Showing papers in "Clinical and Experimental Immunology in 2010"

The ‘hygiene hypothesis’ for autoimmune and allergic diseases: an update

Abstract: According to the 'hygiene hypothesis', the decreasing incidence of infections in western countries and more recently in developing countries is at the origin of the increasing incidence of both autoimmune and allergic diseases. The hygiene hypothesis is based upon epidemiological data, particularly migration studies, showing that subjects migrating from a low-incidence to a high-incidence country acquire the immune disorders with a high incidence at the first generation. However, these data and others showing a correlation between high disease incidence and high socio-economic level do not prove a causal link between infections and immune disorders. Proof of principle of the hygiene hypothesis is brought by animal models and to a lesser degree by intervention trials in humans. Underlying mechanisms are multiple and complex. They include decreased consumption of homeostatic factors and immunoregulation, involving various regulatory T cell subsets and Toll-like receptor stimulation. These mechanisms could originate, to some extent, from changes in microbiota caused by changes in lifestyle, particularly in inflammatory bowel diseases. Taken together, these data open new therapeutic perspectives in the prevention of autoimmune and allergic diseases.

T cells in multiple sclerosis and experimental autoimmune encephalomyelitis

Abstract: Multiple sclerosis (MS) is a demyelinating inflammatory disorder of the central nervous system (CNS), which involves autoimmune responses to myelin antigens. Studies in experimental autoimmune encephalomyelitis (EAE), an animal model for MS, have provided convincing evidence that T cells specific for self-antigens mediate pathology in these diseases. Until recently, T helper type 1 (Th1) cells were thought to be the main effector T cells responsible for the autoimmune inflammation. However more recent studies have highlighted an important pathogenic role for CD4(+) T cells that secrete interleukin (IL)-17, termed Th17, but also IL-17-secreting γδ T cells in EAE as well as other autoimmune and chronic inflammatory conditions. This has prompted intensive study of the induction, function and regulation of IL-17-producing T cells in MS and EAE. In this paper, we review the contribution of Th1, Th17, γδ, CD8(+) and regulatory T cells as well as the possible development of new therapeutic approaches for MS based on manipulating these T cell subtypes.

Reference values for B cell subpopulations from infancy to adulthood

Abstract: The composition of the peripheral blood lymphocyte compartment underlies developmental changes during ontogeny. Recently, several new B cell populations have been characterized which were suggested to develop in an age-dependent manner. However, age-dependent reference values for distinct B cell populations have rarely been reported. Therefore, we have characterized developmental changes in peripheral B cell populations from infancy to adulthood in order to define age-dependent reference values. Using a flow cytometric approach we analysed the frequencies as well as the absolute counts of naive, switched and non-switched memory B cells, CD27-negative memory B cells, transitional B cells as well as CD21(low) CD38(low) B cells from neonates up to the age of 50 years. Most of the B cell subsets showed age-dependent developmental changes: while the peripheral B cell pool during infancy is characterized predominantly by transitional and naive B cells, the fraction of switched and non-switched memory B cells increases gradually with age. CD21(low) CD38(low) B cells as well as plasmablasts do not exhibit developmental changes. In summary, we could demonstrate particular changes in the peripheral blood B cell compartment during ontogeny. This study provides reference values of different B cell subpopulations offering comparability for studies addressing disturbed peripheral B cell development in immunodeficiency, autoimmunity or B cell reconstitution following cell-depleting therapies.

Translational mini-review series on Th17 cells: function and regulation of human T helper 17 cells in health and disease.

Abstract: T helper (Th) cell have a central role in modulating immune responses. While Th1 and Th2 cells have long been known to regulate cellular and humoral immunity, Th17 cells have been identified only recently as a Th lineage that regulates inflammation via production of distinct cytokines such as interleukin (IL)-17. There is growing evidence that Th17 cells are pathological in many human diseases, leading to intense interest in defining their origins, functions and developing strategies to block their pathological effects. The cytokines that regulate Th17 differentiation have been the focus of much debate, due primarily to inconsistent findings from studies in humans. Evidence from human disease suggests that their in vivo development is driven by specialized antigen-presenting cells. Knowledge of how Th17 cells interact with other immune cells is limited, but recent data suggest that Th17 cells may not be subject to strict cellular regulation by T regulatory cells. Notably, Th17 cells and T regulatory cells appear to share common developmental pathways and both cell types retain significant plasticity. Herein, we will discuss the molecular and cellular regulation of Th17 cells with an emphasis on studies in humans.

How tumour necrosis factor blockers interfere with tuberculosis immunity.

