bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effect of DNA immunization against mycobacterial infection is associated with the early emergence of interferon-gamma (IFN-γ)-secreting lymphocytes
Authors: Kamath A T , Groat N L , Bean A G D , Britton W J
Published: March 2003

The development of more effective anti-tuberculosis (TB) vaccines would contribute to the global control of TB. Understanding the activated/memory T cell response to mycobacterial infection and identifying immunological correlates of protective immunity will facilitate the design and assessment of new candidate vaccines. Therefore, we investigated the kinetics of the CD4+ T cell response and IFN-γ production in an intravenous challenge model of Mycobacterium bovis bacille Calmette–Guérin (BCG) before and after DNA immunization. Activated/memory CD4+ T cells, defined as CD44hiCD45RBlo, expanded following infection, peaking at 3–4 weeks, and decreased as the bacterial load fell. Activated/memory CD4+ T cells were the major source of IFN-γ and the level of antigen-specific IFN-γ-secreting lymphocytes, detected by ELISPOT, paralleled the changes in bacterial load. To examine the effects of a DNA vaccine, we immunized mice with a plasmid expressing the mycobacterial secreted antigen 85B (Ag85B). This led to a significant reduction in mycobacteria in the liver, spleen and lung. This protective effect was associated with the rapid emergence of antigen-specific IFN-γ-secreting lymphocytes which were detected earlier, at day 4, and at higher levels than in infected animals immunized with a control vector. This early and increased response of IFN-γ-secreting T cells may serve as a correlate of protective immunity for anti-TB vaccines.
Human T cells with a type-2 cytokine profile are resistant to apoptosis induced by primary activation: consequences for immunopathogenesis
Authors: Carbonari M , Tedesco T , Del Porto P , Paganelli R , Fiorilli M
Published: March 2003

The mechanisms leading to a relative dominance of T cells producing type 2 cytokines in certain human immune disorders are still unclear. We investigated the relative susceptibility to apoptosis induced by primary in vitro activation of human type 1 (producing interferon-gamma (IFN-γ)) or type 2 (producing IL-4) T cells. Peripheral blood lymphocytes were isolated from patients with immune disorders characterized by expansion of type 2 cells (four with AIDS and hyper-IgE/hypereosinophilia, one with Churg–Strauss syndrome, and one with idiopathic hypereosinophilic syndrome) or from individuals with normal cytokine balances. Cells were stimulated for 16 h with ionomycin and phorbol ester, and apoptosis of cytokine-producing cells was assessed by flow cytometry. T cells with a type-2 cytokine profile, i.e. producing IL-4 alone, were significantly more resistant to activation-induced apoptosis than those producing IFN-γ alone. This was observed in AIDS patients, whose type 2 cells were mostly CD8+, as well as in the patients with Churg–Strauss and with hypereosinophilic syndrome. CD4+ and CD8+ IL-4-producing cells were equally resistant to apoptosis. Lower susceptibility to apoptosis of type-2 T cells was also observed in subjects with normal cytokine balances. Bcl-2 expression was high in type-2 cells and in viable type-1 cells, whereas it was low in apoptotic type-1 cells. Resistance to activation-induced apoptosis may explain the expansion of cells producing type-2 cytokines in certain immune disorders.
Differential effect of cholera toxin on CD45RA+ and CD45RO+ T cells: specific inhibition of cytokine production but not proliferation of human naive T cells
Authors: Eriksson K , Nordström I , Czerkinsky C , Holmgren J
Published: March 2003

We have studied how cholera toxin (CT) and its non-toxic cell-binding B-subunit (CTB) affect the activation of pure human T cells in an anti-CD3-driven system. CT, as opposed to CTB, strongly suppressed the proliferative responses as well as cytokine production in CD4+ and CD8+ T cells. CT however, had a differential effect on naive and activated/memory T cell subsets. Costimulation through exogenous IL-2 or through CD28 cross-linking rescued the proliferation of CT-treated naive CD45RA+ T cells, but not of activated/memory CD45RO+ cells. IL-2 production and IL-2 receptor expression were markedly reduced by CT in all T cell fractions, i.e. also in CD45RA+ cells which had maintained proliferative responses. However, the proliferative responses of CT-treated CD45RA+ T cells were IL-2-dependent, as shown by blocking experiments using anti-IL-2 antibodies. These results indicate (i) that CTB has no cytostatic effect on human T cells, (ii) that CT affects proliferation and cytokine production by two different signal pathways, and (iii) that CT might interact with a signal pathway generated through or influenced by CD45.
The 75-kD tumour necrosis factor (TNF) receptor is specificallyup-regulated in monocytes during Q fever endocarditis
Authors: Ghigo E , Capo C , Amirayan N , Raoult D , Mege J-L
Published: March 2003

Q fever is an infectious disease caused by Coxiella burnetii, an obligate intracellular microorganism that inhabits monocytes/macrophages. The dysregulated production of TNF-α in Q fever endocarditis has been associated with defective killing of C. burnetii by patient monocytes. As soluble receptors for TNF-α (TNF-R55 and TNF-R75) regulate TNF-α activity, we investigated their release by monocytes in Q fever. Spontaneous and C. burnetii-stimulated release of TNF-R75, but not of TNF-R55, was up-regulated in patients with ongoing endocarditis compared with controls. The increase in TNF-R75 release was related to the activity of Q fever endocarditis, since TNF-R75 release was similar in patients with cured endocarditis and controls. While spontaneous release of TNF-R75 by monocytes from patients with ongoing Q fever endocarditis occurred without changes in its membrane expression, C. burnetii increased the surface expression of TNF-R75. In addition, TNF-R75 transcripts were increased in resting and C. burnetii-stimulated monocytes from patients with ongoing endocarditis. On the other hand, TNF-R75 release was not related to TNF-α secretion. These results indicate that the modulation of TNF-R75 is a critical feature of the pathophysiology of Q fever endocarditis.
Induction of CD95 ligand expression on T lymphocytes and B lymphocytes and its contribution to apoptosis of CD95-up-regulated CD4+ T lymphocytes in macaques by infection with a pathogenic simian/human immunodeficiency virus
Authors: Sasaki Y , Ami Y , Nakasone T , Shinohara K , Takahashi E , Ando S , Someya K , Suzaki Y , Honda M
Published: March 2003

