Showing papers in "Immunology and Cell Biology in 2004"

Vaccine adjuvants: current state and future trends.

Abstract: The problem with pure recombinant or synthetic antigens used in modern day vaccines is that they are generally far less immunogenic than older style live or killed whole organism vaccines. This has created a major need for improved and more powerful adjuvants for use in these vaccines. With few exceptions, alum remains the sole adjuvant approved for human use in the majority of countries worldwide. Although alum is able to induce a good antibody (Th2) response, it has little capacity to stimulate cellular (Th1) immune responses which are so important for protection against many pathogens. In addition, alum has the potential to cause severe local and systemic side-effects including sterile abscesses, eosinophilia and myofascitis, although fortunately most of the more serious side-effects are relatively rare. There is also community concern regarding the possible role of aluminium in neurodegenerative diseases such as Alzheimer's disease. Consequently, there is a major unmet need for safer and more effective adjuvants suitable for human use. In particular, there is demand for safe and non-toxic adjuvants able to stimulate cellular (Th1) immunity. Other needs in light of new vaccine technologies are adjuvants suitable for use with mucosally-delivered vaccines, DNA vaccines, cancer and autoimmunity vaccines. Each of these areas are highly specialized with their own unique needs in respect of suitable adjuvant technology. This paper reviews the state of the art in the adjuvant field, explores future directions of adjuvant development and finally examines some of the impediments and barriers to development and registration of new human adjuvants.

Aluminium compounds for use in vaccines

Abstract: Aluminium adjuvants are the most widely used adjuvants in both human and veterinary vaccines. These adjuvants have been used in practical vaccination for more than 60 years and are generally recognized as safe and as stimulators of Th2 immunity. The present review gives a short introduction to the pioneering research at the start of the use of aluminium compounds as adjuvants, including references on the chemistry of these compounds. Analytical methods for identifying the most commonly used aluminium compounds, such as boehmite and aluminium hydroxyphosphate, are mentioned. Emphasis is placed on the important factors for antigen adsorption and on the latest work using gene-deficient mice in the research of the mechanism of aluminium adjuvants in terms of cytokine and T-cell subset stimulation. Key references on the ability of aluminium adjuvants to stimulate IgE and also in vivo clearing of aluminium adjuvants are discussed. Furthermore, the review addresses the issue of local reactions in the context of injection route and local tissue disturbance. Possible new applications of aluminium adjuvants in, for example, combined aluminium-adsorbed protein and DNA oligonucleotide vaccines as well as the possible use of aluminium adjuvants in combination with IL-12 to stimulate Th1-type immune responses are mentioned.

Vaccines that facilitate antigen entry into dendritic cells.

Abstract: Summary Although vaccines have been highly successful in preventing and treating many infectious diseases (including smallpox, polio and diphtheria) diseases prevalent in the developing world such as malaria and HIV, that suppress the host immune system, require new, multiple strategies that will be defined by our growing understanding of specific immune activation. The definition of adjuvants, previously thought of as any substance that enhanced the immunogenicity of antigen, could now include soluble mediators and antigenic carriers that interact with surface molecules present on DC (e.g. LPS, Flt3L, heat shock protein) particulate antigens which are taken up by mechanisms available to APC but not other cell types (e.g. immunostimulatory complexes, latex, polystyrene particles) and viral/ bacterial vectors that infect antigen presenting cells (e.g. vaccinia, lentivirus, adenovirus). These approaches, summarized herein, have shown potential in vaccinating against disease in animal models, and in some cases in humans. Of these, particle-antigen conjugates provide rapid formulation of the vaccine, easy storage and wide application, with both carrier and adjuvant functions that activate DC. Combined vaccines of the future could use adjuvants such as virus-like particles and particles targeted towards a predominant cellular type or immune response, with target cell activation enhanced by growth factors or maturation signals prior to, or during immunization. Collectively, these new additions to adjuvant technology provide opportunities for more specific immune regulation than previously available.

Mucosal adjuvants and delivery systems for protein-, DNA- and RNA-based vaccines.