Abstract: Tumour necrosis factor (TNF) is a potent inflammatory cytokine that plays an important role in immunity to numerous bacterial infections, including Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) in humans. Infliximab, adalimumab, certolizumab pegol and etanercept are anti-TNF agents used to treat a range of inflammatory/autoimmune diseases, such as rheumatoid arthritis. The use of some of these drugs has been linked to reactivation TB. In addition to blocking TNF-mediated immune responses, some anti-TNF drugs have been found to interfere with innate immune responses, such as phagolysosomal maturation and monocyte apoptosis, as well as cell-mediated responses, including interferon-gamma secretion by memory T cells, complement-mediated lysis of Mtb-reactive CD8+ T cells and increased regulatory T cell activity. This review summarizes some of the reported effects of TNF blockers on immune cell responses in the context of the observed clinical data on TB reactivation in patients on anti-TNF therapy.

In vivo manipulation of Vgamma9Vdelta2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients.

Abstract: The potent anti-tumour activities of gammadelta T cells have prompted the development of protocols in which gammadelta-agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vgamma9Vdelta2 T cell agonist, plus low-dose interleukin (IL)-2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vgamma9Vdelta2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vgamma9Vdelta2 T cell numbers emerged, as seven patients who failed to sustain Vgamma9Vdelta2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vgamma9Vdelta2 cell populations showed declining CA15-3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vgamma9Vdelta2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low-dose IL-2 offers a novel, safe and feasible approach to enhance this in a subset of treatment-refractory patients with advanced breast cancer.

99th Dahlem Conference on Infection, Inflammation and Chronic Inflammatory Disorders: Darwinian medicine and the ‘hygiene’ or ‘old friends’ hypothesis

Abstract: The current synthesis of the 'hygiene hypothesis' suggests that the recent increase in chronic inflammatory disorders is at least partly attributable to immunodysregulation resulting from lack of exposure to microorganisms that have evolved an essential role in the establishment of the immune system. This document provides a background for discussion of the following propositions. 1. The essential role of these organisms is an example of 'evolved dependence'. 2. The most relevant organisms are those that co-evolved with mammals, and already accompanied early hominids in the Paleolithic. 3. More recently evolved 'childhood infections' are not likely to have evolved this role, and recent epidemiology supports this contention. 4. This mechanism is interacting with other modern environmental changes that also lead to enhanced inflammatory responses [inappropriate diet, obesity, psychological stress, vitamin D deficiency, pollution (dioxins), etc.]. 5. The range of chronic inflammatory disorders that is affected is potentially larger than usually assumed [allergies, autoimmunity, inflammatory bowel disease, but also vascular disease, some cancers, depression/anxiety (when accompanied by raised inflammatory cytokines), and perhaps neurodegenerative disorders and type 2 diabetes].

Inflammatory conditions affect gene expression and function of human adipose tissue-derived mesenchymal stem cells.

Abstract: There is emerging interest in the application of mesenchymal stem cells (MSC) for the prevention and treatment of autoimmune diseases, graft-versus-host disease and allograft rejection. It is, however, unknown how inflammatory conditions affect phenotype and function of MSC. Adipose tissue-derived mesenchymal stem cells (ASC) were cultured with alloactivated peripheral blood mononuclear cells (PBMC) (mixed lymphocyte reaction: MLR), with proinflammatory cytokines [interferon (IFN)-γ, tumour necrosis factor (TNF)-α and interleukin (IL)-6] or under control conditions, and their full genome expression and function examined. Proinflammatory cytokines mainly increased indoleamine-2,3-dioxygenase expression, whereas ASC cultured with MLR showed increased expression of COX-2, involved in prostaglandin E(2) production. Both conditions had a stimulatory, but differential, effect on the expression of proinflammatory cytokines and chemokines, while the expression of fibrotic factors was decreased only in response to proinflammatory cytokines. Functional analysis demonstrated that inflammatory conditions affected morphology and proliferation of ASC, while their differentiation capacity and production of trophic factors was unaffected. The immunosuppressive capacity of ASC was enhanced strongly under inflammatory conditions. In conclusion, ASC showed enhanced immunosuppressive capacity under inflammatory conditions, while their differentiation capacity was preserved. Therefore, in vitro preconditioning provides ASC with improved properties for immediate clinical immune therapy.

Cyclophilin–CD147 interactions: a new target for anti‐inflammatory therapeutics

Abstract: CD147 is a widely expressed plasma membrane protein that has been implicated in a variety of physiological and pathological activities. It is best known for its ability to function as extracellular matrix metalloproteinase inducer (hence the other name for this protein, EMMPRIN), but has also been shown to regulate lymphocyte responsiveness, monocarboxylate transporter expression and spermatogenesis. These functions reflect multiple interacting partners of CD147. Among these CD147-interacting proteins cyclophilins represent a particularly interesting class, both in terms of structural considerations and potential medical implications. CD147 has been shown to function as a signalling receptor for extracellular cyclophilins A and B and to mediate chemotactic activity of cyclophilins towards a variety of immune cells. Recent studies using in vitro and in vivo models have demonstrated a role for cyclophilin-CD147 interactions in the regulation of inflammatory responses in a number of diseases, including acute lung inflammation, rheumatoid arthritis and cardiovascular disease. Agents targeting either CD147 or cyclophilin activity showed significant anti-inflammatory effects in experimental models, suggesting CD147-cyclophilin interactions may be a good target for new anti-inflammatory therapeutics. Here, we review the recent literature on different aspects of cyclophilin-CD147 interactions and their role in inflammatory diseases.

