Showing papers in "Clinical and Experimental Immunology in 2005"

Macrophage activation switching: an asset for the resolution of inflammation

Abstract: Macrophages play a central role in inflammation and host defence against microorganisms, but they also participate actively in the resolution of inflammation after alternative activation. However, it is not known whether the resolution of inflammation requires alternative activation of new resting monocytes/macrophages or if proinflammatory activated macrophages have the capacity to switch their activation towards anti-inflammation. In order to answer this question, we first characterized differential human macrophage activation phenotypes. We found that CD163 and CD206 exhibited mutually exclusive induction patterns after stimulation by a panel of anti-inflammatory molecules, whereas CCL18 showed a third, overlapping, pattern. Hence, alternative activation is not a single process, but provides a variety of different cell populations. The capacity of macrophages to switch from one activation state to another was then assessed by determining the reversibility of CD163 and CD206 expression and of CCL18 and CCL3 production. We found that every activation state was rapidly and fully reversible, suggesting that a given cell may participate sequentially in both the induction and the resolution of inflammation. These findings may provide new insight into the inflammatory process as well as new fields of investigation for immunotherapy in the fields of chronic inflammatory diseases and cancer.

C1 inhibitor deficiency: consensus document

Abstract: Summary We present a consensus document on the diagnosis and management of C1 inhibitor deficiency, a syndrome characterized clinically by recurrent episodes of angio-oedema. In hereditary angio-oedema, a rare autosomal dominant condition, C1 inhibitor function is reduced due to impaired transcription or production of non-functional protein. The diagnosis is confirmed by the pres- ence of a low serum C4 and absent or greatly reduced C1 inhibitor level or function. The condition can cause fatal laryngeal oedema and features indis- tinguishable from gastrointestinal tract obstruction. Attacks can be precipi- tated by trauma, infection and other stimulants. Treatment is graded according to response and the clinical site of swelling. Acute treatment for severe attack is by infusion of C1 inhibitor concentrate and for minor attack attenuated androgens and/or tranexamic acid. Prophylactic treatment is by attenuated androgens and/or tranexamic acid. There are a number of new products in trial, including genetically engineered C1 esterase inhibitor, kal- likrein inhibitor and bradykinin B2 receptor antagonist. Individual sections provide special advice with respect to diagnosis, management (prophylaxis and emergency care), special situations (childhood, pregnancy, contracep- tion, travel and dental care) and service specification.

The number of human peripheral blood CD4+ CD25high regulatory T cells increases with age.

Abstract: Ageing is associated with evidence of immune deficiency and dysregulation. Key changes in the immune system with ageing include a progressive reduction in naive T cell output associated with thymic involution and peripheral expansion of oligoclonal memory T cells. These features are associated with evidence of impaired immune responsiveness both in vitro and in vivo, termed immune senescence. CD4+ CD25+ T cells have recently been recognized as mediators of peripheral immune regulation and play a role in the control of autoimmune and pathogen-specific immune responses. The significance of CD4+ CD25+ regulatory T cells in the context of immunosenescence is not known. We have investigated the number, phenotype and function of CD4+ CD25+ T cells in healthy volunteers over a wide age range. We demonstrate that the number of CD4+ CD25+ and CD4+ CD25high T cells in healthy volunteers increases with age. In both age groups CD4+ CD25+ T cells showed a phenotype consistent with that described for regulatory T cells. Further analysis of CD4+ CD25high T cells in young and elderly donors showed equivalent expression of intracellular CTLA-4 and surface expression of activation markers. In vitro, functional titration assays of CD4+ CD25high T cells demonstrated equivalent regulatory function in both young and elderly donors, with suppression of proliferation and cytokine production in response to polyclonal T cell stimulation. These observations demonstrate an increase in peripheral blood CD4+ CD25high regulatory T cells associated with ageing. The relevance of these expanded cells in relation to the immune senescence seen in the elderly as yet remains unclear.