Using an established SIV/HIV-C2/1-infected cynomolgus monkey model displaying stable CD4+ T cell depletion, the kinetics of apoptosis and the levels of expression of CD95 membrane-associated CD95L on lymphocytes were investigated to test the involvement of the CD95/CD95L system in CD4+ T lymphocyte loss in vivo. Rapid depletion of CD4+ T cells occurred up to 2 weeks after infection, with chronic CD4+ T lymphopenia thereafter. During the initial CD4+ T cell loss, which was accompanied by viraemia, about 90% of the peripheral CD4+ T cell subset underwent spontaneous apoptotic cell death during 24 h of culture. Increased expression of CD95 was observed on both CD4+ and CD8+ T cell subsets, with CD95 expression on CD8+ cells declining rapidly, but high CD95 expression being maintained on CD4+ cells. Since CD95L was expressed on CD8+ T cells, B cells and to a lesser extent on CD4+ T cells, this suggests that CD95-mediated apoptosis might be controlled in an autocrine/paracrine fashion.
Autoimmunity in ulcerative colitis (UC): a predominant colonic mucosal B cell response against human tropomyosin isoform 5
Authors: Onuma E K , Amenta P S , Ramaswamy K , Lin J J-C , Das K M
Published: March 2003

We set out to examine if the IgG-producing cells in the colonic mucosa in UC are committed to tropomyosin isoform 5 (hTM5), a putative autoantigen in UC. Lamina propria mononuclear cells (LPMC) were isolated from colonoscopic biopsy specimens from recto-sigmoid and proximal colon. Twenty-three patients with UC, eight with Crohn's colitis (CC), and 10 non-inflammatory bowel disease (non-IBD) controls were included. The ELISPOT assays were used to quantify lamina propria B cells producing total immunoglobulin (IgA, IgG, IgM), IgG, IgA, as well as IgG against hTM5 isoform. The median value of percentage of total IgG-producing lymphocytes was similar in UC (12%) and CC (11%), but was significantly (P < 0·0002) higher than non-IBD controls (6%). However, in UC, but not in CC and non-IBD, a large number of lamina propria B cells produced IgG against hTM5 (median values: UC 42%, CC 2·5%, non-IBD 0%). This difference in UC when compared with CC and non-IBD was highly significant (P < 0·00001). Twenty-one of 23 (91%) patients with UC had percentage of anti-hTM5 IgG-producing immunocytes more than 2 s.d. above the mean for non-UC patients. In UC but not in CC and non-IBD controls, the increased number of IgG-producing cells are largely committed to produce IgG against hTM5-related epitope(s).
Thalidomide analogue CC-3052 reduces HIV+ neutrophil apoptosisin vitro
Authors: Guckian M , Dransfield I , Hay P , Dalgleish A G
Published: March 2003

Thalidomide has significant immunomodulatory properties and has been used successfully in the treatment of oral ulcers and wasting in HIV patients. However, its use is limited by its poor bioavailability due to low solubility and short half life in solution, and teratogenic and neurotoxic side-effects. Recently, water-soluble analogues of thalidomide with significantly greater immunomodulatory activity and reduced side-effects have become available. We examined the effect of thalidomide and one analogue, CC-3052, on neutrophil apoptosis following culture for 20 h in vitro. Apoptosis was assessed by reduced CD16 expression and Annexin V binding using flow cytometry. Thalidomide or CC-3052 alone had no effect on neutrophil apoptosis when used at physiological levels. However, when used together with prostaglandin E2 (10−7 m), a potent adenylate cyclase activator, CC-3052 but not thalidomide (both 10−5 m) reduced apoptosis in neutrophils from normal and HIV+ donors. The reduced apoptosis could not be attributed to the ability of CC-3052 to reduce tumour necrosis factor-alpha (TNF-α) production, but may be due to its PDE4 inhibitor properties, as it increased [cAMP]i, and mimicked the effect of increasing [cAMP]i using dibutryl cAMP, a membrane-permeable analogue of cAMP. The results suggest a role for thalidomide analogue CC-3052 in reducing persistent activation of the TNF-α system in HIV without markedly impairing neutrophil viability.
The inhibition of cutaneous T cell apoptosis may prevent resolution of inflammation in atopic eczema
Authors: Orteu C H , Rustin M H A , O'toole E , Sabin C , Salmon M , Poulter L W , Akbar A N
Published: March 2003

Atopic eczema (AE) is characterized by the persistence of infiltrating T lymphocytes in the dermis. To test the hypothesis that dysregulation of normal T cell apoptosis may contribute to the pathogenesis and chronicity of AE we compared patients with a normal resolving immune response (Mantoux reaction (MR)) induced in healthy volunteers by cutaneous PPD injection. Significantly less T cell apoptosis was observed in lesional skin of AE patients compared with either the peak or the resolution phase of the MR (P < 0·0001). The low incidence of T cell apoptosis in AE was associated with significantly increased levels of Bcl-2 relative to Bax (P < 0·0001) and significantly decreased CD95-L expression (P < 0·002) compared with the resolving MR. The cytokines IL-15 and interferon-beta (IFN-β), which prevent activated T cell apoptosis, were expressed maximally on day 7 and day 14 of the MR, respectively. In contrast, AE patients expressed high levels of both IL-15 and IFN-β in cutaneous lesions at the same time. This suggests that the co-expression of two anti-apoptotic cytokines, which are not found together during resolving cutaneous responses, may contribute to excessive T cell survival which leads to the persistence of inflammation in patients with AE.
Intrapulmonary concentrations of inflammatory cytokines in a mouse model of chronic respiratory infection caused byPseudomonas aeruginosa
Authors: Yanagihara K , Tomono K , Kuroki M , Kaneko Y , Sawai T , Ohno H , Miyazaki Y , Higashiyama Y , Maesaki S , Kadota J-I , Kohno S
Published: March 2003