Gut hormones: emerging role in immune activation and inflammation

Abstract: Gut inflammation is characterized by mucosal recruitment of activated cells from both the innate and adaptive immune systems. In addition to immune cells, inflammation in the gut is associated with an alteration in enteric endocrine cells and various biologically active compounds produced by these cells. Although the change in enteric endocrine cells or their products is considered to be important in regulating gut physiology (motility and secretion), it is not clear whether the change plays any role in immune activation and in the regulation of gut inflammation. Due to the strategic location of enteric endocrine cells in gut mucosa, these gut hormones may play an important role in immune activation and promotion of inflammation in the gut. This review addresses the research on the interface between immune and endocrine systems in gastrointestinal (GI) pathophysiology, specifically in the context of two major products of enteric endocrine systems, namely serotonin (5-hydroxytryptamine: 5-HT) and chromogranins (Cgs), in relation to immune activation and generation of inflammation. The studies reviewed in this paper demonstrate that 5-HT activates the immune cells to produce proinflammatory mediators and by manipulating the 5-HT system it is possible to modulate gut inflammation. In the case of Cgs the scenario is more complex, as this hormone has been shown to play both proinflammatory and anti-inflammatory functions. It is also possible that interaction between 5-HT and Cgs may play a role in the modulation of immune and inflammatory responses. In addition to enhancing our understanding of immunoendocrine interaction in the gut, the data generated from the these studies may have implications in understanding the role of gut hormone in the pathogenesis of both GI and non-GI inflammatory diseases which may lead ultimately to improved therapeutic strategies in inflammatory disorders.

Various effects of different probiotic strains in allergic disorders: an update from laboratory and clinical data

Abstract: Epidemiological data show that allergic children have a different intestinal flora from healthy children with higher levels of Clostridia and lower levels of Bifidobacteria. Nonetheless, Bifidobacteria and Lactobacilli are found more commonly in the composition of the intestinal flora of non-allergic children. Probiotics are ingested live microbes that can modify intestinal microbial populations in a way that benefits the host and they are represented mainly by Lactobacilli. Enhanced presence of probiotic bacteria in the intestinal microbiota is found to correlate with protection against atopy. There is also very promising evidence to recommend the addition of probiotics to foods for the prevention and treatment of allergic diseases. Clinical improvement, especially in allergic rhinitis and immunoglobulin (Ig)E-sensitized (atopic) eczema, has been reported in most of the published studies. However, clinical benefit of probiotic therapy depends upon numerous factors, such as type of bacterium, dosing regimen, delivery method and other underlying host factors, e.g. the age and diet of the host. Selection of the most beneficial probiotic strain, the dose and the timing of supplementation still need to be determined. This review helps understanding of the role of probiotics in various allergic diseases, explaining laboratory and clinical data in light of recent literature.

Islet inflammation and CXCL10 in recent-onset type 1 diabetes.

Abstract: Type 1 diabetes results from a T cell-mediated destruction of insulin-producing pancreatic beta cells. Little is known on local factors contributing to migration of T cells to pancreatic tissue. We recently demonstrated evidence of viral infection in beta cells in several recent-onset type 1 diabetes patients. Islet inflammation was analysed in a series of new- or recent-onset type 1 diabetic patients and non-diabetic control subjects. Autoimmune T cell reactivity was studied in lymphocytes derived from pancreas-draining lymph nodes of one recent-onset type 1 diabetes patient in partial clinical remission. Insulitic lesions were characterized by presence of beta cells, elevated levels of the chemokine CXCL10 and infiltration of lymphocytes expressing the corresponding chemokine receptor CXCR3 in all pancreatic lesions of type 1 diabetes patients, regardless of enterovirus infection of beta cells. CXCR3 and CXCL10 were undetectable in pancreata of non-diabetic control subjects. T cells isolated from draining lymph nodes of a recent-onset patient with virally infected beta cells and in clinical remission reacted with multiple islet autoantigens and displayed a mixed interferon (IFN)-gamma/interleukin (IL)-10 cytokine pattern. Our data point to CXCL10 as an important cytokine in distressed islets that may contribute to inflammation leading to insulitis and beta cell destruction, regardless of local viral infection. We demonstrate further pro- and anti-inflammatory islet autoreactivity, indicating that different adaptive and innate immune responses may contribute to insulitis and beta cell destruction.

