferon (IFN) receptor is mediated by the SCFbeta-Trcp (Skp1-Cullin1-F-box protein beta transducin repeat-containing protein) E3 ubiquitin ligase in a phosphorylation-dependent manner. In addition, stability of IFNAR1 is regulated by its binding to Tyk2 kinase. Here we characterize the determinants of IFNAR1 ubiquitination and degradation. We found that the integrity of two Ser residues at positions 535 and 539 within the specific destruction motif present in the cytoplasmic tail of IFNAR1 is essential for the ability of IFNAR1 to recruit beta-Trcp as well as to undergo efficient ubiquitination and degradation. Using an antibody that specifically recognizes IFNAR1 phosphorylated on Ser535 we found that IFNAR1 is phosphorylated on this residue in cells. This phosphorylation is promoted by treatment of cells with IFNalpha. Although the cytoplasmic tail of IFNAR1 contains seven Lys residues that could function as potential ubiquitin acceptor sites, we found that only three (Lys501, Lys525, and Lys526), all located proximal to the destruction motif, are essential for ubiquitination and degradation of IFNAR1. Expression of Tyk2 stabilized IFNAR1 in a manner that was dependent neither on its binding to beta-Trcp nor IFNAR1 ubiquitination. We discuss the complexities and specifics of the ubiquitination and degradation of IFNAR1, which is a beta-Trcp substrate that undergoes degradation via a lysosomal pathway.

Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-alpha/beta induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-alpha/beta production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-alpha/beta in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.

Although the role of natural killer (NK) cells in defense against certain viral infections has been appreciated for a number of years, characterization of the virus-induced endogenous mechanisms regulating NK cell responses and functions has been limited to interferon (IFN)-alpha/beta-mediated activation of NK cell cytotoxicity. Studies of experimental infections have demonstrated that virus-induced NK cells undergo blastogenesis and can be activated to produce IFN-gamma. Recent work has shown that some, but not all, viral infections induce IL-12, the expression of which results in NK cell IFN-gamma production, and that NK cell IFN-gamma production contributes to an antiviral state. IL-12 expression can be regulated by IFN-alpha/beta, and endogenous IFN-alpha/beta is responsible for the lack of IL-12 during viral infections that fail to elicit detectable production of this factor. Once T cell responses are activated, additional mechanisms are in place to turn off NK cell functions. These studies demonstrate that viral infections elicit unique mechanisms for regulating NK cell responses, and suggest that the host requires tight control of NK cells under these conditions.

Interleukin 1 alpha (IL-1 alpha), IL-2, IL-4, IL-6, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and granulocyte macrophage colony-stimulating factor (GM-CSF) were tested for their abilities to alter the growth of Brucella abortus in BALB/c J774A.1 murine macrophages. IL-1 alpha, IL-4, IL-6, tumor necrosis factor alpha, and granulocyte macrophage-colony-stimulating factor had no consistent or significant effect on the growth of the avirulent B. abortus strain 19. In contrast, the addition of either IFN-gamma or IL-2 at 100 U/ml to the macrophage cultures resulted in a significant reduction in the number of intracellular bacteria that was not attributable to decreased infection rates. With IL-2, the reduction was most often apparent only during the first 24 h after infection, while inhibition with IFN-gamma was apparent throughout the culture period of 48 h. The addition of either IL-2 or IFN-gamma to macrophage cultures also resulted in reduced intracellular CFU of the virulent B. abortus strain 2308 and the attenuated rough mutant B. abortus strain RBB. abortus in resident and thioglycolate broth-induced BALB/c peritoneal macrophages and in splenic macrophages. IFN-gamma also inhibited bacterial growth when added after infection of the macrophages, although the magnitude of the antibrucellae effects was less than that when it was added before infection. Furthermore, the maximal inhibitory effect was sustained only when IFN-gamma remained in the cultures after infection of the macrophages.