Clinical uses of intravenous immunoglobulin

Abstract: There has been a rapid expansion in the use of intravenous immunoglobulin (IVIG) for an ever growing number of conditions. It is a product with an excellent safety record without the side effects of steroids or other immunosuppressive agents. There have been numerous recent advances in our understanding of the mechanisms of action of IVIG in many of the conditions for which it is being used, but there is still much to be learned. IVIG has had a major impact in neurology, haematology, immunology, rheumatology and dermatology. The limitations for IVIG are cost of the preparation itself and the logistical problems associated with its administration. Many of the side effects are managed easily by careful screening of the patient prior to treatment, premedication with analgesics and antihistamines and adjustment of infusion rate. It is likely, however, that side effects will be more frequent as the dose given increases and effects on plasma viscosity become greater [122]. It will be important to increase the evidence base for IVIG in many conditions to define its therapeutic role. In addition, there have been very few dose-ranging studies for hdIVIG and fewer still of which adjunctive agents (including biological agents such as rituximab and daclizumab) might offer the best therapeutic combinations. Although not established, the use of IVIG is being studied in a range of conditions including heart failure, mycobacterial infection, adult respiratory distress syndrome, transplantation, fibrosis, connective tissue disease, encephalitis, epilepsy and even Alzheimer's disease. Should IVIG prove to be of efficacy in these settings it is likely to have major implications for drug budgets as many of the conditions are very common and place great strain on the world's supply of IVIG itself. Future studies will therefore need to address questions of efficacy, pharmacoeconomics, dose, adjunctive therapies and mechanism of action which may point the way to novel treatment strategies beyond IVIG. It is also clear, however, that controlled trials in many of the rare conditions may be very difficult to carry out and approaches such as prospective disease registries may be needed.

CD4+ CD25+ T cells with the phenotypic and functional characteristics of regulatory T cells are enriched in the synovial fluid of patients with rheumatoid arthritis.

Abstract: CD4(+) CD25(+) regulatory T (T(reg)) cells play a critical role in the maintenance of peripheral tolerance and the prevention of autoimmunity. In the present study, we have explored the characteristics of CD4(+) CD25(+) T(reg) cells in patients with rheumatoid arthritis (RA). The frequency and phenotype of CD4(+) CD25(+) T cells in paired samples of synovial fluid (SF) and peripheral blood (PB) from patients with RA and PB from normal controls were analysed. An increased frequency of CD4+ cells T cells expressing CD25 was detected in SF compared to PB from patients with RA. No significant difference was observed in the numbers of CD4(+) CD25(+) T cells in PB from patients and controls. SF CD4(+) CD25(+) T cells expressed high levels of CTLA-4 (both surface and intracellular), GITR and OX40, as well as Foxp3 transcripts. Functionally, SF CD4(+) CD25(+) T cells were impaired in their proliferative responses and could suppress the proliferation of their CD4(+) CD25(-) counterparts. In conclusion, these data demonstrate that CD4(+) CD25(+) T(reg) cells, with the potential to regulate the function of effector T cells and antigen-presenting cells, accumulate in the synovium of patients with RA.

The paradox of matrix metalloproteinases in infectious disease

Abstract: Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that perform multiple roles in the normal immune response to infection. MMPs facilitate leucocyte recruitment, cytokine and chemokine processing, defensin activation and matrix remodelling. However, excess MMP activity following infection may lead to immunopathology that causes host morbidity or mortality and favours pathogen dissemination or persistence. Here, we review the normal functions of MMPs in immunity and then discuss viral and bacterial infections where excess MMP activity has been implicated in pathology, specifically examining HIV, HTLV-1, hepatitis B, endotoxin shock, Helicobacter pylori and Mycobacterium tuberculosis. Tissue destruction may be exacerbated further by bacterial-derived enzymes which activate the host pro-MMPs. Finally, the potential for therapeutic targeting of excess MMP activity in infection is considered.

Histological analysis of CD11c-DTR/GFP mice after in vivo depletion of dendritic cells

Abstract: To investigate the dependence of individual immunological processes on DC, a transgenic mouse system (CD11c-DTR/GFP mice) has been developed that allows conditional depletion of CD11c+ DC in vivo through administration of diphtheria toxin. We have performed careful histological analysis of CD11c-DTR/GFP mice at different time points after diphtheria toxin injection and confirmed the transient depletion of CD11c+ cells from lymph nodes and spleen. Unexpectedly, the injection of diphtheria toxin completely depleted marginal zone and metallophilic M(Phi) from the spleen and their sinusoidal counterparts from the lymph nodes. This finding limits the use of CD11c-DTR/GFP mice for the analysis of the role of DC to models and read outs that are proven to be independent of marginal zone and sinusoidal M(Phi).

Probiotic Lactobacillus-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines in lamina propria mononuclear cells.