We investigated the role of inflammatory cytokines in a mouse model of chronic Pseudomonas aeruginosa infection mimicking diffuse panbronchiolitis (DPB), and determined the effects of clarithromycin therapy on the production of these cytokines. The concentrations of IL-1β, IL-2, IL-4, IL-5, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) were measured serially in the lungs of mice with experimentally induced chronic respiratory P. aeruginosa infection until 60 days after inoculation. The concentrations of these cytokines during the course of the disease were significantly higher than baseline (before inoculation, P < 0.01 for all cytokines). Clarithromycin significantly inhibited the production of IL-1β and TNF-α in the lung (P < 0.01). The same treatment also reduced the levels of other cytokines, albeit insignificantly. Treatment with anti-TNF-α antibody significantly reduced the number of pulmonary lymphocytes and concentration of IL-1β in the lung (P < 0.01), but did not change the number of viable bacteria. Our findings resemble those detected in bronchoalveolar lavage fluid of patients with DPB and indicate that inflammatory cytokines play an important role in chronic P. aeruginosa lung infection. Our results also show that macrolides modulated the production of these cytokines, ultimately reducing lymphocyte accumulation in the lung. Our data suggest that anti-TNF-α antibody might be a useful new strategy for the treatment of chronic respiratory P. aeruginosa infection.
Apoptosis and apoptosis-associated perturbations of peripheral blood lymphocytes during HIV infection: comparison between AIDS patients and asymptomatic long-term non-progressors
Authors: Moretti S , Marcellini S , Boschini A , Famularo G , Santini G , Alesse E , Steinberg S M , Cifone M G , Kroemer G , De Simone C
Published: March 2003

This study was designed to compare the degree of lymphocyte apoptosis and Fas–Fas ligand (FasL) expression in AIDS patients and long-term non-progressors (LTNPs) and correlate these parameters with apoptosis-associated perturbations in lymphocyte function. LTNPs had a lower frequency of apoptotic CD4+ and CD8+ T cells compared with subjects with AIDS. This correlated with a lower frequency of cells expressing Fas and FasL. The frequency of selected lymphocyte populations exhibiting a disrupted mitochondrial transmembrane potential (ΔΨm) and increased superoxide generation was lower in LTNPs than in patients with AIDS; these abnormalities were associated with lower levels of caspase-1 activation in LTNPs. The results indicate a significantly reduced level of apoptosis and apoptosis-associated parameters in LTNPs than in patients developing AIDS. Based on these findings, a crucial role for mitochondria can be predicted in the process of lymphocyte apoptosis during the evolution of AIDS.
Reduced expression of the complement receptor type 2 (CR2, CD21) by synovial fluid B and T lymphocytes
Authors: Illges H , Braun M , Peter H H , Melchers I
Published: March 2003

The expression of CR2 (CD21) by synovial B and T lymphocytes of patients suffering from various forms of arthritis was analysed with cytofluorometry and with reverse transcriptase-polymerase chain reaction. CR2 (CD21) cell surface protein was detected in normal quantities on peripheral B cells, but was almost absent on synovial B lymphocytes of the same patients. This reduction was most severe in patients with rheumatoid arthritis, but also observed in all other cases. CR2 (CD21) did not reappear after in vitro culture. CR2 (CD21) mRNA was also strongly reduced in synovial B and T lymphocytes. Synovial fluid B lymphocytes were larger than peripheral blood B lymphocytes, while T cells from the same patients showed no size differences. We conclude that synovial fluid B lymphocytes have undergone an irreversible step towards terminal differentiation. The presence or absence of CR2 (CD21) mRNA in peripheral versus synovial T cells indicates that CR2 (CD21) is also differentially expressed by T lymphocytes.
Roles of tumour necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), and IL-10 in the modulation of intercellular adhesion molecule-1 (ICAM-1) expression by macrophages during mycobacterial infection
Authors: Tomioka H , Shimizu T , Maw W W , Ogasawara K
Published: March 2003

Profiles of ICAM-1 expression on cultured murine peritoneal macrophages infected with Mycobacterium avium complex (MAC) were examined, with special reference to modulating roles of TNF-α, TGF-β, and IL-10. When macrophages were infected with MAC, ICAM-1 expression, measured by microscopic counting of ICAM-1+ macrophages stained with anti-ICAM-1 antibody, ELISA, and flow cytometric analysis, was rapidly increased, peaking at day 3 (early-phase up-regulation) due to endogenous TNF-α, and thereafter gradually declined to the normal level within 1 week or more (late-phase down-regulation). The late-phase ICAM-1 down-regulation was also seen in macrophages phagocytosing heat-killed MAC and those stimulated with lipopolysaccharide but not in macrophages phagocytosing latex beads. ICAM-1 mRNA expression was augmented markedly at day 1 after MAC infection and thereafter decreased. While TNF-α and IL-10 production by MAC-infected macrophages was observed during the first 3 days, TGF-β production was initiated from day 3 and continued until day 14. Exogenously added TGF-β strongly inhibited the early-phase increase in ICAM-1 expression by infected macrophages, and the blockade of endogenous TGF-β with anti-TGF-β antibody markedly inhibited late-phase ICAM-1 down-regulation. Moderate blocking effect was also observed for anti-IL-10 antibody. On the other hand, late-phase ICAM-1 down-regulation was not prevented by the addition of exogenous TNF-α. Therefore, TGF-β and IL-10, especially the former, appear to play active roles in the late-phase down-regulation of ICAM-1 in MAC-infected macrophages during long-term cultivation.
Increase of chemokine interferon-inducible protein-10 (IP-10) in the serum of patients with autoimmune liver diseases and increase of its mRNA expression in hepatocytes
Authors: Nishioji K , Okanoue T , Itoh Y , Narumi S , Sakamoto M , Nakamura H , Morita A , Kashima K
Published: March 2003