Knock-out mice with defined major histocompatibility complex (MHC) deficiencies were infected intravenously with Mycobacterium bovis bacille Calmette Guérin (M. bovis BCG) to assess the relative impact of MHC class I- and II-dependent immune responses. Heterozygous control mice were capable of controlling growth of M. bovis BCG, although infection progressed chronically, as assessed by determination of colony-forming units. Furthermore, infected controls developed granulomatous lesions at the site of mycobacterial growth and delayed-type hypersensitivity (DTH) reactions after challenge with purified protein derivative of tuberculin. In vitro, spleen cells from heterozygous control mice produced high concentrations of interferon-gamma (IFN-gamma) after restimulation with mycobacterial antigens. In contrast, the MHC class II-deficient A beta-/- mice, which are virtually devoid of functional CD4 T cells, succumbed to M. bovis BCG infection. Furthermore, A beta-/- mice lacked DTH reactions to tuberculin and only few minute picnotic lesions were formed in livers of infected mice. Finally, spleen cells from infected A beta-/- mice failed to produce measurable IFN-gamma concentrations after restimulation in vitro with various mycobacterial antigen preparations. The capacity of beta 2-microglobulin (beta 2m)-deficient mice, which are devoid of CD8 alpha/beta T cells, to inhibit growth of M. bovis BCG was only slightly affected at low inocula, although significantly higher colony-forming units were detected in spleens. These knock-out mice developed strong DTH responses to tuberculin and their spleen cells produced high levels of IFN-gamma once reactivated by mycobacterial antigens. Furthermore, in livers of infected beta 2m-deficient mice, extravascular infiltrates developed which were more diffuse than those in infected control littermates. Remarkably, the beta 2m-deficient mice were substantially more susceptible to higher inocula of M. bovis BCG than their control littermates. Our data formally prove the essential role of MHC class II-dependent immune mechanisms in all relevant aspects of immunity to M. bovis BCG. In addition, our findings emphasize an important contribution of MHC class I-dependent immunity to effective anti-mycobacterial protection. We assume that CD4 T cells are highly effective in containing M. bovis BCG within distinct granulomatous lesions, but fail to eradicate their intracellular pathogens. It appears most likely that CD8 T cells are also required to achieve this goal.

BACKGROUND

Interferon (IFN)-alpha is an innate immune cytokine that causes high rates of depression in humans and therefore has been used to study the impact of cytokines on the brain and behavior. To establish a nonhuman primate model of cytokine-induced depression, we examined the effects of IFN-alpha on rhesus monkeys.

METHODS

Eight rhesus monkeys were administered recombinant human (rHu)-IFN-alpha (20 MIU/m(2)) or saline for 4 weeks in counterbalanced fashion, and videotaped behavior, as well as plasma and cerebrospinal fluid (CSF), were obtained at regular intervals to assess behavioral, neuroendocrine, immune, and neurotransmitter parameters. Additionally, expression and activity of IFN-alpha/beta receptors in monkey peripheral blood mononuclear cells (PBMCs) were assessed.

RESULTS

Compared with saline treatment, IFN-alpha administration was associated with persistent increases in anxiety-like behaviors and decreases in environmental exploration. In addition, IFN-alpha induced significant increases in plasma concentrations of corticotropin (ACTH), cortisol, and interleukin-6 that tended to diminish after chronic administration, especially in dominant animals. Interestingly, in three animals, depressive-like, huddling behavior was observed. Monkeys that displayed huddling behavior exhibited significantly higher plasma concentrations of ACTH and lower CSF concentrations of the dopamine metabolite homovanillic acid. Rhesus monkey PBMCs were found to express mRNA and protein for the IFN-alpha/beta receptor. Moreover, treatment of PBMCs with rHu-IFN-alpha led to induction of STAT1, one of the primary IFN-alpha-induced signaling molecules.

CONCLUSIONS

IFN-alpha evoked behavioral, neuroendocrine, and immune responses in rhesus monkeys that are similar to humans. Moreover, alterations in hypothalamic-pituitary-adrenal axis responses and dopamine metabolism may contribute to IFN-alpha-induced depressive-like huddling behavior.