CD23/FcεRII: molecular multi-tasking

Abstract: CD23 is the low-affinity receptor for immunoglobulin (Ig)E and plays important roles in the regulation of IgE responses. CD23 can be cleaved from cell surfaces to yield a range of soluble CD23 (sCD23) proteins that have pleiotropic cytokine-like activities. The regions of CD23 responsible for interaction with many of its known ligands, including IgE, CD21, major histocompatibility complex (MHC) class II and integrins, have been identified and help to explain the structure-function relationships within the CD23 protein. Translational studies of CD23 underline its credibility as a target for therapeutic intervention strategies and illustrate its involvement in mediating therapeutic effects of antibodies directed at other targets.

Role of inflammatory cells, cytokines and matrix metalloproteinases in neutrophil-mediated skin diseases.

Abstract: Pyoderma gangrenosum (PG) is a rare, immune-mediated inflammatory skin disease presenting with painful ulcers having undermined edges. Less commonly, bullous and vegetative variants exist. Histology consists of a neutrophil-rich dermal infiltrate. We characterized immunohistochemically the infiltrate in different variants of PG and in another neutrophilic dermatosis as Sweet's syndrome. We studied 21 patients with PG, eight with Sweet's syndrome and 20 controls, evaluating skin immunoreactivity for inflammatory cell markers (CD3, CD163 and myeloperoxidase), cytokines [tumour necrosis factor (TNF)-α, interleukin (IL)-8 and IL-17], metalloproteinases (MMP-2 and MMP-9) and vascular endothelial growth factor (VEGF). Immunoreactivities of CD3, CD163, myeloperoxidase, TNF-α, IL-8, IL-17, MMP-2, MMP-9 and VEGF were significantly higher in both PG and Sweet's syndrome than in controls (P=0·0001). Myeloperoxidase (neutrophil marker), IL-8 (cytokine chemotactic for neutrophils) and MMP-9 (proteinase-mediating tissue damage) were expressed more significantly in both ulcerative and bullous PG than in vegetative PG as well as in Sweet's syndrome (P=0·008-P=0·0001). In ulcerative PG, the expression of CD3 (panT cell marker) and CD163 (macrophage marker) were significantly higher in wound edge than wound bed (P=0·0001). In contrast, the neutrophil marker myeloperoxidase was expressed more significantly in wound bed than wound edge (P=0·0001). Our study identifies PG as a paradigm of neutrophil-mediated inflammation, with proinflammatory cytokines/chemokines and MMPs acting as important effectors for the tissue damage, particularly in ulcerative and bullous PG where damage is stronger. In ulcerative PG, the wound bed is the site of neutrophil-recruitment, whereas in the wound edge activated T lymphocytes and macrophages pave the way to ulcer formation.

The T helper type 17/regulatory T cell imbalance in patients with acute Kawasaki disease

Abstract: The study is designed to investigate the changes and roles of T helper type 17/regulatory T cells (Th17/Treg) in the immunological pathogenesis of Kawasaki disease (KD) In addition, we explore the alteration and significance of Th17 cells in patients with intravenous immune globulin-resistant KD Real-time polymerase chain reaction (PCR) was used to evaluate the mRNA levels of interleukin (IL)-17A/F, retinoic acid-related orphan receptor (ROR)-γt and forkhead box P3 (FoxP3) in CD4-positive cells The proportions of Th17 cells and CD4+CD25+FoxP3high Tregs were analysed by flow cytometry Plasma cytokine [IL-17A, IL-6, IL-23 and transforming growth factor (TGF)-β] concentrations were measured by sandwich enzyme-linked immunosorbent assay Our data demonstrate that Th17 proportions and expression levels of cytokines (IL-17, IL-6 and IL-23) and transcription factors (IL-17A/F, ROR-γt) were up-regulated significantly, while Treg proportions and expression levels of Tregtranscription factor (FoxP3) were down-regulated significantly in children with acute KD (P < 0·01) Compared with the sensitive group, the Th17 proportions were up-regulated significantly during the acute phase in immune globulin-resistant KD (P < 0·01) The plasma IL-17A, IL-6 and IL-23 concentrations in patients with KD were significantly higher compared with the concentrations in normal controls (NC) and infectious disease (ID) Plasma TGF-β concentrations were markedly lower in the KD group than the NC and ID groups (P < 0·05) These results suggest that Th17/Treg cells imbalance exists in the patients with KD Th17/T cells imbalance may be important factors causing disturbed immunological function and resulting in immunoglobulin-resistant KD

Translational Mini-Review Series on Th17 Cells: Induction of interleukin-17 production by regulatory T cells