To clarify the role of IP-10 in autoimmune liver diseases, we studied the serum levels of IP-10 in 14 patients with autoimmune hepatitis (AIH), 23 patients with primary biliary cirrhosis (PBC), and 65 patients with chronic viral hepatitis (20 type B and 45 type C). The hepatic expression of IP-10 mRNA and the correlation between the serum levels of IP-10 and clinical parameters were also evaluated. In addition to 20 healthy controls, 16 rheumatoid arthritis (RA) patients were included as an extrahepatic inflammatory disease. The serum level of IP-10 was significantly (P < 0·02) higher in patients with AIH, PBC, and chronic hepatitis B and C than in healthy controls, and it was significantly correlated (P < 0·05) with the serum levels of aspartate aminotransferase and alanine aminotransferase in patients with AIH, PBC, and chronic hepatitis B and C. The serum level of IP-10 was not elevated in RA patients. After successful treatment of AIH and chronic hepatitis C, the serum level of IP-10 decreased to the same level as in healthy volunteers. As we previously showed in cases with chronic hepatitis B or C, in situ hybridization in both AIH and PBC cases demonstrated the expression of IP-10 mRNA in hepatocytes around focal or lobular necrosis surrounded by infiltrating mononuclear cells, whereas IP-10 mRNA was not expressed in areas around the damaged bile ducts in PBC cases. The present results suggest that IP-10 is specifically produced by hepatocytes in inflammatory areas irrespective of the aetiology of hepatitis, and that IP-10 may help to recruit T cells to the hepatic lesions in autoimmune liver diseases as well as in chronic viral hepatitis.
Failure to detect HLA-A*6802-restricted CD8+ T cells specific forChlamydia trachomatisantigens in subjects from trachoma-endemic communities
Authors: Mahdi O S M , Whittle H C , Joof H , Mabey D C W , Bailey R L
Published: March 2003

The role of HLA-A*6802-restricted CD8+ T cells in chlamydial disease was investigated in human ocular infections. Peptides with predicted binding motifs for HLA-A*6802 were synthesized using sequences based on chlamydial antigens, major outer membrane protein (MOMP), macrophage infectivity potentiator (MIP) and heat shock protein (hsp70). Peptides were pooled according to Chlamydia trachomatis protein type and serovar, and were tested in 51Cr-release cytotoxic T lymphocyte (CTL) and enzyme-linked immunospot (ELISPOT) assays, using peripheral blood mononuclear cells (PBMC) isolated from subjects living in trachoma-endemic communities in The Gambia. Significant CTL activity or interferon-gamma release was not detected in any of the subjects, suggesting either that HLA-A*6802 CD8+ T cells may not be important in ocular infections or that the peptides chosen did not represent epitopes.
Prevention of murine acute graft-versus-host disease by staphylococcalenterotoxin B treatment
Authors: Takenaka K , Fujiyama Y , Andoh A , Sasaki T , Amakata Y , Matsubara H , Hodohara K , Bamba T
Published: March 2003

Retroviral superantigens such as minor lymphocyte stimulating (Mls) antigen play an important role in the pathogenesis of acute graft-versus-host disease (GVHD). However, it remains unclear how exogenous bacterial superantigens modulate acute GVHD. In this study, we tested the effects of staphylococcal enterotoxin B (SEB) on the development of acute GVHD in a model involving the systemic transfer of parental C57Bl/6 spleen cells into BDF1 mice. SEB treatment suppressed the expansion of donor-derived T cells and blocked the decrease in the number of host cells. Impaired haematopoiesis was actually rescued by treatment with SEB. In SEB-treated mice, both spontaneous proliferation and IL-2 production in T cells were suppressed on day 2 after parental cell infusion. On day 21, the number of donor-derived CD4+ Vβ8+ T cells markedly decreased in the spleen of SEB-treated mice. Donor-derived CD4+ T cells failed to proliferate in response to host alloantigens, and both donor- and host-derived T cells were unable to produce IL-2 in response to concanavalin A stimulation, suggesting that SEB treatment induced a general immunosuppressive state. Our results indicate that SEB treatment prevents the development of acute GVHD by leading to unresponsiveness of donor-derived T cells against host alloantigens in a Vβ-restricted and unrestricted manner.
Poor immune reconstitution after four or five major HLA antigens mismatched T cell-depleted allogeneic and autologous stem cell transplantation
Authors: Mattsson J , Uzunel M , Remberger M , Tammik L , Omazic B , Levitsky V , Zou J-Z , Hentschke P , Ringdén O
Published: March 2003