Previous studies have established that the cytokine interleukin 1 (IL-1) is selectively cytotoxic for isolated human and rat pancreatic beta-cells. This observation raises the possibility that insulin-dependent diabetes mellitus is in part due to immunologically mediated mechanisms involving IL-1. However, other cytokines are produced during immunologic responses. To study possible modulatory effects of other cytokines on IL-1-mediated beta-cell cytotoxicity, we added human recombinant IL-1 alpha and beta (rIL-1 alpha, rIL-1 beta), tumor necrosis factor (rTNF), lymphotoxin (rLT), and interferon-gamma (rIFN-gamma) separately or in combinations to the culture medium of isolated rat islets of Langerhans. A half-maximal inhibition of glucose-stimulated insulin release after 7 days of culture was obtained with 100 pg/ml of rIL-1 beta, whereas 1000 pg/ml of rIL-1 alpha were necessary to obtain an equivalent effect. While ineffective in causing inhibition of beta-cell function or morphologic damage to islets alone 2.5 to 25 ng/ml of rTNF, but not 40 ng/ml of rLT, or 25 ng/ml of rIFN-gamma markedly potentiated the inhibition of beta-cell secretory response and dissolution of islet integrity caused by rIL-1 alpha and beta. The potentiating effect of rTNF was more pronounced if the rTNF was added after 60 min of preincubation of the islets with rIL-1 beta, than if rIL-1 beta was added after 60 min of preincubation with rTNF. rTNF did not interfere with the activity of rIL-1 alpha or beta on lymphocytes. Combinations of rIFN-gamma and rTNF or rLT did not affect beta-cell function. In conclusion, rTNF strongly potentiates the functional inhibition of beta-cells and the morphologic disintegration of islets caused by rIL-1 in vitro. These data, seen in context with previous observations of rIL-1-mediated beta-cell cytotoxicity, suggest that macrophages present in the intra-islet mononuclear cell infiltrate in insulin-dependent diabetes mellitus may secrete monokines that could be important effector molecules in beta-cell destruction.

BACKGROUND

Prospective data on incidence, characteristics, and risk factors for cryptococcal meningitis immune reconstitution inflammatory syndrome (CM-IRIS) are lacking.

METHODS

Prospective study of 65 antiretroviral therapy (ART)-naive HIV-infected cryptococcal meningitis (CM) patients, who started ART after initiation of antifungal treatment. CM-IRIS definition: (1) cerebrospinal fluid (CSF) culture-confirmed CM, (2) symptom resolution before starting ART, (3) adherence to fluconazole and ART, (4) recurrence of CM symptoms after starting ART, (5) immunologic and/or virologic response to ART, (6) no alternative diagnosis.

RESULTS

ART was started at a median of 47 days from CM diagnosis. CM-IRIS developed in 11 of 65 (17%), at a median 29 days from starting ART. No factors at first CM episode (fungal burden, rate of clearance, CSF, or HIV parameters) predicted those at risk of CM-IRIS. At 6 months on ART, IRIS patients had greater CD4 rise from baseline (220 vs. 124 x 10 cells /L in non-IRIS, P = 0.01), and 4 of 11 CM-IRIS patients died compared with 14 of 54 non-IRIS patients (P = 0.5). For those developing CM-IRIS, CSF proinflammatory cytokines interferon gamma, tumour necrosis factor alpha, and interleukin 6, did not differ between first CM and CM-IRIS episode.

CONCLUSIONS

Patients with CM-IRIS had greater immune restoration in response to ART. Although common and potentially fatal, larger prospective studies are needed to determine whether CM-IRIS, in patients treated initially with amphotericin B, is associated with any increase in overall mortality.