Abstract: Uncommitted (naive) CD4(+) T helper cells (Thp) can be induced to differentiate to specific lineages according to the local cytokine milieu, towards T helper type 1 (Th1), Th2, Th17 and regulatory T cell (T(reg)) phenotypes in a mutually exclusive manner. Each phenotype is characterized by unique signalling pathways and expression of specific transcription factors, notably T-bet for Th1, GATA-3 for Th2, forkhead box P3 (FoxP3) for T(regs) and receptor-related orphan receptor (ROR)alpha and RORgammat for Th17 cells. T(regs) and Th17 cells have been demonstrated to arise from common precursors in a reciprocal manner based on exposure to transforming growth factor (TGF)-beta or TGF-beta plus interleukin (IL)-6 and carry out diametrically opposing functions, namely suppression or propagation of inflammation, respectively. However, while epigenetic modifications in Th1 and Th2 differentiated cells prevents their conversion to other phenotypes, Th17 cells generated in vitro using TGF-beta and IL-6 are unstable and can convert to other phenotypes, especially Th1, both in vitro and in vivo. T(regs) are generated from naive precursors both in the thymus (natural, nT(regs)) and in the periphery (induced, iT(regs)). The highly suppressive function of T(regs) enables them to control many inflammatory diseases in animals and makes them particularly attractive candidates for immunotherapy in humans. The stability of the T(reg) phenotype is therefore of paramount importance in this context. Recent descriptions of T(reg) biology have suggested that components of pathogens or inflammatory mediators may subvert the suppressive function of T(regs) in order to allow propagation of adequate immune responses. Unexpectedly, however, a number of groups have now described conversion of T(regs) to the Th17 phenotype induced by appropriate inflammatory stimuli. These observations are particularly relevant in the context of cell therapy but may also explain some of the dysregulation seen in autoimmune diseases. In this paper, we review T(reg) to Th17 conversion and propose some potential mechanisms for this phenomenon.

Laser microdissection-based analysis of cytokine balance in the kidneys of patients with lupus nephritis.

Abstract: To determine the cytokine balance in patients with lupus nephritis (LN), we analysed kidney-infiltrating T cells Renal biopsy samples from 15 systemic lupus erythematosus (SLE) patients were used In accordance with the classification of International Society of Nephrology/Renal Pathology Society, they were categorized into Class III, Class III+V (Class III-predominant group, n = 4), Class IV, Class IV+V (Class IV-predominant group, n = 7) and Class V (n = 4) groups The single-cell samples of both the glomelular and interstitial infiltrating cells were captured by laser-microdissection The glomerular and interstitial infiltrating T cells produced interleukin (IL)-2, IL-4, IL-10, IL-13 and IL-17 cytokines in the Class III-predominant, Class IV-predominant and Class V groups Interferon-gamma was detected only in the glomeruli of the Class III-predominant and Class V group samples The expression level of IL-17 was correlated closely with clinical parameters such as haematuria, blood urea nitrogen level, SLE Disease Activity Index scores in both glomeruli and interstitium, urine protein level in glomeruli and serum creatinine and creatinine clearance levels in interstitium This suggests that the glomerular infiltrating T cells might act as T helper type 1 (Th1), Th2 and Th17 cells while the interstitial infiltrating T cells, act as Th2 and Th17 cells in the Class III-predominant and Class V groups In contrast, both the glomerular and interstitial infiltrating T cells might act as Th2 and Th17 cells in the Class IV-predominant group The cytokine balances may be dependent upon the classification of renal pathology, and IL-17 might play a critical role in SLE development

Interferon signature gene expression is correlated with autoantibody profiles in patients with incomplete lupus syndromes

Abstract: Interferon (IFN) signature genes have been shown to be expressed highly in peripheral blood of patients with systemic lupus erythematosus (SLE), especially in the presence of active disease. However, the expression of this gene signature in individuals with incomplete forms of lupus and the pathogenic relationship between IFN signature genes and autoantibody production have not been explored fully. In the present study, we examined the gene expression and autoantibody profiles of patients diagnosed with incomplete lupus erythematosus (ILE) to determine correlations of the gene expression signature with autoantibody production. Gene expression analysis was carried out on the 24K Illumina Human Refseq-8 arrays using blood samples from 84 subjects, including patients with SLE (n = 27) or ILE (n = 24), first-degree relatives (FDR) of these patients (n = 22) and non-autoimmune control (NC) individuals (n = 11). Autoantibody expression was measured using standard immunoassays and autoantigen proteomic arrays. Up-regulation of a set of 63 IFN signature genes was seen in 83% of SLE patients and 50% of ILE patients. High levels of IFN gene expression in ILE and SLE showed significant correlations with the expression of a subset of IgG autoantibodies, including chromatin, dsDNA, dsRNA, U1snRNP, Ro/SSA, La/SSB, topoisomerase I and Scl 70, while low IFN levels were correlated with immunoglobulin (Ig)M autoreactivity. These studies suggest that in patients with ILE the IFN gene expression signature may identify a subset of these individuals who are at risk for disease progression. Furthermore, high levels of alpha IFN may promote autoantibody class-switch from IgM to the more pathogenic IgG class.

