bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular interferon-gamma (IFN-γ) production in normal children and children with atopic dermatitis
Authors: CAMPBELL D E , FRYGA A S , BOL S , KEMP A S
Published: March 2003

A reduction in the in vitro production of IFN-γ has been consistently described in atopic dermatitis (AD). Whether this reduction is due to a decrease in the population of peripheral blood mononuclear cells (PBMC) producing IFN-γ or reduced IFN-γ production per cell, or a combination of both is not clear. We have examined the intracellular production of IFN-γ in children with AD and in healthy non-atopic controls. As Staphylococcus aureus colonization is a feature of childhood AD, and is postulated to contribute to the cutaneous inflammation in atopic dermatitis, S. aureus and Staphylococcal enterotoxin B (SEB) were used to activate PBMC. Stimulated PBMC from subjects with AD had significantly fewer IFN-γ-containing cells in response to SEB (P < 0.001) and S. aureus (P < 0.01) than normal non-atopic children. In addition, SEB-stimulated PBMC from children with AD had less IFN-γ per cell than normal non-atopic children (P < 0.01). Reduction in the proportion of cells containing IFN-γ was seen in CD4+, CD8+ and natural killer (NK) cells in PBMC from children with AD. Our findings indicate that reduced production of IFN-γ observed in childhood AD is due to both a decrease in the number of IFN-γ-producing cells and a reduced amount of IFN-γ production per cell. Furthermore, we found that this defect was not confined to CD4+ T cells, suggesting a more generalized defect in IFN-γ production in childhood AD.
Reduced naive and increased activated CD4 and CD8 cells in healthy adult Ethiopians compared with their Dutch counterparts
Authors: MESSELE T , ABDULKADIR M , FONTANET A L , PETROS B , HAMANN D , KOOT M , ROOS M T L , SCHELLEKENS P T A , MIEDEMA F , RINKE DE WIT T F
Published: March 2003

To assess possible differences in immune status, proportions and absolute numbers of subsets of CD4+ and CD8+ T cells were compared between HIV− healthy Ethiopians (n = 52) and HIV− Dutch (n = 60). Both proportions and absolute numbers of naive CD4+ and CD8+ T cells were found to be significantly reduced in HIV− Ethiopians compared with HIV−Dutch subjects. Also, both proportions and absolute numbers of the effector CD8+ T cell population as well as the CD4+ CD45RA− CD27− and CD8+ CD45RA− CD27− T cell populations were increased in Ethiopians. Finally, both proportions and absolute numbers of CD4+ and CD8+ T cells expressing CD28 were significantly reduced in Ethiopians versus Dutch. In addition, the possible association between the described subsets and HIV status was studied by comparing the above 52 HIV− individuals with 32 HIV+ Ethiopians with CD4 counts > 200/μl and/or no AIDS-defining conditions and 39 HIV+ Ethiopians with CD4 counts < 200/μl or with AIDS-defining conditions. There was a gradual increase of activated CD4+ and CD8+ T cells, a decrease of CD8+ T cells expressing CD28 and a decrease of effector CD8+ T cells when moving from HIV− to AIDS. Furthermore, a decrease of naive CD8+ T cells and an increase of memory CD8+ T cells in AIDS patients were observed. These results suggest a generally and persistently activated immune system in HIV−Ethiopians. The potential consequences of this are discussed, in relation to HIV infection.
Imbalance of IL-1β and IL-1 receptor antagonist mRNA in liver tissue from hepatitis C virus (HCV)-related chronic hepatitis
Authors: GRAMANTIERI L , CASALI A , TRERÈ D , GAIANI S , PISCAGLIA F , CHIECO P , COLA B , BOLONDI L
Published: March 2003