Data on the long-term effects of interferon alfa (IFN) treatment on disease progression and mortality in patients with chronic hepatitis B (CHB) are limited. To evaluate factors that influence clinical outcome and survival, we performed a follow-up study on 165 hepatitis B e antigen (HBeAg) positive CHB patients treated with IFN between 1978 and 2002. The median IFN dose was 30 megaunits (MU)/week (range, 2-70 MU/week), and the median duration of therapy was 16 weeks (range, 1-92 weeks). Response to treatment was defined as HBeAg loss within 12 months after the end of IFN therapy. Median follow-up was 8.8 years (range, 0.3-24 years). Fifty-four patients (33%) responded to IFN treatment. Relapse (HBeAg reactivation) occurred in 7 of the 54 (13%) responders. Fifty-two percent of the responders lost hepatitis B surface antigen (HBsAg) as compared with 9% of the nonresponders (P <.001). Liver histology showed a decreased necroinflammatory activity and less progression of fibrosis in responders. Twenty-six patients died during follow-up. Hepatocellular carcinoma (HCC) was found in 8 patients, 6 of whom were nonresponders. Of the two responders who developed HCC, one patient had relapsed after discontinuation of therapy. Multivariate analysis showed significantly improved survival (relative risk (RR) of death 0.28, 95% CI 0.10-0.78) and reduced risk of developing HCC (RR 0.084, 95% CI 0.09-0.75) in responders. In conclusion, response to IFN therapy results in a prolonged clinical remission with an increased rate of HBsAg seroconversion and improved liver histology. Our results indicate that after correction for baseline factors, response to IFN therapy increases survival and reduces the risk of developing HCC.

The current knowledge about early innate immune responses at mucosal sites of human immunodeficiency virus (HIV) entry is limited but likely to be important in the design of effective HIV vaccines against heterosexual transmission. This study examined the temporal and anatomic relationship between virus replication, lymphocyte depletion, and cytokine gene expression levels in mucosal and lymphoid tissues in a vaginal-transmission model of HIV in rhesus macaques. The results of the study show that the kinetics of cytokine gene expression levels in the acute phase of infection are positively correlated with virus replication in a tissue. Thus, cytokine responses after vaginal simian immunodeficiency virus (SIV) inoculation are earliest and strongest in mucosal tissues of the genital tract and lowest in systemic lymphoid tissues. Importantly, the early cytokine response was dominated by the induction of proinflammatory cytokines, while the induction of cytokines with antiviral activity, alpha/beta interferon, occurred too late to prevent virus replication and dissemination. Thus, the early cytokine response favors immune activation, resulting in the recruitment of potential target cells for SIV. Further, unique cytokine gene expression patterns were observed in distinct anatomic locations with a rapid and persistent inflammatory response in the gut that is consistent with the gut being the major site of early CD4 T-cell depletion in SIV infection.

Among its pleiotropic actions, ghrelin modulates insulin secretion and glucose metabolism. Herein we investigated the role of ghrelin in pancreatic beta-cell proliferation and apoptosis induced by serum starvation or interferon (IFN)-gamma/TNF-alpha, whose synergism is a major cause for beta-cell destruction in type I diabetes. HIT-T15 beta-cells expressed ghrelin but not ghrelin receptor (GRLN-R), which binds acylated ghrelin (AG) only. However, both unacylated ghrelin (UAG) and AG recognized common high-affinity binding sites on these cells. Either AG or UAG stimulated cell proliferation through Galpha(s) protein and prevented serum starvation- and IFN-gamma/TNF-alpha-induced apoptosis. Antighrelin antibody enhanced apoptosis in either the presence or absence of serum but not cytokines. AG and UAG even up-regulated intracellular cAMP. Blockade of adenylyl cyclase/cAMP/protein kinase A signaling prevented the ghrelin cytoprotective effect. AG and UAG also activated phosphatidyl inositol 3-kinase (PI3K)/Akt and ERK1/2, whereas PI3K and MAPK inhibitors counteracted the ghrelin antiapoptotic effect. Furthermore, AG and UAG stimulated insulin secretion from HIT-T15 cells. In INS-1E beta-cells, which express GRLN-R, AG and UAG caused proliferation and protection against apoptosis through identical signaling pathways. Noteworthy, both peptides inhibited cytokine-induced NO increase in either HIT-T15 or INS-1E cells. Finally, they induced cell survival and protection against apoptosis in human islets of Langerhans. These expressed GRLN-R but showed also UAG and AG binding sites. Our data demonstrate that AG and UAG promote survival of both beta-cells and human islets. These effects are independent of GRLN-R, are likely mediated by AG/UAG binding sites, and involve cAMP/PKA, ERK1/2, and PI3K/Akt.