Abstract: IL-6/STAT-3 signals play key roles in inflammatory bowel disease (IBD). It is known that Lactobacillus casei strain Shirota (LcS) improves inflammatory disorders. This study aimed to elucidate the effect of LcS on murine chronic IBD and to clarify the mechanism. We focused the inhibitory effect of LcS on the production of IL-6 in lipopolysaccharide (LPS)-stimulated large intestinal lamina propria mononuclear cells (LI-LPMC) isolated from mice with chronic colitis and in RAW264·7 cells in vitro. We also determined in vivo the effect of LcS on murine chronic IBD models induced with dextran sodium sulphate and SAMP1/Yit mice. Finally, we examined the cellular determinants of LcS for the down-regulation of IL-6 secretion by LI-LPMC, RAW264·7 cells and peripheral blood mononuclear cells (PBMC) derived from patients with ulcerative colitis (UC). LcS, but not other strains of Lactobacillus, inhibited the production of IL-6 in LPS-stimulated LI-LPMC and RAW264·7 cells, down-regulating the nuclear translocation of NF-κB. The LcS-diet-improved murine chronic colitis is associated with the reduction of IL-6 synthesis by LI-LPMC. LcS also improved chronic ileitis in SAMP1/Yit mice. The release of IL-6 in vitro in LPS-stimulated LI-LPMC, RAW 264·7 cells and UC-PBMC was inhibited by a polysaccharide-peptidoglycan complex (PSPG) derived from LcS. This probiotic-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines such as IL-6 and IFN-γ production in LPMC. Therefore, LcS may be a useful probiotic for the treatment of human inflammatory bowel disease.

The Bisphosphonate Acute Phase Response: Rapid and Copious Production of Proinflammatory Cytokines by Peripheral Blood Gd T Cells in Response to Aminobisphosphonates Is Inhibited by Statins

Abstract: The bisphosphonates are a novel class of drug that have been registered for various clinical applications worldwide. Bisphosphonates, and in particular the aminobisphosphonates (nBPs), are known to have a number of side-effects including a rise in body temperature and accompanying flu-like symptoms that resemble a typical acute phase response. The mechanism for this response has been partially elucidated and appears to be associated with the release of tumour necrosis factor (TNF)α and interleukin (IL)6, although the effector cells that release these cytokines and the mechanism of action remain enigmatic. Here, we show that the nBP-induced acute phase response differs from the typical acute phase response in that CD14+ cells such as monocytes and macrophages are not the primary cytokine producing cells. We show that by inhibiting the mevalonate pathway, nBPs induce rapid and copious production of TNFα and IL6 by peripheral blood γδ T cells. Prior treatment with statins, which inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, blocks nBP-induced production of these proinflammatory cytokines by γδ T cells and may offer a means of avoiding the associated acute phase response. In addition, our findings provide a further mechanism for the anti-inflammatory effects attributed to inhibitors of HMG CoA reductase.

Mammalian Toll-like receptors: to immunity and beyond.

Abstract: Toll-like receptors (TLRs) constitute an archetypal pattern recognition system. Their sophisticated biology underpins the ability of innate immunity to discriminate between highly diverse microbial pathogens and self. However, the remarkable progress made in describing this biology has also revealed new immunological systems and processes previously hidden to investigators. In particular, TLRs appear to have a fundamental role in the generation of clonal adaptive immune responses, non-infectious disease pathogenesis and even in the maintenance of normal mammalian homeostasis. Although an understanding of TLRs has answered some fundamental questions at the host-pathogen interface, further issues, particularly regarding therapeutic modulation of these receptors, have yet to be resolved.

The immune recognition of gluten in coeliac disease.

Abstract: Coeliac disease, the most common intestinal disorder of western populations, is an autoimmune enteropathy caused by an abnormal immune response to dietary gluten peptides that occurs in genetically susceptible individuals carrying the HLA-DQ2 or -DQ8 haplotype. Despite the recent progresses in understanding the molecular mechanisms of mucosal lesions, it remains unknown how increased amounts of gluten peptides can enter the intestinal mucosa to initiate the inflammatory cascade. Current knowledge indicates that different gluten peptides are involved in the disease process in a different manner, some fragments being 'toxic' and others 'immunogenic'. Those defined as 'toxic' are able to induce mucosal damage either when added in culture to duodenal endoscopic biopsy or when administered in vivo, while those defined as 'immunogenic' are able to specifically stimulate HLA-DQ2- or DQ8-restricted T cell clones isolated from jejunal mucosa or peripheral blood of coeliac patients. These peptides are able to trigger two immunological pathways: one is thought to be a rapid effect on the epithelium that involves the innate immune response and the other represents the adaptive immune response involving CD4+ T cells in the lamina propria that recognize gluten epitopes processed and presented by antigen presenting cells. These findings are the subject of the present review.

T cell-mediated immune responses in human newborns: ready to learn?

Abstract: Infections with intracellular pathogens are often more severe or more prolonged in young infants suggesting that T cell-mediated immune responses are different in early life. Whereas neonatal immune responses have been quite extensively studied in murine models, studies of T cell-mediated immunity in human newborns and infants are scarce. Qualitative and quantitative differences when compared with adult immune responses have been observed but on the other hand mature responses to certain vaccines and infectious pathogens were demonstrated during the postnatal period and even during foetal life. Herein, we review the evidence suggesting that under appropriate conditions of stimulation, protective T cell-mediated immune responses could be induced by vaccines in early life.

Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis.

Abstract: Rituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic B-cell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca(2+) levels and downstream apoptotic signalling.

Cytokine responses correlate differentially with age in infancy and early childhood

Abstract: The functional differentiation of immune cells at early age plays a central role in immune physiology, e.g. for the sufficient eradication of pathogens. However, imbalances in effector cell responses may also have an impact in the pathophysiology of childhood diseases such as atopy and autoimmune disorders. As information on immune cell responses in infancy and early childhood is scarce, we conducted an observational, cross-sectional study in healthy newborns (n = 18), infants and young children (n = 54) aged 1-96 months and adult controls (n = 19) to assess cytokine mRNA and protein expression upon phorbol 12-myristate 13-actate/ionomycin stimulation and LPS-induced IL-12 expression in monocytes. The intracellular expression of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha (R = 0.748, P < 0.0001; R = 0.784, P < 0.0001, respectively) and interleukin (IL)-2 protein expression (R = 0.384, P = 0.008) was demonstrated to increase progressively with age. While a correlation between IL-4 protein expression and age was noted (R = 0.342, P = 0.007), the levels of IL-5 and IL-10 protein expression tended to be regulated on an individual basis during infancy and early childhood. An age correlation was also observed for intracellular IL-12 expression (R = 0.331, P = 0.009) in monocytes. These findings are valuable for further assessment of normal variations and maturation processes in immune cell responses and for the clinical-therapeutic monitoring of immunological status in various childhood diseases.

Expression of Toll-like receptor 9 and response to bacterial CpG oligodeoxynucleotides in human intestinal epithelium*

Abstract: Recognition of repeat CpG motifs, which are common in bacterial, but not in mammalian, DNA, through Toll-like receptor (TLR)9 is an integral part of the innate immune system. As the role of TLR9 in the human gut is unknown, we determined the spectrum of TLR9 expression in normal and inflamed colon and examined how epithelial cells respond to specific TLR9 ligand stimulation. TLR9 expression was measured in human colonic mucosal biopsies, freshly isolated human colonic epithelial cells and HT-29 cells by reverse transcriptase-polymerase chain reaction or Western blotting. Colonic epithelial cell cultures were stimulated with a synthetic CpG-oligodeoxynucleotide (ODN), exhibiting strong immunostimulatory effects in B cells. Interleukin (IL)-8 secretion was determined by enzyme-linked immunosorbent assay, nuclear factor-kappaB (NF-kB) activity by electrophoretic mobility shift assay and IkB phosphorylation by Western blotting. TLR9 mRNA was equally expressed in colonic mucosa from controls (n = 6) and patients with ulcerative colitis or Crohn's disease disease (n = 13). HT-29 cells expressed TLR9 mRNA and protein and responded to CpG-ODN (P < 0.01), but not to non-CpG-ODN stimulation, by secreting IL-8, apparently in the absence of NF-kB activation. Primary epithelial cells isolated from normal human colon expressed TLR9 mRNA, but were completely unresponsive to CpG-ODN stimulation in vitro. In conclusion, differentiated human colonic epithelial cells are unresponsive to TLR9 ligand stimulation in vitro despite spontaneous TLR9 gene expression. This suggests that the human epithelium is able to avoid inappropriate immune responses to luminal bacterial products through modulation of the TLR9 pathway.

Immunological profile of peripheral blood lymphocytes and monocytes/macrophages in Kawasaki disease

Abstract: Kawasaki disease (KD) is an acute illness of early childhood characterized by prolonged fever, diffuse mucosal inflammation, indurative oedema of the hands and feet, a polymorphous skin rash and nonsuppurative lymphadenopathy. The histopathological findings in KD comprise panvasculitis with endothelial necrosis, and the infiltration of mononuclear cells into small and medium-sized blood vessels. The levels of many proinflammatory cytokines, chemokines and adhesion molecules can be elevated in sera from children with KD at the acute stage. Although many immunological studies on KD involving peripheral blood have been reported, the data obtained remain controversial. This review focuses on the immune response of peripheral blood lymphocytes and monocytes/macrophages during acute KD.

Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens.