Two adults with primary liver cancer underwent liver transplantation from 5/6 and 4/6 major HLA-antigen mismatched unrelated donors. They were then conditioned with 4 × 2 Gy of total lymphoid irradiation, 120 mg/kg cyclophosphamide, 7·5 Gy total body irradiation and anti-T cell antibodies. Thereafter, the patients received T cell-depleted autologous: unrelated mismatched bone marrow in a proportion of 0·5:3·0 and 0·35:1·1 × 106 CD34+ cells/kg, respectively. After allogeneic stem cell transplantation (ASCT), both became mixed chimeras, as determined with polymerase chain reaction amplification of variable number tandem repeats from DNA obtained from CD3+, CD19+ and CD45+ magnetic bead-separated cells. Due to a reduction in donor T cells, the first patient was given 105 donor T cells/kg and became a complete donor chimera within 3 months. The second patient rejected all donor cells within 1 month after ASCT. Leucocytes normalized in both patients within 1 month. CD8+ cells normalized after 4 and 2 months in the two patients, respectively. However, CD4+, CD56+ and CD19+ cells remained low, except for a transient increase in patient 2. Lymphocyte responses to mitogens were negative in patient 1 from 1 to 5 months after ASCT. This patient also showed an oligoclonal pattern of the B cell repertoire, performed by CDR3 spectratyping. Epstein–Barr virus DNA in lymphocytes increased by 4–5 log in both patients. Prior to ASCT, recipients and donors were mutually reactive in mixed lymphocyte cultures (MLC). In the first patient, who became a complete donor chimera, the chimera cells showed no response to recipient or donor, but a positive response to third party. In the other patient, recipient cells reacted vigorously against donor lymphocytes at the time of rejection. Both patients suffered from overwhelming bacterial, fungal and viral infections, and died of pneumonia 5 and 3 months after ASCT, respectively. To conclude, with a major HLA-mismatch barrier, stable mixed chimerism seems difficult to achieve. The first patient became a full donor chimera and the second one rejected the graft. Both suffered from immune incompetence.
Characterization of the cellular immune function of patients with chronic mucocutaneous candidiasis
Authors: De Moraes-Vasconcelos D , Orii N M , Romano C C , Iqueoka R Y , Duarte A J Da S
Published: March 2003

Chronic mucocutaneous candidiasis (CMC) is a rare syndrome characterized by persistent and refractory infections of the skin, nails and mucosal tissues by yeasts of the genus Candida. Defects in the cellular limb of the immune system are well documented in CMC patients, but non-specific immune defects, such as myeloperoxidase deficiency or phagocyte chemotaxis disorders, have also been described. Nonetheless, the underlying defect(s) remains poorly understood, and further studies are required. We studied eight CMC patients without endocrinopathies, who showed (i) low normal proliferative response to phytohaemagglutinin (PHA), (ii) partially defective response to pokeweed mitogen (PWM), and (iii) impaired response to Candida and PPD antigens. Furthermore, peripheral blood mononuclear cells (PBMC) from CMC patients produced lower levels of type-1 cytokines (IL-2 and interferon-gamma) in response to Candida antigens, compared with control individuals. Conversely, we did not observe an enhancement of IL-4 and IL-10 in the patients, suggesting that, even though Th1 cytokines are decreased, the Th2 response is not increased in CMC. Nevertheless, the synthesis of these cytokines was normal when induced by PHA. We also observed an increased antigen-induced apoptosis in lymphocytes from the patients compared with controls, and this applied both to Candida and PPD antigens. Lastly, innate immunity defects were investigated. We observed an impairment of natural killer activity against K-562 target cells in half of the studied patients. These findings corroborate the extensive clinical and laboratory variability of CMC, which requires further studies on a larger number of patients to be better understood.
Increase in tonsillar germinal centre B-1 cell numbers in IgA nephropathy (IgAN) patients and reduced susceptibility to Fas-mediated apoptosis
Authors: Kodama S , Suzuki M , Arita M , Mogi G
Published: March 2003

IgAN is a common form of primary glomerulonephritis and also a disease of tonsillar focal infection. The comprehensive mechanism underlying this disease remains to be defined. To better understand its pathogenesis, we investigated tonsillar CD5+ B cells (B-1 cells) with respect to IgA synthesis. Germinal centre (GC) B cells were isolated from the tonsils of IgAN patients and the number of B-1 cells in the GC determined by flow cytometry. GC B-1 and B-2 (CD5− B) cells were purified by cell sorter, the cells were incubated with agonist anti-CD40 MoAb and the ability for antibody production by B-1 and B-2 cells determined by ELISPOT assay. GC B-1 cells and B-2 cells were incubated with agonist anti-Fas MoAb, and apoptosis in GC B-1 cells and B-2 cells was analysed by flow cytometry. Although B-1 cells do not usually take part in the GC reaction, an increase in B-1 cell numbers was observed in the GC of tonsils from IgAN patients. These B-1 cells were likely IgA1 antibody-producing cells, since the prominent IgA subclass in IgAN is generally considered to be IgA1. Although Fas-dependent apoptosis is essential for the elimination of activated B cells, these B-1 cells showed a reduced susceptibility to Fas-mediated apoptosis. It is conceivable that activated B-1 cells may survive in the GC due to impaired apoptosis and thus produce abnormal antibodies. These findings suggest that the immune responses of B-1 cells in the tonsillar GC could thus have an impact on the pathogenesis of IgAN.
Paired cloning of the T cell receptorαandβgenes from a single T cell without the establishment of a T cell clone
Authors: Kurokawa M , Tong J , Matsui T , Masuko-Hongo K , Yabe T , Nishioka K , Yamamoto K , Kato T
Published: March 2003

T cell receptors, which recognize antigen peptides on MHC molecules, are essential probes for the analysis of T cell antigen specificity. The identification of paired T cell receptor (TCR) chains, α/β or γ/δ, usually requires the establishment of T cell clones, which is not always available. In this study, we tried, as an alternative method, the paired cloning of TCR α/β genes directly from a single T cell. T cells were sorted as a single cell from which RNA was extracted. Then, TCR α/β CDR3 regions were amplified from the single cell-derived cDNA by reverse transcriptase-polymerase chain reaction to determine their sequences. We successfully identified pairs of TCR α/β genes, and reconstructed the TCR molecule by a bacterial expression system. This strategy makes it possible to obtain recombinant TCR molecules from a single T cell without cellular cloning and promotes the investigation of T cell antigen specificity.
Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin
Authors: Hilger C , Grigioni F , De Beaufort C , Michel G , Freilinger J , Hentges F
Published: March 2003

The aim of this study was to investigate the recognition pattern of bovine serum albumin (BSA), a major dietary protein by serum IgG and IgA antibodies. Anti-BSA IgG and IgA antibodies were measured by ELISA technique in 3 different cohorts: 578 unselected persons, 84 new-onset insulin-dependent diabetes mellitus (IDDM) patients and 103 atopic persons. In order to characterize the recognition pattern of the different BSA domains, recombinant BSA and recombinant fragments covering the 3 BSA domains were produced. BSA digestion was monitored in simulated gastric fluid experiments by means of domain specific monoclonal antibodies.