Calcineurin inhibitors affect B cell antibody responses indirectly by interfering with T cell help

Abstract: In general, humoral immune responses depend critically upon T cell help. In transplantation, prevention or treatment of humoral rejection therefore require drugs that ideally inhibit both B cell and T helper cell activity. Here, we studied the effects of commonly used immunosuppressive drugs [tacrolimus, cyclosporin, mycophenolic acid (MPA) and rapamycin] on T cell helper activity and on T cell-dependent B cell responses. T cells were activated polyclonally in the presence of immunosuppressive drugs in order to analyse the effect of these drugs on T cell proliferation, co-stimulatory ligand expression and cytokines. The impact of immunosuppressive drugs on T cell-dependent immunoglobulin production by B cells was addressed in T-B cell co-cultures. All drugs affected T cell proliferation and attenuated T cell co-stimulatory ligand (CD154 and CD278) expression when T cells were activated polyclonally. Tacrolimus, cyclosporin and rapamycin also attenuated B cell stimulatory cytokine mRNA levels in T cells. As a consequence, a decrease in immunoglobulin levels was observed in autologous T-B cell co-cultures, where T cell help is essential for immunoglobulin production. In contrast, when pre-activated T cells were used to stimulate autologous B cells, calcineurin inhibitors failed to inhibit B cell immunoglobulin production, whereas MPA and rapamycin did show inhibition. From these studies, it is evident that calcineurin inhibitors affect the humoral immune response by interfering with T helper signals, but not by targeting B cells directly. Furthermore, our studies support the necessity of intervening in T cell helper function to attenuate humoral responses.

Recent insights into the role of Toll‐like receptors in viral infection

Abstract: Toll-like receptors (TLRs) have a central role in innate immunity as they detect conserved pathogen-associated molecular patterns (PAMPs) on a range of microbes, including viruses, leading to innate immune activation and orchestration of the adaptive immune response. To date, a large number of viruses have been shown to trigger innate immunity via TLRs, suggesting that these receptors are likely to be important in the outcome to viral infection. This suggestion is supported by the observation that many viruses have evolved mechanisms not only to evade the innate immune system, but also to subvert it for the benefit of the virus. In this review we will discuss earlier evidence, mainly from knock-out mice studies, implicating TLRs in the innate immune response to viruses, in light of more recent clinical data demonstrating that TLRs are important for anti-viral immunity in humans.

Developing combination immunotherapies for type 1 diabetes: recommendations from the ITN–JDRF Type 1 Diabetes Combination Therapy Assessment Group

Abstract: Like many other complex human disorders of unknown aetiology, autoimmune-mediated type 1 diabetes may ultimately be controlled via a therapeutic approach that combines multiple agents, each with differing modes of action. The numerous advantages of such a strategy include the ability to minimize toxicities and realize synergies to enhance and prolong efficacy. The recognition that combinations might offer far-reaching benefits, at a time when few single agents have yet proved themselves in well-powered trials, represents a significant challenge to our ability to conceive and implement rational treatment designs. As a first step in this process, the Immune Tolerance Network, in collaboration with the Juvenile Diabetes Research Foundation, convened a Type 1 Diabetes Combination Therapy Assessment Group, the recommendations of which are discussed in this Perspective paper.

The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease.

Abstract: Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa alpha-chain linked to a 70-kDa beta-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of I kappaB alpha. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17.

Association of functional polymorphisms related to the transcriptional level of FOXP3 with prognosis of autoimmune thyroid diseases.

Abstract: The severity of Hashimoto's disease (HD) and intractability (or inducibility to remission) of Graves' disease (GD) varies among patients. Forkhead box P3 (FoxP3) is a crucial regulatory factor for the development and function of regulatory T (T(reg) ) cells, and deficiency of the FoxP3 gene (FOXP3) suppresses the regulatory function of T(reg) cells. To clarify the association of the functional polymorphisms of the FOXP3 with the prognosis of GD and HD, we genotyped -3499A/G, -3279C/A and -2383C/T polymorphisms in FOXP3 gene obtained from 38 patients with severe HD, 40 patients with mild HD, 65 patients with intractable GD, in whom remission was difficult to induce, 44 patients with GD in remission and 71 healthy volunteers. The -3279CA genotype was more frequent in patients with GD in remission than in patients with intractable GD, and the -3279AA genotype, which correlates to defective transcription of FOXP3, was absent in patients with GD in remission. The -2383CC genotype was more frequent in patients with severe HD than in those with mild HD. In conclusion, the -3279A/C polymorphism is related to the development and intractability of GD and the -2383CC genotype to the severity of HD.

Circulating CD14(+) CD16(+) monocyte levels predict tissue invasive character of cholangiocarcinoma.

