The anti-tumor activity of IL-12: Mechanisms of innate immunity that are model and dose dependent

IL-12 has been demonstrated to have potent anti-tumor activities in a variety of mouse tumor models, but the relative roles of NK, NKT, and T cells and their effector mechanisms in these responses have not been fully addressed. Using a spectrum of gene-targeted or Ab-treated mice we have shown that for any particular tumor model the effector mechanisms downstream of IL-12 often mimic the natural immune response to that tumor. For example, metastasis of the MHC class I-deficient lymphoma, EL4-S3, was strictly controlled by NK cells using perforin either naturally or following therapy with high-dose IL-12. Intriguingly, in B16F10 and RM-1 tumor models both NK and NKT cells contribute to natural protection from tumor metastasis, In these models, a lower dose of IL-12 or delayed administration of IL-12 dictated a greater relative role of NKT cells in immune protection from tumor metastasis. Overall, both NK and NKT cells can contribute to natural and IL-12-induced immunity against tumors, and the relative role of each population is turner and therapy dependent.

Therapeutic activation of V alpha 24(+)V beta 11(+) NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity

Human Valpha24(+)Vbeta11(+) natural killer T (NKT) cells are a distinct CD1d-restricted lymphoid subset specifically and potently activated by alpha-galactosylceramide (alpha-GalCer) (KRN7000) presented by CD1 d on antigen-presenting cells. Preclinical models show that activation of Valpha24(+)Vbeta11(+) NKT cells induces effective antitumor immune responses and potentially important secondary immune effects, including activation of conventional T cells and NK cells. We describe the first clinical trial of cancer immune therapy with alpha-GalCer-pulsed CD1d-expressing dendritic cells. The results show that this therapy has substantial, rapid, and highly reproducible specific effects on Valpha24(+)Vbeta11(+) NKT cells and provide the first human in vivo evidence that Valpha24(+)Vbeta11(+) NKT cell stimulation leads to activation of both innate and acquired immunity, resulting in modulation of NK, T-, and B-cell numbers and increased serum interferon-gamma. We present the first clinical evidence that Valpha24(+)Vbeta11(+) NKT cell memory produces faster, more vigorous secondary immune responses by innate and acquired immunity upon restimulation.

Alternatives to conventional vaccines - Mediators of innate immunity

Vaccines have been described as weapons of mass protection. The eradication of many diseases is testament to their utility and effectiveness. Nevertheless, many vaccine preventable diseases remain prevalent because of political and economic barriers. Additionally, the effects of immaturity and old age, therapies that incapacitate the adaptive immune system and the multitude of strategies evolved by pathogens to evade immediate or sustained recognition by the mammalian immune system are barriers to the effectiveness of existing vaccines or development of new vaccines. In the front line of defence against the pervasiness of infection are the elements of the innate immune system. Innate immunity is under studied and poorly appreciated. However, in the first days after entry of a pathogen into the body, our entire protective response is dependant upon the various elements of our innate immune repertoire. In spite of, its place as our initial defence against infection, attention is only now turning to strategies which enhance or supplement innate immunity. This review examines the need for and potential of innate immune therapies.

Mechanisms of hematopoietic stem cell mobilization: When innate immunity assails the cells that make blood and bone

Mobilization is now used worldwide to collect large numbers of hematopoietic stem and progenitor cells (HSPCs) for transplantation. Although the first mobilizing agents were discovered largely by accident, discovery of more efficient mobilizing agents will require a better understanding of the molecular mechanisms responsible. During the past 5 years, a number of mechanisms have been identified, shedding new light on the dynamics of the hematopoietic system in vivo and on the intricate relationship between hematopoiesis, innate immunity, and bone. After briefly reviewing the mechanisms by which circulating HSPCs home into the bone marrow and what keeps them there, the current knowledge of mechanisms responsible for HSPC mobilization in response to hematopoietic growth factors such as granulocyte colony-stimulating factor, chemotherapy, chemokines, and polyanions will be discussed together with current strategies developed to further increase HSPC mobilization. (c) 2006 International Society for Experimental Hematology.

Bovine mammary epithelial cells, initiators of innate immune responses to mastitis

Innate immunity plays a vital role in the protection of the bovine mammary gland against mastitis. Until recently, the migration of effector cells such as neutrophils and monocytes into the mammary gland was thought to provide the only defence against invading pathogens. However, mammary epithelial cells may also play an important role in the immune response, contributing to the innate defence of the mammary tissue through secretion of antimicrobial peptides and attraction of circulating immune effector cells. This paper reviews the innate immune pathways in mammary epithelial cells and examines their role in the initiation of an innate immune response to Gram-positive and Gram-negative bacteria.