Abstract: Almost all vaccinations today are delivered through parenteral routes. Mucosal vaccination offers several benefits over parenteral routes of vaccination, including ease of administration, the possibility of self-administration, elimination of the chance of injection with infected needles, and induction of mucosal as well as systemic immunity. However, mucosal vaccines have to overcome several formidable barriers in the form of significant dilution and dispersion; competition with a myriad of various live replicating bacteria, viruses, inert food and dust particles; enzymatic degradation; and low pH before reaching the target immune cells. It has long been known that vaccination through mucosal membranes requires potent adjuvants to enhance immunogenicity, as well as delivery systems to decrease the rate of dilution and degradation and to target the vaccine to the site of immune function. This review is a summary of current approaches to mucosal vaccination, and it primarily focuses on adjuvants as immunopotentiators and vaccine delivery systems for mucosal vaccines based on protein, DNA or RNA. In this context, we define adjuvants as protein or oligonucleotides with immunopotentiating properties co-administered with pathogen-derived antigens, and vaccine delivery systems as chemical formulations that are more inert and have less immunomodulatory effects than adjuvants, and that protect and deliver the vaccine through the site of administration. Although vaccines can be quite diverse in their composition, including inactivated virus, virus-like particles and inactivated bacteria (which are inert), protein-like vaccines, and non-replicating viral vectors such as poxvirus and adenovirus (which can serve as DNA delivery systems), this review will focus primarily on recombinant protein antigens, plasmid DNA, and alphavirus-based replicon RNA vaccines and delivery systems. This review is not an exhaustive list of all available protein, DNA and RNA vaccines, with related adjuvants and delivery systems, but rather is an attempt to highlight many of the currently available approaches in immunopotentiation of mucosal vaccines.

Helper T cells, dendritic cells and CTL Immunity

Abstract: In this review, we examine the emerging view that all CTL responses depend on CD4 T-cell help for the generation of efficient memory. We further review the evidence that CD4 and CD8 T cells must recognize antigen on the same dendritic cell, and examine why this corecognition is required. Earlier studies have suggested that CD4 T cells must activate the dendritic cell via CD40 to license it for the capacity to prime CTL immunity. More recently, however, CD40 signalling of the CTL has been reported. Here, we argue that the main reason for corecognition of antigen on the dendritic cell may be related to the time taken to activate and release CD4 and CD8 T cells from their priming dendritic cell. CD4 T cells may only be capable of activating one dendritic cell during the period that CD8 T cells are primed. In this case, corecognition of this same dendritic cell would be essential.

Donor treatment with pegylated-G-CSF augments the generation of IL-10 producing regulatory T cells and promotes transplant tolerance

Abstract: We investigated whether the protection from graft-versus-host disease (GVHD) afforded by donor treatment with granulocyte colony-stimulating factor (G-CSF) could be enhanced by dose escalation. Donor treatment with human G-CSF prevented GVHD in the B6 --> B6D2F1 murine model in a dose-dependent fashion, and murine G-CSF provided equivalent protection from GVHD at 10-fold lower doses. Donor pretreatment with a single dose of pegylated G-CSF (peg-G-CSF) prevented GVHD to a significantly greater extent than standard G-CSF (survival, 75% versus 11%, P <.001). Donor T cells from peg-G-CSF-treated donors failed to proliferate to alloantigen and inhibited the responses of control T cells in an interleukin 10 (IL-10)-dependent fashion in vitro. T cells from peg-G-CSF-treated IL-10(-/-) donors induced lethal GVHD; T cells from peg-G-CSF-treated wild-type (wt) donors promoted long-term survival. Whereas T cells from peg-G-CSF wt donors were able to regulate GVHD induced by T cells from control-treated donors, T cells from G-CSF-treated wt donors and peg-G-CSF-treated IL-10(-/-) donors did not prevent mortality. Thus, peg-G-CSF is markedly superior to standard G-CSF for the prevention of GVHD following allogeneic stem cell transplantation (SCT), due to the generation of IL-10-producing regulatory T cells. These data support prospective clinical trials of peg-G-CSF-mobilized allogeneic blood SCT.

Inulin-derived adjuvants efficiently promote both Th1 and Th2 immune responses.

Abstract: There has been a recent resurgence of interest into new and improved vaccine adjuvants. This interest has been stimulated by the need for new vaccines to combat problematic pathogens such as SARS and HIV, and to counter potential bioterrorist attacks. A major bottleneck in vaccine development is the low immunogenicity of purified subunit or recombinant proteins, creating the need for safe human adjuvants with high potency. A major problem in the search for the ideal adjuvant is that adjuvants that promote cell-mediated (Th1) immunity (e.g. Freund's complete adjuvant) generally have unacceptable local or systemic toxicity that precludes their use in human vaccines. There is a need for a safe, non-toxic adjuvant that is able to stimulate both cell-mediated and humoral immunity. Inulin-derived adjuvants that principally stimulate the innate immune system through their ability to activate the alternative complement pathway have proven ability to induce both cellular and humoral immunity. With their excellent tolerability, long shelf-life, low cost and easy manufacture, they offer great potential for use in a broad range of prophylactic and therapeutic vaccines. Based on successful animal studies in a broad range of species, human trials are about to get underway to validate the use of inulin-based adjuvants in prophylactic vaccines against hepatitis B, malaria and other pathogens. If such trials are successful, then it is possible that inulin-derived adjuvants will one day replace alum as the adjuvant of choice in most human prophylactic vaccines.