Abstract: Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14(dim)CD16(+) monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll-like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis-relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14(dim)CD16(+) monocyte population expanded at 20 h post-stimulation, after a rise in tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 at 2 h. A strong positive correlation between the percentage of CD14(dim)CD16(+) monocytes and secreted TNF-alpha was demonstrated (r = 0.72). Furthermore, we were able to induce expansion of the CD14(dim)CD16(+) population to approximately 35% of all monocytes with the addition of recombinant TNF-alpha to the whole blood culture. TLR4 was found to be expressed 2.5 times higher on CD14(dim)CD16(+) compared to CD14(+) CD16(-) monocytes, while TLR2 expression was similar in both subpopulations. The CD14(dim)CD16(+) and CD14(+) CD16(-) monocyte populations were different in their response to various antigens. LPS down-regulated TLR4 by 4.9 times in CD16(+) monocytes compared to only 2.3 times in CD16(-) monocytes at 2 h. LPS was able to up-regulate TLR2 by 6.2 times after 2 h, with no difference between the subpopulations. LPS further up-regulated TLR2 by 18.4 times after 20 h only in the CD14(+) CD16(-) population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF-alpha drives a differentiation of monocytes into a CD14(dim)CD16(+) subpopulation.

Involvement of Th1 cells and heat shock protein 60 in the pathogenesis of intestinal Behcet's disease.

Abstract: Involvement of excessive Th1 cell functions and heat shock protein expression in the pathogenesis of Behcet's disease (BD) has been reported. In this study we have characterized immune responses in intestinal lesions of BD. Peripheral blood lymphocytes (PBL) of BD and healthy controls (HC) and tissue specimens of intestinal Behcet's disease (intestinal BD), Crohn's disease (CD) and ulcerative colitis (UC) were analysed for mRNA and protein expression by reverse transcriptase-polymerase chain reaction (PCR) and immunohistochemistry, respectively. PBL of BD patients expressed the Th1-related chemokine receptor, CCR5 and CXCR3 preferentially compared with those of healthy controls. Intestinal lesions of BD expressed interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12 mRNA, indicating Th1 skewed responses in vivo. mRNA of Txk, a Tec family tyrosine kinase specific to Th1 cells, was expressed in the lesions, suggesting its contribution to the Th1-dominant responses. In the intestinal samples, CCR5 was detected in all the cases with BD, whereas Th2-related CCR3 and CCR4 were detected randomly, mainly in the cases with inactive BD and those receiving large amounts of prednisolone, indicating the Th1-dominant immune responses in the intestinal lesions. As the ligands of CCR5, MIP1alpha and MIP1beta were detected, whereas RANTES was not. Heat shock protein (HSP) 60 was expressed in PBL and intestinal tissues of BD. Th1-dominant immune responses and HSP60 expression may induce the inflammatory responses and thus be associated with the pathogenesis of intestinal BD.

Chronic fatigue syndrome is associated with diminished intracellular perforin.

Abstract: Chronic fatigue syndrome (CFS) is an illness characterized by unexplained and prolonged fatigue that is often accompanied by abnormalities of immune, endocrine and cognitive functions. Diminished natural killer cell cytotoxicity (NKCC) is a frequently reported finding. However, the molecular basis of this defect of in vitro cytotoxicy has not been described. Perforin is a protein found within intracellular granules of NK and cytotoxic T cells and is a key factor in the lytic processes mediated by these cells. Quantitative fluorescence flow cytometry was used to the intracellular perforin content in CFS subjects and healthy controls. A significant reduction in the NK cell associated perforin levels in samples from CFS patients, compared to healthy controls, was observed. There was also an indication of a reduced perforin level within the cytotoxic T cells of CFS subjects, providing the first evidence, to our knowledge, to suggest a T cell associated cytotoxic deficit in CFS. Because perforin is important in immune surveillance and homeostasis of the immune system, its deficiency may prove to be an important factor in the pathogenesis of CFS and its analysis may prove useful as a biomarker in the study of CFS.

Differential pattern of cytokine expression by macrophages infected in vitro with different Mycobacterium tuberculosis genotypes

Abstract: It has been shown recently that different genotypes of Mycobacterium tuberculosis induce distinct immune responses in the host, as reflected by variations in cytokine and iNOS expression. Because these molecules are probably regulated by multiple factors in vivo this complex phenomenon was partially analysed by assessing cytokine and iNOS expression by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) in an in vitro model of bone marrow-derived macrophages infected with three different M. tuberculosis genotypes: Canetti, H37 Rv and Beijing. Although the three genotypes induced production of iNOS and the different cytokines tested at 24 h post-infection, macrophages infected with the Beijing isolate expressed the highest levels of mRNA for iNOS, interleukin (IL)-1beta, tumour necrosis factor (TNF)-alpha, IL-12 cytokines and lower levels of IL-10 compared with cells infected with other genotypes. This expression pattern has been associated with infection control, but during infection in vivo with the Beijing genotype it is lost upon progression to chronic phase. The failure to control infection is likely to be influenced by cytokines produced by other cell types and bacterial molecules expressed during the course of disease. Results presented in this work show that each genotype has the ability to induce different levels of cytokine expression that could be related to its pathogenesis during infection.