IgG and IgA antibody titres to native BSA were highest in IDDM patients. The three major BSA domains were equally well recognized by IgG antibodies of the three cohorts. Interestingly all three study groups showed a dissociation of their IgG and IgA antibody response to the first BSA domain. The ratio of IgG to IgA antibodies recognizing this domain was 93%/42% in controls, 92%/37% in IDDM patients and 80%/47% in atopic persons. In simulated gastric fluid experiments, the first BSA domain was the first to become undetectable to specific monoclonal antibodies during digestion.

In conclusion humoral IgG and IgA antibodies recognize the major BSA domains with different frequencies. The N-terminal domain of BSA, the first to be degraded during simulated gastric digestion is less well recognized by IgA antibodies. This suggests that early digestion is negatively correlated to the IgA antibody response and that the IgA response associated to the gut associated lymphoid tissue (GALT) and the systemic IgG antibody responses are independent.
Membrane cofactor protein (MCP; CD46) expression in transgenic mice
Authors: Kemper C , Leung M , Stephensen C B , Pinkert C A , Liszewski M K , Cattaneo R , Atkinson J P
Published: March 2003

Human membrane cofactor protein (MCP; CD46) is a widely distributed complement regulator. In the mouse, expression of MCP is largely restricted to the testis while a related, widely expressed protein (Crry) appears to perform MCP's (CD46) regulatory activity. We have developed two mouse strains transgenic for human MCP (CD46) utilizing an ∼ 400 kb YAC clone carrying the complete gene. A third mouse strain was generated using an overlapping YAC clone isolated from a second library. The expression of human MCP (CD46) in these mouse strains was characterized by immunohistochemistry, FACS, Western blotting and RT-PCR. No differences were detected in the isoform pattern or distribution among the three strains, although the expression level varied according to how many copies of the gene were integrated. The expression profile closely mimicked that observed in humans, including the same pattern of isoform expression as the donor. In addition, tissue-specific isoform expression in the kidney, salivary gland and brain paralleled that observed in man. The transgenic mice expressed low levels of MCP (CD46) on their E, in contrast to humans but in line with most other primates. These mice should be a useful tool to analyse tissue-specific expression, to establish animal models of infections and to characterize the role of MCP (CD46) in reproduction.
Lack of evidence for the Th2 predominance in patients with chronic hepatitis C
Authors: Bergamini A , Bolacchi F , Cerasari G , Carvelli C , Faggioli E , Cepparulo M , Demin F , Uccella I , Bongiovanni B , Niutta P , Capozzi M , Lupi M , Piscitelli E , Rocchi G , Angelico M
Published: March 2003

A T helper (Th)1 to Th2 shift has been proposed to be a critical pathogenic determinant in chronic hepatitis C. Here, we evaluated mitogen-induced and hepatitis C virus (HCV) core antigen-induced cytokine production in 28 patients with biopsy-proven chronic hepatitis C. Flow cytometry demonstrated that after mitogenic stimulation the percentage of Th2 cells (IL-4 + or IL-13 +) and Th0 cells (IFN-γ/IL-4 + or IL-2/IL-13 +) did not differ between patients and controls. In contrast, the percentage of Th1 cells (IFN-γ + or IL-2 +) was significantly increased in CD4 + , CD8 + , ‘naive’-CD45RA + and ‘memory’-CD45RO + T-cell subsets from patients versus controls. Similar results were obtained by ELISA testing supernatants from mitogen-stimulated, unfractionated peripheral blood mononuclear cell (PBMC) cultures. Interferon-alpha treatment was associated with a reduction in the mitogen-induced Th1 cytokine response in those patients who cleared their plasma HCV-RNA. Analysis of cytokine expression by CD4 + T cells after HCV core antigen stimulation in a subgroup of 13 chronic hepatitis C patients demonstrated no cytokine response in 74% of these patients and an IFN-γ-restricted response in 26%. Finally, no Th2 shift was found in lipopolysaccharide-stimulated monocytes. These data indicate that a Th1 to Th2 shift does not occur in chronic hepatitis C.
IncreasedMycobacterium tuberculosisgrowth in HIV-1-infected human macrophages: role of tumour necrosis factor-α
Authors: Imperiali F G , Zaninoni A , La Maestra L , Tarsia P , Blasi F , Barcellini W
Published: March 2003