B, NK and NKT cells contribute to suppression of immunity by dendritic cells

Among the population of antigen presenting cells, dendritic cells (DCs) are considered the sentinels of the immune system. Besides activating naı¨ ve T cells, DC can directly activate naı¨ ve and memory B cells and are also able to regulate effectors of innate immunity such as NK cells and NKT cells. Increasing evidence indicates that DCs are not only decisive for T cell priming, but are also key players to maintain self-tolerance in vivo. Previous results in our lab have shown that DCs treated with a pharmacological NFkB inhibitor (BAY11–7082) confer suppression to a previously immune response. This suppression was IL-10 dependent and results from the induction of Ag specific CD4+ regulatory T cells. To elucidate the mechanism of suppression induced by administration of Bay treated DC, we used a model of infectious tolerance transfer from DC treated mice to primed recipient mice. Our results show that both CD4 + splenic cells and non T cells from animals injected with Bay treated DC, but not from untreated DC, were capable of transferring the suppression. Moreover, sorted B cells and NK cells could transfer antigenspecific infectious tolerance after administration of Bay treated DC. In addition, this suppressive effect could not be seen either in mice depleted of NK cells nor in NKT deficient mice. These observations highlight the role of several immune cells in the maintenance of tolerance, and impact on the design of immunotherapeutic suppression of autoimmune diseases in which NKT cells are deficient or defective, such as diabetes and lupus.

Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity

Dendritic cells (DCs) regulate various aspects of innate immunity, including natural killer (NK) cell function. Here we define the mechanisms involved in DC - NK cell interactions during viral infection. NK cells were efficiently activated by murine cytomegalovirus ( MCMV) - infected CD11b(+) DCs. NK cell cytotoxicity required interferon-alpha and interactions between the NKG2D activating receptor and NKG2D ligand, whereas the production of interferon-gamma by NK cells relied mainly on DC-derived interleukin 18. Although Toll-like receptor 9 contributes to antiviral immunity, we found that signaling pathways independent of Toll-like receptor 9 were important in generating immune responses to MCMV, including the production of interferon-alpha and the induction of NK cell cytotoxicity. Notably, adoptive transfer of MCMV-activated CD11b(+) DCs resulted in improved control of MCMV infection, indicating that these cells participate in controlling viral replication in vivo.

Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that Of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity:for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not,necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within I h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

Perforin and interferon-gamma activities independently control tumor initiation, growth, and metastasis

Perforin (pfp) and interferon-gamma (IFN-gamma) together in C57BL/6 (B6) and BALB/c mouse strains provided optimal protection in 3 separate tumor models controlled by innate immunity. Using experimental (B6, RM-1 prostate carcinoma) and spontaneous (BALB/c, DA3 mammary carcinoma) models of metastatic cancer, mice deficient in both pfp and IFN-gamma were significantly less proficient than pfp- or IFN-gamma -deficient mice in preventing metastasis of tumor cells to the lung. Pfp and IFN-gamma -deficient mice were as susceptible as mice depleted of natural killer (NK) cells in both tumor metastasis models, and IFN-gamma appeared to play an early role in protection from metastasis, Previous experiments in a model of fibrosarcoma induced by the chemical carcinogen methylcholanthrene indicated an important role for NK1.1(+) T cells, Herein, both pfp and IFN-gamma played critical and independent roles in providing the host with protection equivalent to that mediated by NK1.1+ T cells, Further analysis demonstrated that IFN-gamma, but not pfp, controlled the growth rate of sarcomas arising in these mice. Thus, this is the first study to demonstrate that host IFN-gamma, and direct cytotoxicity mediated by cytotoxic lymphocytes expressing pfp independently contribute antitumor effector functions that together control the initiation, growth, and spread of tumors in mice, (C) 2001 by The American Society of Hematology.

Disturbances in microbiota - cause or effect?

IBD are a group of complex polygenetic diseases also involving environmental factors. Evidence for a role for bacteria in IBD include an increased abundance of mucosa-associated bacteria in IBD (which occurs even where there is no intestinal inflammation), and the positive impact of antibiotics on the progress of both Crohn's disease (CD) and ulcerative colitis (UC) of the pouch - pouchitis. Bacteria are necessary for most animal models of IBD. The increased abundance of mucosal bacteria in IBD is not non-specific because while some mucosal bacteria are more abundant this is not the case for all mucosal bacteria including the very abundant Bacteroides vulgatus. On the other hand, antibiotic treatments are not curative, and the humoral immune Ig response to bacterial antigens which is more evident in CD, appears to be polyclonal. While this argues against a role for specific bacteria causing a classical infection, certain mucosal bacteria may damage the mucosal barrier. This would promote invasion by other commensal mucosal bacteria triggering an immune response. Altered adaptive, and to a lesser extent, innate immunity have been extensively studied, and genetic defects in the CARD15 (or NOD2) gene that encodes a bacterial sensing protein modulating innate and adaptive immunity are strongly associated with ileal CD. However, the penetrance of the homozygous CARD15 frameshift mutation, which is the most strongly CD-associated genotype, is very low with only 4% of humans with this developing CD. Furthermore, mice with the same defects in CARD15 do not develop spontaneous ileitis or colitis. Therefore, there have to be other aetiological factor(s). Altered permeability is a consistent finding in subclinical CD. There are other data to suggest that altered mucin is an early event in UC. We propose that the pathogenesis of IBD is multifactorial involving specific mucosal bacteria, defective barrier function and altered mucosal immunity in an aetiology triangle.