Phenotypic and functional changes of circulating monocytes and polymorphonuclear leucocytes from elderly persons

Abstract: The function and phenotype of monocytes and granulocytes in the elderly is consistently remodelled. Because leucocyte adhesion molecules play important roles in mediating a wide variety of leucocyte functions, age-related changes in their expression on granulocyte and monocyte surfaces could be partially responsible for immune dysfunctions during senescence. Considering the central role of innate immunity in the process of immunosenescence and the involvement of cell adhesion molecules (CAM) in the great majority of leucocyte functions, we studied the expression of CD50 and CD62L adhesion molecules in peripheral blood granulocytes and monocytes from healthy elderly and young subjects. We show here that the percentage of granulocytes and monocytes expressing CD62L is decreased in the elderly, whereas its density expression is unchanged on both cell types. A downregulation of the density expression of CD50 at a per cell level characterizes granulocytes in the elderly, whereas CD50 expression on monocytes from old subjects shows a peculiar attitude: its density expression decreases whereas the number of positive cells is expanded. The downregulation of this receptor on granulocytes from aged people could determine a state of hyperactivation contributing to the proinflammatory status of the elderly, while the lower expression on monocytes could therefore contribute to the impaired antigen presentation in the elderly. On the other hand, the increased number of CD50 positive monocytes in the elderly, despite its decreased density expression at a per cell level, could be interpreted as an attempt to counteract the inability to mount strong immune responses. Both CD50 and CD62L changes in ageing polymorphonuclear (PMN) cells allow recognition as non-self or senescent self to permit macrophages in the liver and spleen to remove them from the circulation. The increased proportion of granulocytes and monocytes lacking CD62L and the downregulation of CD50 intensity expression on both cell types may suggest a state of in vivo activation. Therefore, CD50 and CD62L shedding from the cell surface of activated granulocytes and monocytes could be interpreted as a tentative to counteract the dangerous effects of an excessive chronic inflammation in the elderly. However, the increased proportion of CD62L negative granulocytes in the elderly leads to an impairment in cell adhesion which is the first line of response to acute inflammatory stimuli. This phenomenon likely contributes to the increased susceptibility to acute infections of elderly people.

Shaping of adaptive immune responses to soluble proteins by TLR agonists: A role for IFN-α/β

Abstract: Toll-like receptors (TLR) are believed to play a major role in the recognition of invading organisms, although their ability to shape immune responses is not completely understood. Our aim was to investigate in vivo the effect of different TLR stimuli on the generation of antibody responses and the induction of CD8+ T-cell cross-priming after immunization with soluble protein antigens. While all TLR agonists tested elicited the production of immunomodulatory cytokines, marked differences were observed in their ability to stimulate antigen-specific immune responses. Zymosan, poly(I:C) and CpG DNA, which signal through TLR2/6, 3 and 9, respectively, were found to strongly induce the production of IgG2a antibodies, whereas R-848 (TLR7) and LPS (TLR4) did so much more weakly. In contrast, LPS, poly(I:C) and CpG DNA, but not zymosan, induced functional CD8+ T-cell responses against OVA; peptidoglycan (TLR2/?) and R-848 were also ineffective in stimulating cross-priming. Experiments using IFN-alpha/beta R-deficient mice showed that the induction of cross-priming by LPS and poly(I:C) was abrogated in the absence of IFN-alpha/beta signalling, and induction by CpG DNA was greatly reduced. Overall, our results identify LPS as another TLR agonist that is able to generate functional cross-priming against a soluble protein antigen. In addition, our results demonstrate that the ability of TLR stimuli to initiate CD8+ T-cell responses against soluble protein antigens is largely dependent on the IFN-alpha/beta signalling pathway.

Interleukin-17: the missing link between T-cell accumulation and effector cell actions in rheumatoid arthritis?

Abstract: The prominence of T cells and monocyte/macrophages in rheumatoid synovium suggests T cells may localize and amplify the effector functions of monocyte/macrophages in rheumatoid disease. However, while T cells are abundant in rheumatoid joints, classic T-cell derived cytokines are scarce, especially when compared to the levels of monokines IL-1 beta and TNF-alpha. For this reason, it has been speculated that monocyte/macrophages may act independently of T cells in rheumatoid disease and that the role of T cells may be more or less irrelevant to core disease mechanisms. The question of T-cell influence requires re-evaluation in light of the characterization of IL-17, a T-cell derived cytokine that is abundant in rheumatoid synovium and synovial fluid. IL-17 has a number of pro-inflammatory effects, both directly and through amplification of the effects of IL-1 beta and TNF-alpha. IL-17 is able to induce expression of pro-inflammatory cytokines and stimulate release of eicosanoids by monocytes and synoviocytes. Furthermore, IL-17 has been implicated in the pathogenesis of inflammatory bone and joint damage through induction of matrix metalloproteinases and osteoclasts, as well as inhibition of proteoglycan synthesis. In animal models of arthritis, intra-articular injection of IL-17 results in joint inflammation and damage. The recognition of IL-17 as a pro-inflammatory T cell derived cytokine, and its abundance within rheumatoid joints, provides the strongest candidate mechanism to date through which T cells can capture and localize macrophage effector functions in rheumatoid arthritis. As such, IL-17 warrants consideration for its potential as a therapeutic target in rheumatoid arthritis.