Synergism between Mycobacterium tuberculosis (M. tuberculosis) and HIV-1 infections was demonstrated in several in vitro models and clinical studies. Here, we investigated their reciprocal effects on growth in chronically HIV-1-infected promonocytic U1 cells and in acutely infected monocyte-derived macrophages (MDM). Phagocytosis of M. tuberculosis induced HIV-1 expression in U1 cells, together with increased TNF-α production. M. tuberculosis growth, evaluated by competitive PCR, was greater in HIV-1-infected MDM compared to uninfected cells. M. tuberculosis phagocytosis induced greater TNF-α and IL-10 production in HIV-1-infected MDM than in uninfected cells. In uninfected MDM, addition of TNF-α and IFN-γ decreased, whereas IL-10 increased M. tuberculosis growth. On the contrary, in HIV-1-infected MDM, addition of TNF-α and IFN-γ increased, whereas IL-10 has no effect on M. tuberculosis growth. TNF-α seems to play a pivotal role in the enhanced M. tuberculosis growth observed in HIV-1-infected MDM, being unable to exert its physiological antimycobacterial activity. Here, for the first time we demonstrated an enhanced M. tuberculosis growth in HIV-1-infected MDM, in line with the observed clinical synergism between the two infections.
Identification of Th2-type suppressor T cells amongin vivoexpanded ocular T cells in mice with experimental autoimmune uveoretinitis
Authors: Keino H , Takeuchi M , Suzuki J , Kojo S , Sakai J , Nishioka K , Sumida T , Usui M
Published: March 2003

Experimental autoimmune uveoretinitis (EAU), which is a T cell mediated organ specific autoimmune disease, is induced by immunization with interphotoreceptor retinoid binding protein (IRBP) in susceptible strains of mice. It has been found that IRBP-derived peptide 518–529 (p518–529) generates Th2-type responses and inhibits IRBP-induced EAU, indicating that the p518–529 might be an epitope for suppressor T cells in IRBP-induced EAU. First, we observed that there were T cells producing the Th2 type cytokines such as IL-4 and IL-10 in late phase of EAU. Furthermore, to examine whether p518–529-reactive T cells expand in the eye during EAU, T cell receptor (TCR) of ocular T cells was compared with that of p518–529 reactive T cells in spleen from mice with EAU by PCR-single strand conformation polymorphism (PCR-SSCP) and nucleotide sequence analysis. SSCP and sequence analyses indicated that p518–529 reactive TCR BV10+ T cells bearing amino acid motif(PWG) and TCR BV13+ T cells bearing amino acid motif(PGLGGY) in their complementary-determining region 3 (CDR3) region were clonally expanding in ocular tissues on day 28 after immunization, although these T cells were not detected on day 14. These findings demonstrate that p518–529 reactive Th2-type T cells expand oligoclonally in the uveitic eyes in the late stage of EAU and may function as Th2-type suppressor T cells for improvement of the disease.
Altered phenotype and function of blood dendritic cells in multiple sclerosis are modulated by IFN-βand IL-10
Authors: Huang Y-M , Stoyanova N , Jin Y-P , Teleshova N , Hussien Y , Xiao B-G , Fredrikson S , Link H
Published: March 2003

Multiple sclerosis (MS) is assumed to result from autoaggressive T cell-mediated immune responses, in which T helper type 1 (Th1) cells producing cytokines, e.g. IFN-γ and lymphotoxin promote damage of oligodendrocyte-myelin units. Dendritic cells (DCs) as potent antigen presenting cells initiate and orchestrate immune responses. Whether phenotype and function of DCs with respect to Th1 cell promotion are altered in MS, are not known. This study revealed that blood-derived DCs from MS patients expressed low levels of the costimulatory molecule CD86. In addition, production of IFN-γ by blood mononuclear cells (MNCs) was strongly enhanced by DCs derived from MS patients. IFN-β and IL-10 inhibited the costimulatory capacity of DCs in mixed lymphocyte reaction (MLR) and showed additive effects on suppression of IL-12 production by DCs. Correspondingly, DCs pretreated with IFN-β and IL-10 significantly suppressed IFN-γ production by MNCs. IFN-β in vitro also upregulated CD80 and, in particular, CD86 expression on DCs. In vitro, anti-CD80 antibody remarkably increased, while anti-CD86 antibody inhibited DC-induced IL-4 production in MLR. We conclude that DC phenotype and function are altered in MS, implying Th1-biased responses with enhanced capacity to induce Th1 cytokine production. In vitro modification of MS patients' DCs by IFN-β and IL-10 could represent a novel way of immunomodulation and of possible usefulness for future immunotherapy of MS.
Differential requirement for interferon-γto restrict the growth of or eliminate some recently identified species of nontuberculous mycobacteriain vivo
Authors: Ehlers S , Richter E
Published: March 2003

In recent years, a number of newly identified species of the genus Mycobacterium (M.) have been isolated from tissues of both immunocompetent and immunocompromised patients, e.g. M. celatum, M. intermedium, M. interjectum, M. bohemicum, M. conspicuum, M. confluentis, M. heidelbergense, M. lentiflavum, and M. branderi. Little is known about their in vivo virulence characteristics and the host factors predisposing to infection with these strains. In an effort to elucidate the pathogenesis of these nontuberculous mycobacterial species, BALB/c and syngeneic IFNγ-deficient (GKO) mice were intravenously infected with 106 colony forming units of each of these species, and bacterial growth in infected organs and the development of splenomegaly and granulomatous liver lesions were examined for a period of 3 months. Based on their in vivo virulence, mycobacterial strains could be divided into three major groups: (i) Most species examined either grew progressively or persisted at plateau levels in the livers and spleens of immunocompetent mice, and their growth was increased in GKO mice. (ii) M. heidelbergense, M. intermedium and another species not officially accorded separate taxonomical status were eliminated in BALB/c mice, but persisted in GKO mice. (iii) M. confluentis, M. lentiflavum and another novel species were eradicated even in the absence of IFNγ. Nontuberculous mycobacterial species differed widely in their capacity to induce splenomegaly and lymphadenopathy in GKO mice. In conclusion, IFNγ is a crucial determinant of infection outcome with most, but not all opportunistic mycobacterial species.
Chemokines in human periodontal disease tissues
Authors: Gemmell E , Carter C L , Seymour G J
Published: March 2003