Analysis of intestinal haem-oxygenase-1 (HO-1) in clinical and experimental colitis.

Abstract: Haem-oxygenase-1 (HO-1) has been shown to exert anti-inflammatory, anti-apoptotic and anti-proliferative effects. We investigated HO-1 expression in patients with inflammatory bowel disease (IBD) and could demonstrate a scattered expression of HO-1 in the intestinal epithelium of severely inflamed colonic mucosa of patients with IBD compared to control specimens such as diverticulitis, suggesting dysregulated expression in IBD. To further analyse potential mechanisms of HO-1 induction in the intestine we employed an in vitro epithelial cell apoptosis model and an experimental colitis model. In vitro induction of HO-1 by the HO-1 inducer cobalt protoporphyrin (CoPP) resulted in a dose-dependent down-regulation of caspase-3 activation in HT-29 cells, indicating an anti-apoptotic function of HO-1 in the intestine. In vivo, preventive HO-1 induction by CoPP in acute dextran sodium sulphate (DSS)-induced colitis led to a significant down-regulation of colonic inflammation (P < 0.01) with a concomitant reduction in interferon (IFN)-gamma - but unaffected interleukin (IL)-10-secretion by isolated mesenteric lymph nodes (P < 0.01). Additionally, TUNEL staining of colonic sections demonstrated fewer apoptotic epithelial cells in the colon of CoPP treated animals. No beneficial effects were observed if HO-1 was induced by CoPP after the onset of acute colitis or in chronic DSS-induced colitis. In conclusion, the data suggest a protective role of HO-1 if it is induced before the onset of inflammation. However, as shown by the lack of effects in established acute or in chronic colitis, the induction of HO-1 may not be a promising approach for the treatment of IBD.

Dendritic cell immunizations alone or combined with low doses of interleukin‐2 induce specific immune responses in melanoma patients

Abstract: Dendritic cell (DC)-based therapy has proved to be effective in patients with a variety of malignancies. However, an optimal immunization protocol using DCs and the best means for delivering antigens has not yet been described. In this study, 20 patients with malignant melanoma in stages III or IV were vaccinated with autologous DCs pulsed with a melanoma cell lysate, alone (n = 13) or in combination with low doses of subcutaneous (s.c.) interleukin (IL)-2 injections (n = 7), to assess toxicity, immunological and clinical responses. Monocyte-derived DCs were morphological, phenotypic and functionally characterized in vitro. Peripheral blood mononuclear cells (PBMC), harvested from patients either prior to and after the treatment, were analysed using enzyme-linked immunosorbent spot (ELISPOT). After vaccination, 50% of the patients tested (seven of 13) from the first group and (three of seven) from the second, showed an increase in interferon (IFN)-gamma production in response to allogeneic melanoma cell lines but not to controls. Four of five tested human leucocyte antigen (HLA)-A2(+) patients with anti-melanoma activity also showed specific T cell responses against peptides derived from melanoma-associated antigens. Delayed type IV hypersensitivity reaction (DTH) against melanoma cell lysate was observed in six of 13 patients from the group treated with DC vaccines only and four of seven from the group treated with the combination of DCs and IL-2. Significant correlations were found between DTH-positive responses against tumour lysate and both disease stability and post-vaccination survival on the stage IV patients. There were no toxicities associated with the vaccines or evidence of autoimmunity including vitiligo. Furthermore, no significant enhancement was observed as a result of combining DC vaccination with IL-2. Our data suggest that autologous DCs pulsed with tumour lysate may provide a standardized and widely applicable source of melanoma specific antigens for clinical use. It is safe and causes no significant side effects and has been demonstrated to be partially efficient at triggering effective anti-melanoma immunity.

Lower expression of Th1-related cytokines and inducible nitric oxide synthase in mice with streptozotocin-induced diabetes mellitus infected with Mycobacterium tuberculosis.

Abstract: Diabetes mellitus is an important predisposing factor for tuberculosis. The aim of this study was to investigate the mechanism underlying this association using a murine model. Mice with streptozotocin-induced diabetes mellitus were prone to Mycobacterium tuberculosis infection, as indicated by increased numbers of live bacteria in lung, liver and spleen. In diabetic mice, the levels of IL-12 and IFN-gamma in the lung, liver and spleen were lower than those in control animals on day 14 postinfection, while the opposite was true for IL-4 levels in the lung and liver. The expression pattern of inducible nitric oxide synthase (iNOS), in the two mice types was as for IL-12 and IFN-gamma. In addition, peritoneal exudate cells obtained from diabetic mice produced lower amounts of IL-12 and NO than those from control mice, when stimulated in vitro with M. bovis BCG. Spleen cells from diabetic mice infected with M. tuberculosis produced a significantly lower amount of IFN-gamma upon restimulation with purified protein derivatives (PPD) than those from infected nondiabetic mice. Interestingly, addition of high glucose levels (33 mM) to the cultures of PPD-restimulated spleen cells reduced the synthesis of IFN-gamma only in diabetic mice, and not in nondiabetic mice. Finally, control of blood glucose levels by insulin therapy resulted in improvement of the impaired host protection and Th1-related cytokine synthesis. Our results suggest that the reduced production of Th1-related cytokines and NO account for the hampered host defense against M. tuberculosis infection under diabetic conditions.