Abstract: Chronic inflammation as a risk factor for cancer development is driven in part by monocyte/macrophages, which in many cancers exhibit pro-tumorigenic activity. In this study we identified elevation in CD14(+) CD16(+) , a minor blood monocyte subpopulation in cholangiocarcinoma (CCA) patients, compared to normal and biliary disease patient specimens. Tumour association was suggested by the observation that this elevated level decreased to normal after tumour resection. Moreover, the elevated level of CD14(+) CD16(+) monocytes in CCA patient blood correlated with degree of MAC387-positive (recent blood-derived macrophage migrant-specific marker) tumour-associated macrophage infiltration as determined by immunohistochemistry. These CD14(+) CD16(+) monocytes were suggested to enhance tumour progression as this subpopulation possesses (i) high expression of adhesion molecules (CD11c, CD49d, and CD54) and scavenger receptor (CD163), which enable them to adhere strongly to endothelial cells, and (ii) that peripheral blood monocytes from CCA patients express high levels of growth and angiogenic factor-related genes (epiregulin, VEGF-A and CXCL3). Elevation of peripheral CD14(+) CD16(+) monocyte levels was associated with features associated with poor prognosis CCA parameters (non-papillary type and high number of tissue macrophages). These data indicate that the CD14(+) CD16(+) monocytes from CCA patients with pro-tumorigenic characteristics may associate with rapid tumour progression and poor patient outcome. If confirmed in subsequent studies, the level of CD14(+) CD16(+) monocytes may serve as a marker for disease activity in CCA patients and serve as a target for pathogenic macrophage specific drug development.

CD6 synergistic co-stimulation promoting proinflammatory response is modulated without interfering with the activated leucocyte cell adhesion molecule interaction

Abstract: Summary The CD6 membrane-proximal scavenger receptor cysteine-rich domain (SRCR3) includes the activated leucocyte cell adhesion molecule (ALCAM) binding site CD6-ALCAM mediates a low-affinity interaction and their long- term engagement contributes to the immunological synapse Their ligation may play a dual function, facilitating stable adhesion between the antigen- presenting cells and T cells during the early activation phase and later in the proliferative phase of the immune response This study explored the strength of the CD6 co-stimulatory effect and whether CD6 co-stimulation with its natural ligand ALCAM also contributes to the lymphocyte effector differentiationIt was found that CD6-ALCAM interaction in vitro induced a synergistic co-stimulation of normal human peripheral blood mononuclear cells, defined by Bliss analysis CD6 co-stimulation enhanced the CD3 prolif- erative efficacy by 23-34% Moreover, a fivefold increment in the CD25 mol- ecules number with a distinct gene transcription profile associated with cell activation, differentiation, survival and adhesion molecules was observed over CD3 single activation Additionally, CD6 co-stimulation in excess inter- leukin (IL)-2 promotes a preferentially proinflammatory response Besides, a CD6 membrane-distal domain (SRCR1)-specific non-depleting monoclonal antibody (mAb) inhibited the induced proliferation in the presence of ALCAM, reducing interferon-g, IL-6 and tumour necrosis factor-a production These results suggest that CD6 co-stimulation enhances the intrinsic activity of the CD3 activation pathway and contributes to the T helper type 1 subset commitment, enhancing the IL-2 sensitivity of recent activated human lymphocytes It supports the role of CD6 as a susceptibility gene for pathological autoimmunity leading to tissue inflammation, and its relevance for targeted therapy

Impact of lipoteichoic acid modification on the performance of the probiotic Lactobacillus rhamnosus GG in experimental colitis

Abstract: While some probiotic strains might have adjuvant effects in the therapy for inflammatory bowel diseases (IBD), these effects remain controversial and cannot be generalized. In this study, a dltD mutant of the model probiotic Lactobacillus rhamnosus GG (LGG), having a drastic modification in its lipoteichoic acid (LTA) molecules, was analysed for its effects in an experimental colitis model. Dextran sulphate sodium (DSS) was used to induce either moderate to severe or mild chronic colitis in mice. Mice received either phosphate-buffered saline (PBS), LGG wild-type or the dltD mutant via the drinking water. Macroscopic parameters, histological abnormalities, cytokine and Toll-like receptor (TLR) expression were analysed to assess disease activity. LGG wild-type did not show efficacy in the different experimental colitis set-ups. This wild-type strain even seemed to exacerbate the severity of colitic parameters in the moderate to severe colitis model compared to untreated mice. In contrast, mice treated with the dltD mutant showed an improvement of some colitic parameters compared to LGG wild-type-treated mice in both experimental models. In addition, treatment with the dltD mutant correlated with a significant down-regulation of Toll-like receptor-2 expression and of downstream proinflammatory cytokine expression in the colitic mice. These results show that molecular cell surface characteristics of probiotics are crucial when probiotics are considered for use as supporting therapy in IBD.