Lymphoid organ dendritic cells: beyond the Langerhans cells paradigm

Abstract: The immune system has developed mechanisms to detect and initiate responses to a continual barrage of immunological challenges. Dendritic cells (DC), a heterogeneous population of leucocytes, play a major role as immunosurveillance agents. To accomplish this function, DC are equipped with highly efficient mechanisms to detect pathogens, to capture, process and present antigens, and to initiate T-cell responses. These mechanisms are developmentally regulated during the DC life cycle in a process termed 'maturation', which was originally defined using Langerhans cells (LC), a DC type of the epidermis. LC exist in the skin in an immature state dedicated to capturing antigens, and in the subcutaneous lymph nodes in a mature state dedicated to presenting those antigens to T cells. The phenotypic changes undergone by LC during maturation, and the correlation of these changes with tissue localization, have been generally considered a paradigm for all DC. However, studies of the multiple DC types found in the lymphoid organs of mice and humans have revealed that most DC subsets do not follow the life cycle typified by LC. In this review we discuss the limitations of the 'LC paradigm' and suggest that this model should be revised to accommodate the heterogeneity of the DC system. We also discuss the implications of the maturational status of the DC subsets contained in the lymphoid organs for their putative roles in the induction of immune responses and the maintenance of peripheral tolerance.

Innate Versus Adaptive Immunity in Candida Albicans Infection

Abstract: Candida albicans is a common opportunistic pathogen, causing both superficial and systemic infection. Clinical observations indicate that mucocutaneous infections are commonly associated with defective cell-mediated immune responses, whereas systemic infection is more frequently seen in patients with deficiencies in neutrophil number or function. Analysis of mechanisms of host resistance against gastrointestinal and oral infection in mouse models has demonstrated an absolute dependence on CD4(+) T cells, although clearance also involves phagocytic cells. Both IL-12 and TNF-alpha appear to be important mediators, but mouse strain-dependent variations in susceptibility to infection may be related to T-cell enhancement of production of phagocytic cells by the bone marrow. In murine systemic infection, the role of innate and adaptive responses is less well defined. Studies in immunodeficient and T-cell-depleted mice suggest that clearance of the yeast may be predominantly a function of the innate response, whereas the adaptive response may either limit tissue damage or have the potential to cause immunopathology, depending on the host genetic context in which the infection takes place.

Challenges facing adjuvants for cancer immunotherapy.

Abstract: An adjuvant is defined as a product that increases or modulates the immune response against an antigen (Ag). Based on this general definition many authors have postulated that the ideal adjuvant should increase the potency of the immune response, while being non-toxic and safe. Although dozens of different adjuvants have been shown to be effective in preclinical and clinical studies, only aluminium-based salts (Alum) and squalene-oil-water emulsion (MF59) have been approved for human use. However, for the development of therapeutic vaccines to treat cancer patients, the prerequisites for an ideal cancer adjuvant differ from conventional adjuvants for many reasons. First, the patients that will receive the vaccines are immuno-compromised because of, for example, impaired mechanisms of antigen presentation, non-responsiveness of activated T cells and enhanced inhibition of self-reactivity by regulatory T cells. Second, the tumour Ag are usually self-derived and are, therefore, poorly immunogenic. Third, tumours develop escape mechanisms to avoid the immune system, such as tumour editing, low or non-expression of MHC class I molecules or secretion of suppressive cytokines. Thus, adjuvants for cancer vaccines need to be more potent than for prophylactic vaccines and consequently may be more toxic and may even induce autoimmune reactions. In summary, the ideal cancer adjuvant should rescue and increase the immune response against tumours in immuno-compromised patients, with acceptable profiles of toxicity and safety. The present review discusses the role of cancer adjuvants at the different phases of the generation of antitumour immunity following vaccination.

Effect of retinoic acid in experimental diabetic nephropathy.