Exposure to Mycobacterium avium induces low-level protection from Mycobacterium bovis infection but compromises diagnosis of disease in cattle.

Abstract: We assessed the effect of exposure to Mycobacterium avium on the development of immune responses and the pathogenesis of disease observed following Mycobacterium bovis challenge. A degree of protection against M. bovis was observed in calves which were pre-exposed to M. avium as assessed by the extent of lesions and bacterial load compared to the M. bovis alone group. The immune response following M. bovis challenge in cattle previously inoculated with M. avium was biased towards antigens (PPD) present in M. avium, whereas the response following M. bovis alone was biased towards antigens present in M. bovis, indicating an imprinting of memory to avian antigens on T lymphocytes. A consequence of the memory to M. avium antigens was failure to diagnose M. bovis infection by the skin test or the IFN(gamma) assay in some of the animals which had lesions of tuberculosis at necropsy. The use of M. bovis specific antigens ESAT-6 and CFP-10 increased IFN(gamma) test specificity in animals previously exposed to M. avium but the responses to these antigens were lower than those observed in animals exposed to M. bovis alone. The implication is that responses to M. avium, although providing some immunity, may mask diagnosis of M. bovis infection, even when specific antigens are employed, potentially contributing to disease transmission in the field.

Vaccination of neonatal calves with Mycobacterium bovis BCG induces protection against intranasal challenge with virulent M. bovis.

Abstract: Summary Vaccination of neonates with Mycobacterium bovis bacillus Calmette–Guerin (BCG) may be a strategy that overcomes reduced vaccine efficacy associated with exposure to environmental mycobacteria in humans and cattle. Preliminary comparisons indicated that 2-week-old calves produced an immune response to vaccination at least as intense as that observed in adults. Subsequently, five gnotobiotic hysterotomy derived calves aged 1 day were inoculated with BCG and 3 months later were challenged intranasally with virulent M. bovis. The number of tissues with lesions and the pathological extent of these lesions was reduced significantly in vaccinates. Furthermore, lesions were evident in the lung or associated chest lymph nodes of four of five controls but none of five vaccinates. BCG vaccination reduced significantly the level of bacterial colonization. However, lesions in the head associated lymph nodes were observed in three of five BCG-vaccinated cattle. Levels of interferon gamma (IFN-γ) detected by enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot (ELISPOT) in individual vaccinated animals at challenge did not correlate with subsequent resistance and in general immune responses post-challenge were lower in vaccinated calves. Low IL-10 responses were evident but IL-4 was not detected. Responses to ESAT-6 and/or CFP-10 were evident in four of four control calves that had lesions. Two of the BCG vaccinates with lesions did not produce a response to ESAT-6 and CFP-10, indicating that these antigens did not distinguish vaccinated immune animals from vaccinated animals with lesions. Overall, vaccination of neonatal calves with BCG induced significant protection against disease and has potential as a strategy for the reduction of the incidence of bovine tuberculosis.

Predominance of type 1 CD4+ T cells in human abdominal aortic aneurysm.

Abstract: The functional repertoire of T cells in abdominal aortic aneurysm (AAA) and the exact nature of aortic wall adaptive cellular immune responses still remains a matter of debate. In this study, we sought to determine whether type 1 or type 2 responses occur predominantly in human aneurysmal aortic lesions. We first examined the phenotype and cytokine secretion profile of T lymphocytes freshly isolated from aneurysmal aortic wall for comparison with their circulating counterparts using flow cytometry. We found that both populations of infiltrating CD4(+) and CD8(+)T cells displayed a unique activated memory phenotype. In addition, we identified the presence in human aneurysmal aortic lesion of CD4(+)T cells producing high levels of interferon (IFN)-gamma but not interleukin (IL)-4, reflecting their type 1 nature. Quantitative analysis of cytokine gene expression confirmed increased IFN-gamma transcript levels in infiltrating cells compared to controls. We next analysed aortic wall responses using LightCycler-based quantitative real-time reverse transcription-polymerase chain reaction. Compared to control non-diseased aortic samples, we demonstrated that whole AAA tissues exhibited high mRNA levels of IFN-gamma but not IL-4. Overexpression of the transcription factor T-bet in the absence of significant GATA-3 expression further assessed the type 1 polarization of aortic wall immune responses. These findings indicate that type 1 CD4(+)T cells predominate in human AAA lesions. This study has important implications for the pathogenesis of aneurysm disease. Through the production of IFN-gamma, T cells may indeed contribute to orchestrate extracellular matrix remodelling.