Gene expression during early ascidian metamorphosis requires signaling by Hemps, an EGF-like protein

Hemps, a novel epidermal growth factor (EGF)-like protein, is expressed during larval development and early metamorphosis in the ascidian Herdmania curvata and plays a direct role in triggering metamorphosis. In order to identify downstream genes in the Hemps pathway we used a gene expression profiling approach, in which we compared post-larvae undergoing normal metamorphosis with larval metamorphosis blocked with an anti-Hemps antibody. Molecular profiling revealed that there are dynamic changes in gene expression within the first 30 minutes of normal metamorphosis with a significant portion of the genome (approximately 49%) being activated or repressed. A more detailed analysis of the expression of 15 of these differentially expressed genes through embryogenesis, larval development and metamorphosis revealed that while there is a diversity of temporal expression patterns, a number of genes are transiently expressed during larval development and metamorphosis. These and other differentially expressed genes were localised to a range of specific cell and tissue types in Herdmania larvae and post-larvae. The expression of approximately 24% of the genes that were differentially expressed during early metamorphosis was affected in larvae treated with the anti-Hemps antibody. Knockdown of Hemps activity affected the expression of a range of genes within 30 minutes of induction, suggesting that the Hemps pathway directly regulates early response genes at metamorphosis. In most cases, it appears that the Hemps pathway contributes to the modulation of gene expression, rather than initial gene activation or repression. A total of 151 genes that displayed the greatest alterations in expression in response to anti-Hemps antibody were sequenced. These genes were implicated in a range of developmental and physiological roles, including innate immunity, signal transduction and in the regulation of gene transcription. These results suggest that there is significant gene activity during the very early stages of H. curvata metamorphosis and that the Hemps pathway plays a key role in regulating the expression of many of these genes.

Challenge-induced plasma exudation and mucinous secretion in human airways

Secretion of mucins and exudation of plasma are distinct processes of importance to innate immunity and inflammatory disease. Yet, little is known about their relation in human airways. The objective of the present study was to use the human nasal airway to determine mucinous secretion and plasma exudation in response to common challenge agents and mediators. Ten healthy volunteers were subjected to nasal challenge-lavage procedures. Thus, the nasal mucosa was exposed to increasing doses of histamine (40 and 400 mu g ml(-1)), methacholine (12.5 and 25 mg) and capsaicin (30 and 300 ng ml(-1)). Fucose was selected as a global marker of mucinous secretion and alpha(2)-macroglobulin as an index of exudation of bulk plasma. All challenge agents increased the mucosal output of fucose to about the same level (P < 0.01-0.05). Once significant secretion had been induced the subsequently increased dose of the challenge agent, in the case of histamine and methacholine, failed to further increase the response. Only histamine increased the mucosal output of alpha(2)-macroglobulin (P < 0.01). We conclude that prompt but potentially rapidly depleted mucinous secretion is common to different kinds of airway challenges, whereas inflammatory histamine-type mediators are required to produce plasma exudation. Along with the acknowledged secretion of mucins, a practically non-depletable, pluripotent mucosal output of plasma emerges as an important component of the innate immunity of human airways.

Mechanisms of inducible resistance against Bacillus thuringiensis endotoxins in invertebrates

Abstract Resistance in insect pests against the endotoxin of Bacillus thuringiensis (Berliner) (Bt) is a major threat to the usefulness of this biopesticide, both used as traditional formulations and in transgenic crops. A crucial requirement for the development of successful resistance management strategies is a molecular understanding of the nature and inheritance of resistance mechanisms. This information can be used to design management strategies that will delay or counteract Bt resistance. The best known Bt resistance mechanism is inactivation of brush border membrane receptors. This type of resistance has a largely recessive mode of inheritance, which has enabled the design of resistance management approaches involving high dose and refuge strategies. Recent observations suggest that other resistance mechanisms are possible, including a mechanism that sequesters the toxin in the gut lumen through inducible immune reactions. The elevated immune status associated with tolerance to the toxin can be transmitted to subsequent generations by a maternal effect, which has implications for resistance management in the field. The high dose/refuge strategy may not be appropriate for the management of these alternative resistance mechanisms and other strategies have to be developed if inducible dominant resistance or tolerance mechanisms occur frequently in the field.