Abstract: Although the pathogenetic mechanism of diabetic nephropathy has not been elucidated, an inflammatory mechanism has been suggested to contribute to its progression. Monocyte chemoattractant peptide (MCP)-1 attracts macrophages and T cells, and ultimately injures renal tissue. In early diabetic nephropathy, urinary excretion of MCP-1 was elevated, and increased as renal damage became more severe. Podocytes are expected to have an inflammatory role in diabetic nephropathy, as the surface expression of chemokine receptors such as CCR and CXCR on these cells has been recently reported. Although retinoid (retinal), a known anti-inflammatory agent, has been reported to be beneficial in some experimental models of renal disease, it has not been determined to prevent disease progression in diabetic nephropathy. We investigated the effects of all-trans retinoic acid on the production of MCP-1 under high glucose conditions in cultured mouse podocytes. We also evaluated whether all-trans retinoic acid inhibits inflammatory changes and improves renal function during the early stages of diabetic nephropathy in streptozotocin-induced diabetic rats. In cultured podocytes, high glucose stimuli rapidly upregulated the MCP-1 mRNA transcript and protein release. Treatment with retinoic acid tended to suppress the MCP-1 gene transcript, and significantly inhibited MCP-1 protein synthesis induced by high glucose stimulation. Urinary protein excretion and the urinary albumin : creatinine ratio (ACR) were significantly higher in diabetic rats 4 weeks after the induction of diabetes mellitus compared with control rats, and retinoic acid treatment markedly decreased both proteinuria and urinary ACR (proteinuria: 1.25+/-0.69 vs 0.78+/-0.72 mg/mgCr, P=0.056; urinary ACR: 0.47+/-0.25 vs 0.21+/-0.06 mg/mgCr, P=0.088). Urinary excretion of MCP-1 was rapidly increased 2 days after induction of diabetes mellitus in diabetic rats, and further increased until rats were 4 weeks of age, compared with control rats. Retinoic acid treatment resulted in 30% reduction of the urinary level of MCP-1 compared with vehicle-treated diabetic rats (119.3+/-74.2 vs 78.1+/-62.7 pg/mgCr, P=0.078). Immunohistochemistry revealed a significant increase in staining for MCP-1 and anti-monocyte/macrophage (ED-1) protein in the diabetic kidney, and retinoic acid treatment significantly suppressed intrarenal MCP-1 and ED-1 protein synthesis. In conclusion, podocytes are involved in the inflammatory reaction under diabetic circumstances, and these reactions were suppressed by retinoic acid. Retinoic acid also suppressed inflammatory changes in the diabetic rat kidney, and decreased proteinuria in diabetic rats. These results suggest that retinoic acid may have renoprotective effects in the early stages of diabetic nephropathy through an anti-inflammatory activity.

Development of a nasal vaccine for chronic hepatitis B infection that uses the ability of hepatitis B core antigen to stimulate a strong Th1 response against hepatitis B surface antigen.

Abstract: There are estimated to be 350 million chronic carriers of hepatitis B infection worldwide. Patients with chronic hepatitis B are at risk of liver cirrhosis with associated mortality because of hepatocellular carcinoma and other complications. An important goal, therefore, is the development of an effective therapeutic vaccine against chronic hepatitis B virus (HBV). A major barrier to the development of such a vaccine is the impaired immune response to HBV antigens observed in the T cells of affected patients. One strategy to overcome these barriers is to activate mucosal T cells through the use of nasal vaccination because this may overcome the systemic immune downregulation that results from HBV infection. In addition, it may be beneficial to present additional HBV epitopes beyond those contained in the traditional hepatitis B surface antigen (HbsAg) vaccine, for example, by using the hepatitis B core antigen (HBcAg). This is advantageous because HBcAg has a unique ability to act as a potent Th1 adjuvant to HbsAg, while also serving as an immunogenic target. In this study we describe the effect of coadministration of HBsAg and HBcAg as part of a strategy to develop a more potent and effective HBV therapeutic vaccine.

Novel adjuvant based on a proteoliposome-derived cochleate structure containing native lipopolysaccharide as a pathogen-associated molecular pattern.

Abstract: Proteoliposomes (PL) from Neisseria meningitidis B have been widely used as a core antigen for antimeningococcal vaccination. PL contain major outer membrane proteins, LPS and phospholipids, and they induce a strong Th1 immune response, but they have low stability in solution. Attending to the need for new vaccine adjuvants, we developed a highly stable cochleate structure (CS) from PL using a technology that allows easy incorporation of new antigens. We explored the ability of PLCS to activate the immune system and its possible application as an adjuvant for parenteral and mucosal routes. Our results showed that PLCS were able to upregulate the expression of MHC class II and costimulatory molecules on human dendritic cells, as well as being able to stimulate the production of soluble mediators of a Th1 response, such as IL-12 and nitric oxide. High levels of anti-PL IgG were detected in serum after i.m. or mucosal (oral and nasal) administration, but also anti-PL secretory IgA was produced in saliva following nasal delivery. The immune response polarization to a Th1 pattern was confirmed by the induction of IgG2a antibodies, positive delayed type hypersensitivity reactions, and IFN-gamma production by splenocytes from immunized mice. The adjuvant potential was explored using PLCS containing ovalbumin (Ova). PLCS-Ova was able to elicit a substantial increase in anti-Ova IgG compared with Ova alone. In addition, a significant reduction in lesion size was observed in mice immunized with Leishmania major antigens in PLCS after challenge with virulent protozoa, suggesting at least partial modulation of the Th2 environment induced by this parasite. In conclusion, our results support the use of PLCS as a potent Th1 adjuvant for parenteral and mucosal vaccines.