An immunoperoxidase technique was used to examine IP-10 (interferon-gamma inducible protein 10), RANTES (regulated on activation normal T cell expressed and secreted), MCP-1 (monocyte chemoattractant protein-1), and MIP-1alpha (macrophage inflammatory protein-1alpha) in gingival biopsies from 21 healthy/gingivitis and 26 periodontitis subjects. The samples were placed into 3 groups according to the size of infiltrate. MIP-1alpha+ cells were more abundant than the other chemokines with few MCP-1+ cells. The mean percent MIP-1alpha+ cells was higher than the percent MCP-1+ cells (P = 0·02) in group 2 (intermediate size infiltrates) lesions from periodontitis subjects, other differences not being significant due to the large variations between tissue samples. Analysis of positive cells in relation to CD4/CD8 ratios showed that with an increased proportion of CD8+ cells, the mean percent MIP-1alpha+ cells was significantly higher in comparison with the mean percent RANTES+ and MCP-1+ cells (P < 0·015). Endothelial cells were MCP-1+ although positive capillaries were found on the periphery of infiltrates only. Keratinocyte expression of chemokines was weak and while the numbers of healthy/gingivitis and periodontitis tissue sections positive for IP-10, RANTES and MCP-1 reduced with increasing inflammation, those positive for MIP-1alpha remained constant for all groups. In conclusion, fewer leucocytes expressed MCP-1 in gingival tissue sections, however, the percent MIP-1alpha+ cells was increased particularly in tissues with increased proportions of CD8 cells and B cells with increasing inflammation and also in tissues with higher numbers of macrophages with little inflammation. Further studies are required to determine the significance of MIP-1alpha in periodontal disease.
Differential recognition of obligate anaerobic bacteria by human mannose-binding lectin
Authors: Townsend R , Read R C , Turner M W , Klein N J , Jack D L
Published: March 2003

Deficiency of the innate, humoral immune component mannose-binding lectin (MBL) predisposes individuals to a variety of infections, but the importance of MBL in infection by anaerobes has not been addressed. The attachment of MBL to a wide range of anaerobic bacteria associated with human disease and colonization was surveyed. The results suggest that for the species we examined, resistance to MBL binding may be associated with organisms that are more commonly pathogenic and that MBL binding to some bacteria may be phase variable.
Subtractive screening reveals up-regulation of NADPH oxidase expression in Crohn's disease intestinal macrophages
Authors: Hausmann M , Spöttl T , Andus T , Rothe G , Falk W , Schölmerich J , Herfarth H , Rogler G
Published: March 2003

Macrophages play a central role during the pathogenesis of inflammation. In normal intestinal mucosa surface expression of typical macrophage markers such as CD14, CD16, CD11b or T-cell co-stimulatory molecules such as CD80 or CD86 is low indicating anergy and low pro-inflammatory activity of these cells. During inflammatory bowel disease (IBD) the mucosa is invaded by a population of macrophages displaying these markers, secreting higher cytokine levels and representing an activated cell population. CD33+ cells (macrophages) were isolated from normal and Crohn's disease mucosa and mRNA was isolated by polyT magnetic beads. A subtractive screening was performed subtracting mRNA from normal macrophages from those of Crohn's disease macrophages. Oxidative burst activity was determined by flow cytometry. Seventy clones were obtained by the subtractive mRNA screening. Sequencing showed > 99% homology to mRNA of monocyte chemoattractant protein-1 (MCP-1) for three clones. Five clones obtained by subtraction revealed > 99% homology to mRNA of cytochrome b (subunit gp91). Differential expression of the cytochrome b subunit gp91 and the cytosolic NADPH oxidase subunit p67 was confirmed by RT-PCR and ‘virtual’ Northern blots. The fluorescence ratio of stimulated versus unstimulated cells was 0·9 ± 0·16 in control macrophages indicating a lack of oxidative burst activity. In Crohn's disease this ratio was significantly increased to 1·80 ± 0·8 (P = 0·004) confirming the molecular data. In conclusion NADPH oxidase mRNA is down-regulated or absent in macrophages from normal mucosa correlating with a lack of oxidative burst activity. In IBD macrophage-oxidative burst activity is increased and NADPH oxidase mRNA induced. Inhibition of NADPH oxidase could be a new therapeutical target in IBD and reduce mucosal tissue damage in active IBD.
Analysis of mercury-induced immune activation in nonobese diabetic (NOD) mice
Authors: Brenden N , Rabbani H , Abedi-Valugerdi M
Published: March 2003

In susceptible mice, the heavy metal ion mercury is able to induce a strong immune activation, which resembles a T helper 2 (Th2) type of immune response and is characterized by a polyclonal B cell activation, formation of high levels of IgG1 and IgE antibodies, production of autoantibodies of different specificities and development of renal IgG deposits. In the present study, we analysed the in vivo effects of mercury in nonobese diabetic (NOD) mice, which is believed to develop a spontaneous Th1 cell-mediated autoimmune diabetes similar to type 1 diabetes in humans. Three weeks of treatment with mercury induced a strong Th2 like immune/autoimmune response in NOD mice. This response was characterized by an intensive increase in splenic IgG1 antibody secreting cells, a marked elevation in serum IgE levels, a substantial increase in splenic IL-4 mRNA, but a significant decrease in splenic IFN-γ mRNA. Mercury-induced IgG1 antibodies were mainly against ssDNA, TNP and thyroglobulin, but not against nucleolar antigen. Moreover, mercury-injected NOD mice developed high titres of IgG1 deposits in the kidney glomeruli. We further tested if the generated Th2 response could interfere with the development of insulitis and diabetes in NOD mice. We found that three weeks of treatment with mercury was also able to significantly suppress the development of insulitis and postpone the onset of diabetes in these mice. Thus, mercury-induced immune activation can counter-regulate the Th1 cell-mediated autoimmune responses and confer a partial protection against autoimmune diabetes in NOD mice.