Measles virus (MV), a member of the family Paramyxoviridae, encodes C and V non-structural proteins. To clarify the functions of MV C and V proteins, HeLa cell lines constitutively expressing C or V protein were established. We found that expression of V protein inhibited interferon (IFN)-alpha/beta signaling but not IFN-gamma signaling. C protein had no inhibitory effect on IFN signaling in our experimental condition. Degradation of selective signal transducers and activators of transcription (STAT) proteins was not observed in HeLa cells expressing V protein. In contrast, tyrosine phosphorylation of both STAT1 and STAT2 was inhibited in these cells after IFN-beta stimulation.

Several receptors and counter-receptor pairs on T cells and on antigen-presenting cells (APCs) deliver costimulatory signals to T cells during antigen presentation. The CD28 receptor on T cells with its ligand <em>B</em>7 represents one of the best characterized and most important examples of this costimulation. We show here that interleukin 12 (IL-12), a cytokine also produced by APCs (monocyte/macrophages and <em>B</em> cells) and active on T and natural killer cells, has a strong synergistic effect with the <em>B</em>7/CD28 interaction in inducing proliferation and cytokine production in both mitogen-activated and freshly isolated peripheral blood T cells. Together with anti-CD28 antibodies, IL-12 induces proliferation of T cells to levels higher than those obtained with IL-2 stimulation and it is effective at IL-12 concentrations 100- to 1,000-fold lower than effective concentrations of IL-2. The proliferative effect of anti-CD28 and IL-12 is resistant to moderate doses of cyclosporin A and is largely independent of endogenous IL-2, IL-12, in synergy with anti-CD28 or <em>B</em>7-transfected cells, is most effective in inducing <em>interferon</em> gamma (IFN-gamma) production, but production of tumor necrosis factor <em>alpha</em> and granulocyte/macrophage colony-stimulating factor is also observed. IL-12-induced IFN-gamma production in peripheral blood mononuclear cells is inhibited by the chimeric molecule CTLA-4 immunoglobulin, which prevents binding of CD28 to <em>B</em>7, suggesting that endogenous <em>B</em>7 on the mononuclear cells and IL-12 cooperate in inducing IFN-gamma production. IL-10 inhibits both IL-12 production and <em>B</em>7 expression on monocytes. These two effects are largely responsible for the ability of IL-10, acting on accessory cells, to inhibit IFN-gamma production by lymphocytes, because anti-CD28 antibodies and IL-12 can reverse the inhibitory effect of IL-10 on IFN-gamma production. Our results in vitro suggest that the synergy between <em>B</em>7 and IL-12, a surface antigen and a soluble product of APCs, respectively, plays a role in regulating T cell activation and immune response in the microenvironment of inflamed tissues.

P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm.