Regulation of antitumour immunity by CD1d-restricted NKT cells.

Abstract: An understanding of the complex interactions occurring between tumours and the immune system is a prerequisite for the rational design of effective cancer immunotherapies. To date, attention has focused mainly on the role the adaptive immune system plays in controlling tumourigenesis, with conventional T cells, which recognize peptide antigens presented by classical MHC molecules, coming under close scrutiny. Accumulating reports now suggest that an additional T-cell subset, known as CD1d-restricted natural killer T (NKT) cells, also plays a pivotal role in modulating antitumour responses. Found in both humans and mice, CD1d-restricted NKT cells are a highly specialized cell type that, in contrast to conventional T cells, recognize lipid/glycolipid antigens presented by the non-classical MHC molecule CD1d. Several features of NKT cells, including their ability to rapidly produce large quantities of cytokines upon primary stimulation, make them ideal targets for developing anticancer immunotherapies. This intriguing cell type is the focus of this review.

Cytokines as adjuvants for avian vaccines.

Abstract: The worldwide trend towards a reduced reliance on in-feed antibiotics has increased the pressure to develop alternative strategies to manage infectious diseases in poultry. With this in mind, there is a great emphasis on vaccine use and the enhancement of existing vaccines to provide long-term protection. Currently existing adjuvants for poultry can have deleterious side-effects, such as inflammation, resulting in the down-grading of meat quality and a subsequent reduction in profits. Therefore, to enhance the use of vaccination, alternative adjuvants must be developed. The use of recombinant cytokines as adjuvants in poultry is attracting considerable attention, and their potential role as such has been addressed by several studies. The recent identification of a number of chicken cytokine genes has provided the possibility to study their effectiveness in enhancing the immune response during infection and vaccination. This review focuses on the recent studies involving the assessment of cytokines as vaccine adjuvants.

Natural killer T cells as targets for immunotherapy of autoimmune diseases

Abstract: CD1d-restricted natural killer T (NKT) cells are innate lymphocytes that play a regulatory role during an immune response. The identification of alpha-galactosylceramide (alpha-GalCer), a marine sponge-derived glycosphingolipid, as a potent stimulator of NKT cells led many laboratories to investigate the effects of NKT cell activation on the regulation of immune responses. These studies revealed that alpha-GalCer induces rapid and robust cytokine production by NKT cells, secondary activation of a variety of innate and adaptive immune cells, and modulation of Th cell responses. Further, alpha-GalCer influences disease progression in a variety of experimental models of autoimmunity and inflammation in mice, including models for type 1 diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, and atherosclerosis. While these studies have raised significant enthusiasm for manipulation of NKT cells as a means of preventing autoimmunity in the clinical setting, there are significant concerns regarding the safety of repeated alpha-GalCer injections in human subjects.

Prostanoids and their receptors that modulate dendritic cell-mediated immunity.

Abstract: Dendritic cells (DC) are essential for the initiation of immune responses by capturing, processing and presenting antigens to T cells. In addition to their important role as professional APC, they are able to produce immunosuppressive and pro-inflammatory prostanoids from arachidonic acid (AA) by the action of cyclooxygenase (COX) enzymes. In an autocrine and paracrine fashion, the secreted lipid mediators subsequently modulate the maturation, cytokine production, Th-cell polarizing ability, chemokine receptor expression, migration, and apoptosis of these extremely versatile APC. The biological actions of prostanoids, including their effects on APC-mediated immunity and acute inflammatory responses, are exerted by G protein-coupled receptors on plasma membrane. Some COX metabolites act as anti-inflammatory lipid mediators by binding to nuclear receptors and modulating DC functions. Although the role of cytokines in DC function has been studied extensively, the effects of prostanoids on DC biology have only recently become the focus of investigation. This review summarizes the current knowledge about the role of prostanoids and their receptors in modulating DC function and the subsequent immune responses.

Some Scaling Principles for the Immune System

Abstract: Using recent progress in biological scaling, we explore the way in which the immune system of an animal scales with its mass (M). It is shown that the number of cells in a single clone of B cells should scale as M and that the B-cell repertoire scales as ln (cM), where c is a constant. The time that a B cell needs to circulate once through the organism is shown to scale as M(1/4)ln (cM). It is suggested that the scaling of other cell populations in the immune system could be derived from these scaling relations for B cells.

T cell activation: in vivo veritas.