Haematopoietic stem cell transplantation in the treatment of severe autoimmune disease: results from phase I/II studies, prospective randomized trials and future directions.

Abstract: Around 700 patients have received an autologous haematopoietic stem cell transplant (HSCT) as treatment for a severe autoimmune disease (AD). The majority of these have been within the context of phase I/II clinical trials and following international guidelines proposed 7 years ago. In general, a positive benefit/risk ratio has led to phase III prospective randomized controlled trials in multiple sclerosis (MS), systemic sclerosis (SSc) and rheumatoid arthritis (RA) in Europe. In the US, similar trials are being planned for SSc, MS and systemic lupus erythematosus (SLE). Transplant related mortality (TRM) has fallen in all disease subgroups since the inception due to more appropriate patient selection, and so far a clear advantage of the more intense myeloablative regimens in terms of remission induction and relapse rate has not emerged. Although each AD has a different profile, over a third of patients have sustained a durable remission, often with no further need for immunosuppressive drugs. In those who relapsed, many responded to agents which pre transplant had been ineffective. The study of immune reconstitution and gene expression pre and post HSCT is being undertaken to further understand the mechanism of autoimmunity.

New therapies for rheumatoid arthritis

Abstract: Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease, which continues to cause significant morbidity in affected persons. In the past few years, a number of new exciting therapeutic options have become available. These reflect the application of knowledge obtained from advancements in understanding of disease pathogenesis and underlying molecular mechanisms. A number of these therapies are outlined in the following review, including the various biological modifiers, in particular, anti-tumour necrosis factor-alpha agents and interleukin-1 (IL-1) receptor antagonists, which have been developed in recognition of the role of pro-inflammatory cytokines in RA. Also notable, is the current interest centring on the development and trials with B cell depletion therapies, specifically rituximab, in patients with RA. This demonstrates acknowledgment for a more significant role for B cells in the aetiology of RA, in contrast to the long held view that RA was a predominantly T cell mediated disease. To evaluate this therapeutic option for RA, salient features from recent rituximab trials have been collated. Finally, a selection of other therapeutic alternatives, including anti-IL-6 receptor monoclonal antibody and tacrolimus, and newer anti-rheumatic therapies presently in development are summarized.

Lymphocyte subsets in term and significantly preterm UK infants in the first year of life analysed by single platform flow cytometry.

Abstract: This observational study describes the ranges observed for lymphocyte subsets for significantly preterm infants (<32 weeks) in the first year of life, measured by single platform flow cytometry and compared to identically determined subsets in term infants. After ethical approval 39 term and 28 preterm infants had lymphocyte subset analysis before and after their primary immunization series. Median values with 5th and 95th percentiles of absolute counts and percentages are presented for total lymphocytes, T cells, NK cells, B cells, cytotoxic T cells, helper T cells, dual positive T cells, activated T cells, activated T helper cells (including T regulatory cells), pan memory T cells, pan naive T cells, memory helper T cells, naive helper T cells and the T helper/suppressor ratio. The lymphocyte profile of the preterm infants differed from that of the term infants.

Lymphocyte subset differences in patients with chronic fatigue syndrome, multiple sclerosis and major depression

Abstract: Chronic fatigue syndrome (CFS) is a heterogeneous disorder of unknown aetiology characterized by debilitating fatigue, along with other symptoms, for at least 6 months. Many studies demonstrate probable involvement of the central and autonomic nervous system, as well as a state of generalized immune activation and selective immune dysfunction in patients with CFS. The aim of this study was to compare the lymphocyte subsets of patients with chronic fatigue syndrome to those of patients with major depression and multiple sclerosis as well as those of healthy control subjects. No differences were found in total numbers of T cells, B cells or natural killer (NK) cells. However, differences were found in T, B and NK cell subsets. Patients with major depression had significantly fewer resting T (CD3 + /CD25 - ) cells than the other groups. Patients with major depression also had significantly more CD20 + / CD5 + B cells, a subset associated with the production of autoantibodies. Compared to patients with multiple sclerosis, patients with CFS had greater numbers of CD16 + /CD3 - NK cells. Further study will be required to determine whether these alterations in lymphocyte subsets are directly involved in the pathophysiology of these disorders, or are secondary effects of the causal agent(s).