Class II (or Ia) antigens are highly polymorphic surface molecules which are essential for the cellular interactions involved in the immune response. In man, these antigens are encoded by a complex multigene family which is located in the major histocompatibility complex (MHC) and which comprises up to 12 distinct alpha- and beta-chain genes, coding for the HLA-DR, -DQ and -DP antigens. One form of congenital severe combined immunodeficiency (SCID) in man, which is generally lethal, is characterized by an absence of HLA-DR histocompatibility antigens on peripheral blood lymphocytes (HLA class II-deficient SCID). In these patients, as reported here, we have observed an absence of messenger RNA for the alpha- and beta-chains of HLA-DR, -DQ and -DP, indicating a global defect in the expression of all class II genes. Moreover, the lack of expression of HLA class II mRNAs could not be corrected by gamma-interferon, an inducer of class II gene expression in normal cells. Family studies have established that the genetic defect does not segregate with the MHC. We conclude, therefore, that the expression of the entire family of class II genes is normally controlled by a trans-acting class II regulatory gene which is unlinked to the MHC and which is affected in the patients. This gene controls a function or a product necessary for the action of gamma-interferon on class II genes.

Host responses to infectious challenges include initial events elicited directly by agent structures distinct from host determinants, activation of innate immune system components by the products of initial events, and the shaping of downstream adaptive immunity by these initial/innate responses. The picture emerging from viral infections is that viral structures interact with intracellular signaling pathways to induce expression of the type 1 interferons, IFN-alpha/beta. In addition to mediating direct antiviral effects, these cytokines play dominant roles in regulating innate and adaptive immune responses to infection. In particular, IFN-alpha/beta acts to inhibit interleukin-12 (IL-12) expression and IL-12 activation of innate natural killer (NK) cell IFN-alpha production, while inducing NK cell cytotoxicity and proliferation, and promoting adaptive T cell IFN-alpha responses. Although certain viral infections do elicit initial/innate IL-12 and NK-cell-produced IFN-alpha, endogenous IFN-alpha/beta also controls the magnitudes of these responses. Thus, the pathways activated, to dominantly regulate innate and adaptive immune responses during viral infections, are being defined.

The VP35 protein of Ebola virus is a viral antagonist of interferon. It acts to block virus or double-stranded RNA-mediated activation of interferon regulatory factor 3, a transcription factor that facilitates the expression of interferon and interferon-stimulated genes. In this report, we show that the VP35 protein is also able to inhibit the antiviral response induced by alpha interferon. This depends on the VP35 function that interferes with the pathway regulated by double-stranded RNA-dependent protein kinase PKR. When expressed in a heterologous system, the VP35 protein enhanced viral polypeptide synthesis and growth in Vero cells pretreated with alpha/beta interferon, displaying an interferon-resistant phenotype. In correlation, phosphorylation of PKR and eIF-2alpha was suppressed in cells expressing the VP35 protein. This activity of the VP35 protein was required for efficient viral replication in PKR+/+ but not PKR-/- mouse embryo fibroblasts. Furthermore, VP35 appears to be a RNA binding protein. Notably, a deletion of amino acids 1 to 200, but not R312A substitution in the RNA binding motif, abolished the ability of the VP35 protein to confer viral resistance to interferon. However, the R312A substitution rendered the VP35 protein unable to inhibit the induction of the beta interferon promoter mediated by virus infection. Together, these results show that the VP35 protein targets multiple pathways of the interferon system.

Chronic inflammatory disorders such as inflammatory bowel diseases (IBD) affect bone metabolism and are frequently associated with the presence of osteoporosis. Bone loss is regulated by various mediators of the immune system such as the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6, or interferon-gamma. TNF-alpha, a master cytokine in human IBD, causes bone erosions in experimental models and these effects are exerted by osteoclasts. Other TNF-related cytokines such as receptor activator of nuclear factor kappa B (RANK), its ligand, RANKL, and osteoprotegerin are important mediators in inflammatory processes in the gut and are critically involved in the pathophysiology of bone loss. The awareness and early diagnosis of osteoporosis in states of chronic inflammation, together with applied therapies such as bisphosphonates, may be beneficial in inflammation-associated osteoporosis. Although several mechanisms may contribute to osteoporosis in patients with IBD and coeliac disease, inflammation as an important factor has so far been neglected. As key inflammatory mediators in IBD such as TNF-alpha are involved in the disease process both in gut and bone, we hypothesise that neutralisation of TNF-alpha could prove an efficient strategy in the treatment of inflammation-related osteoporosis in the future.