Abstract: Phenotypic changes in CD4 + T cells undergoing antigen-dependent activation were compared in vivo and in vitro. The most obvious difference was in expression of CD25, the alpha chain of the high affinity receptor for IL-2. High level expression of CD25 in vivo is restricted to a small fraction of the cells at the leading edge of the cell division profile, whereas all activated cells express high levels of CD25 in cultures responding to antigen. Because IL-2 is known to upregulate expression of CD25 in preactivated T cells, this suggests a difference in IL-2 exposure in the two responses. A number of other markers, including CD54, show a similar difference in the pattern of expression in vivo and in vitro. Using 6-colour flow cytometry, it was demonstrated that the small percentage of cells expressing CD25 in vivo coexpresses a very high level of a number of other activation markers, including CD38, CD44 and Ly-6A/E, suggesting that these may also be upregulated by autocrine IL-2.

Human DNA polymerase‐η, an A‐T mutator in somatic hypermutation of rearranged immunoglobulin genes, is a reverse transcriptase

Abstract: We have proposed previously that error-prone reverse transcription using pre-mRNA of rearranged immunoglobulin variable (IgV) regions as templates is involved in the antibody diversifying mechanism of somatic hypermutation (SHM). As patients deficient in DNA polymerase- exhibit an abnormal spectrum of SHM, we postulated that this recently discovered Y-family polymerase is a reverse transcriptase (RT). This possibility was tested using a product-enhanced RT (PERT) assay that uses a real time PCR step with a fluorescent probe to detect cDNA products of at least 27-37 nucleotides. Human pol- and two other Y-family enzymes that are dispensable for SHM, human pols- and -, copied a heteropolymeric DNA-primed RNA template in vitro under conditions with substantial excesses of template. Repeated experiments gave highly reproducible results. The RT activity detected using one aliquot of human pol- was confirmed using a second sample from an independent source. Human DNA pols- and -, and T4 DNA polymerase repeatedly demonstrated no RT activity. Pol- was the most efficient RT of the Y-family enzymes assayed but was much less efficient than an HIV-RT standard in vitro. It is thus feasible that pol- acts as both a RNA- and a DNA-dependent DNA polymerase in SHM in vivo, and that Y-family RT activity participates in other mechanisms of physiological importance.

Systemic NKT cell deficiency in NOD mice is not detected in peripheral blood: implications for human studies.

Abstract: In the diabetes-prone NOD mouse, there is a proven association between a systemic deficiency of NKT cells and the onset of type 1 diabetes. Numerous reports of similar defects within the NKT cell compartment of human type 1 diabetes patients suggested NKT cell levels might be a valuable predictor of susceptibility and could provide a target for therapeutic intervention. Two recent studies, however, found no association between type 1 diabetes and blood NKT cell levels in humans and consequently rejected a link between the onset of diabetes and NKT cell deficiency. This cast considerable doubts on the potential for NKT cell-based clinical applications and challenged the validity of the NOD mouse as a model of human type 1 diabetes. We now report that NKT cell levels in blood are a poor representation of those in other organs. Strikingly, systemic NKT cell deficiencies were identified in NOD mice with normal, or even raised, blood levels. This re-establishes the correlation between NKT cell deficiency and type 1 diabetes and raises important questions regarding the assaying of NKT cell levels in humans.

Mycobacterium tuberculosis induces high production of nitric oxide in coordination with production of tumour necrosis factor-alpha in patients with fresh active tuberculosis but not in MDR tuberculosis.

Abstract: Mycobacterium tuberculosis is an intracellular pathogen that readily survives and replicates in human macrophages. Host cells have developed various mycobactericidal and immunoregulatory mechanisms, such as the production of nitric oxide and inflammatory cytokines to control intracellular replication of M. tuberculosis. Inducible nitric oxide synthase (iNOS) is transcriptionally under the control of IFN-gamma and TNF-alpha. IL-12 provides a crucial link between activated mononuclear phagocytes and T cells by regulating the production of IFN-gamma. In this study, we investigated the production of nitric oxide (NO), TNF-alpha and IL-12 by the peripheral blood monocytes (PB Mn) of patients suffering from multidrug-resistant tuberculosis (MDR-TB). The cells were infected with M. tuberculosis and stimulated with IFN-gamma or activated with mycobacterial subcellular components. The results were compared with those from cases of newly diagnosed TB and healthy controls. Nitric oxide production was significantly depressed in PB Mn from MDR-TB patients. Infected monocytes from newly diagnosed TB patients produced significantly higher levels of NO as compared to those from MDR-TB patients or normal controls. The subcellular fraction of M. tuberculosis-like whole cell lysate (WCL), culture filtrate protein (CFP) and lipoarabinomannan (LAM) induced higher concentrations of NO release in PB Mn from newly diagnosed TB patients as compared to those from MDR-TB patients. Cell culture supernatant from PB Mn assayed at 48 h after infection or stimulation demonstrated significantly depressed release of TNF-alpha and IL-12 from MDR-TB cases as compared to the fresh cases. We observed a definite correlation between nitric oxide release and TNF-alpha production, irrespective of low or high production in MDR-TB or fresh cases, respectively. The present data suggest that peripheral blood monocytes of MDR-TB patients typically show signs of immunosuppression. Whether such immunodepression is the cause or the effect of MDR-TB merits further investigation.