Modulation of immune responses through direct activation of Toll-like receptors to T cells

Abstract: Summary Toll-like receptors (TLRs),which are a family of pattern recognition receptors (PRRs), are involved critically in the generation and regulation of innate immunity as well as initiation of subsequent adaptive immune responses However, recent research results showed that different subsets of T cells express certain types of TLRs during development and activation stages Importantly, TLRs participate in the direct regulation of adaptive immune response, possibly as co-stimulatory molecules In this review we summarize recent studies about the novel regulation of TLRs on the homeostasis and immunity of different T cell subtypes including CD4 + CD25 + T regulatory cells (Treg) and interleukin (IL)-17-producing CD4 + T cells (T helper type 17) The direct involvement of TLRs in T cell-mediated immunity prompted us to reconsider the role of TLRs in the occurrence of autoimmune diseases, infec- tious diseases and graft rejection The important effects of TLRs in T cell- intrinsic components also prompt us to explore novel vaccine adjuvants for modifying desired immune responses in an efficient way

Investigating the role of proinflammatory CD16+ monocytes in the pathogenesis of inflammatory bowel disease

Abstract: Summary Infiltrating monocytes and macrophages contribute to the initiation and perpetuation of mucosal inflammation characteristic for human inflammatory bowel disease (IBD). Peripheral blood monocytes expressing the low-affinity Fcγ receptor CD16 have been identified previously as a major proinflammatory cell population, based on their unique cytokine secretion profile. However, the contribution of these cells to the pathogenesis of inflammatory bowel disease remains to be elucidated. Thus, in this study we investigated whether the peripheral CD16+ monocyte count correlates with common IBD disease parameters, and whether these cells infiltrate the intestinal mucosa under inflammatory conditions. We observed that CD16+ peripheral blood monocytes are increased significantly in active Crohn's disease, particularly in patients with high Crohn's disease activity index and colonic involvement. Furthermore, we found that CD16+ cells are a major contributor to the inflammatory infiltrate in Crohn's disease mucosa, although their spontaneous migration through primary human intestinal endothelial cells is limited. Our data suggest that lamina propria, but not peripheral blood, CD16+ monocytes are a crucial proinflammatory cell population in IBD, and a potential target for anti-inflammatory therapy.

Toll‐like receptor 4 expression is increased in circulating mononuclear cells of patients with immunoglobulin A nephropathy

Abstract: We investigated Toll-like receptors (TLR-3, -4 and -7) expression in circulating mononuclear cells of patients with immunoglobulin A nephropathy (IgAN), a disease with debated relationships with mucosal immunity. TLR-4 expression (detected by fluorescence activated cell sorter) and mRNA transcriptional levels (Taqman) were significantly higher in patients with IgAN than in healthy controls (P = 0.00200 and P = 0.0200). TLR-3 and TLR-7 were not modified significantly. In IgAN patients proteinuria was correlated significantly with TLR-4 expression (P = 0.0312). In a group of nephrotic syndromes, TLR-3, -4 and -7 expression was similar to healthy controls. A significant difference in TLR-4 expression and mRNA levels was found between very active IgAN patients (proteinuria > 1 g/1.73 m(2)/day in association with severe microscopic haematuria) and inactive patients (proteinuria < 0.5 g/1.73 m(2)/day, with absent or minimal haematuria). No correlation with levels of aberrantly glycosylated IgA1, age, renal biopsy features or therapy was found. This study shows for the first time an up-regulation of TLR-4 in circulating mononuclear cells of patients with IgAN, particularly in association with proteinuria and heavy microscopic haematuria.

Gene expression profiles for the human pancreas and purified islets in Type 1 diabetes: new findings at clinical onset and in long-standing diabetes

Abstract: Type 1 diabetes (T1D) is caused by the selective destruction of the insulin-producing beta cells of the pancreas by an autoimmune response. Due to ethical and practical difficulties, the features of the destructive process are known from a small number of observations, and transcriptomic data are remarkably missing. Here we report whole genome transcript analysis validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and correlated with immunohistological observations for four T1D pancreases (collected 5 days, 9 months, 8 and 10 years after diagnosis) and for purified islets from two of them. Collectively, the expression profile of immune response and inflammatory genes confirmed the current views on the immunopathogenesis of diabetes and showed similarities with other autoimmune diseases; for example, an interferon signature was detected. The data also supported the concept that the autoimmune process is maintained and balanced partially by regeneration and regulatory pathway activation, e.g. non-classical class I human leucocyte antigen and leucocyte immunoglobulin-like receptor, subfamily B1 (LILRB1). Changes in gene expression in islets were confined mainly to endocrine and neural genes, some of which are T1D autoantigens. By contrast, these islets showed only a few overexpressed immune system genes, among which bioinformatic analysis pointed to chemokine (C-C motif) receptor 5 (CCR5) and chemokine (CXC motif) receptor 4) (CXCR4) chemokine pathway activation. Remarkably, the expression of genes of innate immunity, complement, chemokines, immunoglobulin and regeneration genes was maintained or even increased in the long-standing cases. Transcriptomic data favour the view that T1D is caused by a chronic inflammatory process with a strong participation of innate immunity that progresses in spite of the regulatory and regenerative mechanisms.