Neonates have an immature immune system, which cannot adequately protect against infectious diseases. Early in life, immune protection is accomplished by maternal antibodies transferred from mother to offspring. However, decaying maternal antibodies inhibit vaccination as is exemplified by the inhibition of seroconversion after measles vaccination. This phenomenon has been described in both human and veterinary medicine and is independent of the type of vaccine being used. This review will discuss the use of animal models for vaccine research. I will review clinical solutions for inhibition of vaccination by maternal antibodies, and the testing and development of potentially effective vaccines. These are based on new mechanistic insight about the inhibitory mechanism of maternal antibodies. Maternal antibodies inhibit the generation of antibodies whereas the T cell response is usually unaffected. B cell inhibition is mediated through a cross-link between B cell receptor (BCR) with the Fcγ-receptor IIB by a vaccine-antibody complex. In animal experiments, this inhibition can be partially overcome by injection of a vaccine-specific monoclonal IgM antibody. IgM stimulates the B cell directly through cross-linking the BCR via complement protein C3d and antigen to the complement receptor 2 (CR2) signaling complex. In addition, it was shown that interferon alpha binds to the CD21 chain of CR2 as well as the interferon receptor and that this dual receptor usage drives B cell responses in the presence of maternal antibodies. In lieu of immunizing the infant, the concept of maternal immunization as a strategy to protect neonates has been proposed. This approach would still not solve the question of how to immunize in the presence of maternal antibodies but would defer the time of infection to an age where infection might not have such a detrimental outcome as in neonates. I will review successful examples and potential challenges of implementing this concept.

We present here the first report of a metalloporphyrin-based antioxidant that can prevent or delay the onset of autoimmune diabetes. Type 1 diabetes is an autoimmune process whereby T-cells recognize pancreatic beta-cell antigens and initiate a leukocyte infiltrate that produces proinflammatory cytokines and reactive oxygen species (ROS), ultimately leading to beta-cell destruction. Because islet beta-cells have a reduced capacity to scavenge free radicals, they are very sensitive to ROS action. Metalloporphyrin-based superoxide dismutase (SOD) mimics scavenge ROS and protect cells from oxidative stress and apoptosis. To investigate the effect of SOD mimics and the role of oxidative stress in the development of autoimmune diabetes in vivo, we used a diabetogenic T-cell clone, BDC-2.5, to induce rapid onset of diabetes in young nonobese diabetic-severe combined immunodeficient mice (NOD.scid). Disease was significantly delayed or prevented altogether by treatment of recipient mice with an SOD mimic, AEOL-10113, before transfer of the BDC-2.5 clone. To investigate the mechanisms of protection, in vitro assays for T-cell proliferation and gamma-interferon (IFN-gamma) production were carried out using the T-cell clone BDC-2.5. We found that the SOD mimic significantly inhibited antigen-presenting cell-dependent T-cell proliferation and IFN-gamma production in vitro. In addition, pretreatment of lipopolysaccharide (LPS)-stimulated peritoneal macrophages with SOD mimic inhibited the LPS-dependent increase in TNF-alpha as well as the NADPH oxidase-dependent release of superoxide. Finally, this compound protected NIT-1 insulinoma cells from interleukin-1beta and alloxan cytotoxicity in vitro.

The IKKbeta and NEMO/IKKgamma subunits of the NF-kappaB-activating signalsome complex are known to be essential for activating NF-kappaB by inflammatory and other stress-like stimuli. However, the IKKalpha subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-kappaB-dependent and -independent functions. In contrast to this generally accepted view of IKKalpha's physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKKbeta and NEMO/IKKgamma, IKKalpha is also a global regulator of tumor necrosis factor alpha- and IL-1-responsive IKK signalsome-dependent target genes including many known NF-kappaB targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx3, beta(2)-microglobulin, IL-1alpha, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-kappaB-dependent target genes were preferentially dependent on IKKalpha or IKKbeta. Constitutive expression of a trans-dominant IkappaBalpha superrepressor (IkappaBalphaSR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-kappaB. A subset of NF-kappaB target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-kappaB in established MEFs. Overall, a sizable number of novel NF-kappaB/IKK-dependent genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-beta and -delta, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, interferon (alpha and beta) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-kappaB/IKK-induced gene, suggesting that IKK-mediated NF-kappaB activation can coordinately illicit negative effects on STAT signaling.