Adjuvant synergy: The effects of nasal coadministration of adjuvants

Abstract: Modern peptide and protein subunit vaccines suffer from poor immunogenicity and require the use of adjuvants. However, none of the currently licensed adjuvants can elicit cell-mediated immunity or are suitable for mucosal immunization. In this study we explored the immunological effect of nasal co-administration of adjuvants with distinct functions: cholera toxin subunit B, a potent mucosal adjuvant that induces strong humoral responses, muramy di-peptide (MDP), an adjuvant known to elicit cell mediated immunity but rarely used nasally, and chitosan, an adjuvant that achieves specific physiological effects on mucosal membranes that improve antigen uptake. Groups of five female BALB/c mice received on days 1 and 56 nasal instillations of the recombinant Helicobacter pylori antigen urease admixed to single or multiple adjuvant combinations. Serum IgG kinetics were followed over 24 weeks. At the conclusion of the experiment, local antibody responses were determined and antigen-specific recall responses in splenocyte cultures were assayed for proliferation and cytokine production. The combination of adjuvants was shown to further contribute to the increased antigenicity of recombinant H. pylori urease. The data presented here outline and support facilitation of increased immunomodulation by an adjuvant previously defined as an effective mucosal adjuvant (chitosan) for another adjuvant (MDP) that is not normally effective via this route.

Parallels and distinctions between T and NKT cell development in the thymus.

Abstract: NKT cells are emerging as an extremely influential regulatory subset of T lymphocytes that are functionally and developmentally distinct from their mainstream counterparts. Like other T cells, NKT cells are thymus-dependent but their apparently unique pathway of differentiation is poorly characterized. Given the strong association between NKT cell deficiency and increased incidences of autoimmunity and cancer it is imperative that the mechanisms by which NKT cells are generated becomes better understood. This review examines what is known about NKT cell development in the thymus and highlights elements of the pathway that differ significantly from mainstream T-cell development. It is here that NKT cell-specific disorders may originate and may best be addressed.

The CD11/CD18 (β 2 ) integrins modulate neutrophil caspase activation and survival following TNF-α or endotoxin induced transendothelial migration

Abstract: The CD11/CD18 (β 2 ) integrins modulate neutrophil caspase activation and survival following TNF-α or endotoxin induced transendothelial migration

Double-stranded RNA enhances the expression of galectin-9 in vascular endothelial cells.

Abstract: Treatment of cells with double-stranded RNA (dsRNA) in vitro mimics viral infection and regulates expression of various genes. We addressed the mechanisms of leucocyte traffic across the vascular endothelium induced by dsRNA. The present study focused on the expression of galectin-9, which is one of key molecules in the regulation of the interaction between vascular wall and white blood cells. Human umbilical vein endothelial cells (HUVEC) in culture were treated with polyinosinic-polycytidylic acid (poly IC), and expression of mRNA and protein of galectin-9 was analysed by reverse transcription polymerase-chain reaction (RT-PCR) and Western blotting. Poly IC enhanced the expression of galectin-9 mRNA and protein in concentration- and time-dependent manners. This effect of poly IC was almost completely suppressed by the pretreatment with 2-aminopurine, an inhibitor of dsRNA-dependent kinase. Poly IC treatment of HUVEC also enhanced the adherence of EoL-1 cells to the cells, which was inhibited by co-treatment with lactose. We conclude that poly IC upregulates galectin-9 expression in the vascular endothelium and this may explain part of the mechanism for leucocyte traffic through the vascular wall.

Exocytosis and Fas mediated cytolytic mechanisms exert protection from West Nile virus induced encephalitis in mice.

Abstract: Infection of mice with the flaviviruses West Nile virus (WNV) and Murray Valley encephalitis (MVE) induces cytolytic T-cell responses which are highly cross-reactive on target cells infected with heterologous flaviviruses. Of C57BL/6 mice infected with low doses (102-106 PFU) of either virus, 30-40% develop encephalitis and die within 10-12 days1, 2. Mice with defects in the Fas or granule exocytosis (perforin and granzymes A and B) pathway of cellular cytotoxicity display reduced mortality and increased survival time when infected with MVE and are protected from encephalitis when deficient in both pathways. This contrasts with infection with WNV where defects in these cytolytic mechanisms increase the percentage of mice that succumb to encephalitis. Thus, no generalizations as to protective or detrimental effects of cytolytic effector functions in recovery from closely related flavivirus infections can be made. Virus-host immune interactions have to be assessed individually and cannot be generalized.