Hepatitis B virus (HBV) genotype and precore/core promoter mutations have been implicated in spontaneous and interferon alfa (IFN-alpha)-related hepatitis B e antigen (HBeAg) seroconversion. We performed a retrospective analysis of a previously reported randomized controlled trial to determine the effects of HBV genotype and precore/core promoter mutations on IFN-alpha response in patients with HBeAg-positive chronic hepatitis. Clinical data and stored sera from 109 (95%) patients in the original trial were analyzed. Seventy-three patients received IFN-alpha and 34 received no treatment (controls). Almost all patients had HBV genotypes B (38%) and C (60%). Antiviral response was achieved in 39% and 17% of IFN-alpha-treated patients (P =.03) and in 10% and 8% of untreated controls (P =.88) with HBV genotype B and C, respectively. Multivariate analysis identified HBV genotype B, elevated pretreatment alanine aminotransferase (ALT) levels, and low pretreatment HBV-DNA levels but not IFN-alpha treatment as independent factors associated with antiviral response. Among the 66 patients with elevated pretreatment ALT level, antiviral response was achieved in 57% and 21% of IFN-alpha-treated patients (P =.019), and in 25% and 8% of untreated controls (P =.45) with HBV genotype B and C, respectively. Multivariate analysis showed that genotype B and low pretreatment HBV-DNA levels were independent predictors of antiviral response. In conclusion, our data showed that HBV genotype B was associated with a higher rate of IFN-induced HBeAg clearance compared with genotype C. Stratification for HBV genotypes should be considered in future clinical trials of antiviral therapy of chronic hepatitis B.

During influenza virus infection innate and adaptive immune defenses are activated to eliminate the virus and thereby bring about recovery from illness. Both arms of the adaptive immune system, antibody neutralization of free virus and termination of intracellular virus replication by antiviral cytotoxic T cells (CTLs), play pivotal roles in virus elimination and protection from disease. Innate cytokine responses, such as alpha/beta interferon (IFN-alpha/beta) or IFN-gamma, can have roles in determining the rate of virus replication in the initial stages of infection and in shaping the initial inflammatory and downstream adaptive immune responses. The effect of these cytokines on the replication of pneumotropic influenza A virus in the respiratory tract and in the regulation of adaptive antiviral immunity was examined after intranasal infection of mice with null mutations in receptors for IFN-alpha/beta, IFN-gamma, and both IFNs. Virus titers in the lungs of mice unable to respond to IFNs were not significantly different from congenic controls for both primary and secondary infection. Likewise the mice were comparably susceptible to X31 (H3N2) influenza virus infection. No significant disruption to the development of normal antiviral CTL or antibody responses was observed. In contrast, mice bearing the disrupted IFN-alpha/beta receptor exhibited accelerated kinetics and significantly higher levels of neutralizing antibody activity during primary or secondary heterosubtypic influenza virus infection. Thus, these observations reveal no significant contribution for IFN-controlled pathways in shaping acute or memory T-cell responses to pneumotropic influenza virus infection but do indicate some role for IFN-alpha/beta in the regulation of antibody responses. Recognizing the pivotal role of CTLs and antibody in virus clearance, it is reasonable to assume a redundancy in IFN-mediated antiviral effects in pulmonary influenza. However, IFN-alpha/beta seems to be a valid factor in determining tissue tropism and replicative rates of highly virulent influenza virus strains as reported previously by others, and this aspect is discussed here.