Increased levels of IL-1β and IL-1 receptor antagonist (IL-1Ra) have been found in serum of patients with chronic liver diseases, although their expression in liver tissue has not been extensively investigated. The aim of this study was therefore to examine the relationship between IL-1β and IL-1Ra at tissue level in patients with HCV-related chronic active hepatitis (CAH) of varying degrees of severity. IL-1β and IL-1Ra mRNA expression was investigated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 31 patients with CAH of varying severity (classified as minimal/mild in 13 cases and moderate/severe activity in 18 cases) and in 12 control subjects. Quantitative evaluation of IL-1β and IL-1Ra corresponding bands was performed by densitometric image analysis, and expressed in arbitrary units. The 12 controls expressed a similar pattern with a mean IL-1β/IL-1Ra ratio of 1.03 (1.03 ± 0.15 (mean ± s.e.m.), median 0.92, range 0.71–1.45). Minimal/mild activity CAH showed a prevalence of IL-1Ra mRNA expression (1.14 ± 0.64, median 0.43, range 0–8.75) when compared with controls (0.27 ± 0.04, median 0.23, range 0.11–0.45) and with moderate/severe activity CAH (0.20 ± 0.04, median 0.12, range 0–0.67; P = 0.01). Since IL-1β expression was similar in the three groups, a significantly different IL-1β/IL-1Ra ratio emerged between controls, patients with moderate/severe CAH (2.22 ± 0.48, median 2.76, range 0–6.12) and those with minimal/mild activity CAH (0.62 ± 0.15, median 0.5, range 0–1.58, P = 0.005). Patients with higher grades of fibrosis showed a higher IL-1β/IL-1Ra ratio (2.49 ± 0.56, median 2.15, range 0.35–6.12) in comparison with lower grade fibrosis (1.06 ± 0.30, median 0.59, range 0.03–4.50) and control patients (P = 0.01). These results suggest that an imbalance between IL-1β and IL-1Ra, at the tissue level, may contribute to the pathogenesis and the activity of chronic active hepatitis C.
Superantigen-induced T cell responses in acute rheumatic fever and chronic rheumatic heart disease patients
Authors: BHATNAGAR A , GROVER A , GANGULY N K
Published: March 2003

CD4+ and CD8+ T cells from healthy donors, acute rheumatic fever (ARF) and chronic rheumatic heart disease (CRHD) patients responded variably to a superantigen from Streptococcus pyogenes—Streptococcal pyrogenic erythrogenic toxin A (SPE-A). In vitro culture of CD4+ T cells from ARF patients (CD4-ARF) with SPE-A exhibited a Th1 type of response as they produced high levels of IL-2, while CD4+ T cells from CRHD patients (CD4-RHD) secreted IL-4 and IL-10 in large amounts, i.e. Th2 type of cytokine profile. The skewing of human CD4+ T cells (in response to SPE-A stimulation) to Th1 or Th2 type reflects the role of the two subsets in a disorder with differing intensities at the two extremes of the spectrum. Moreover, the anergy induction experiments revealed that CD8-ARF and CD8-RHD undergo anergy (to different extents), whereas CD4+ T cells do not, in response to re-stimulation by SPE-A. These results initially demonstrate that both CD4+ and CD8+ T cells respond differentially to SPE-A, and hence it is an important observation with respect to the pathogenesis of ARF/CRHD. Anergy in CD8+ T cells in the presence of SPE-A in vitro goes a step further to show the clinical relevance of these cells and their possible role in suppression of the disease.
Early reduction of the over-expression of CD40L, OX40 and Fas on T cells in HIV-1 infection during triple anti-retroviral therapy: possible implications for lymphocyte traffic and functional recovery
Authors: SOUSA A E , CHAVES A F , DOROANA M , ANTUNES F , VICTORINO R M M
Published: March 2003

Fas, CD40L and OX40 are members of the tumour necrosis factor (TNF) receptor superfamily with critical roles in T cell activation and death, B cell function, dendritic cell maturation and leucocyte traffic regulation. The aim of this study was to evaluate the effects of anti-retroviral therapy (HAART) on CD40L, OX40 and Fas expression on freshly isolated peripheral blood T cells by three-colour flow cytometry and compare them with lymphoproliferative responses, peripheral blood cell counts and viral load. Fourteen asymptomatic HIV-1+ patients treated with Lamivudine, Stavudine and Nelfinavir were prospectively investigated sequentially for 48 weeks. At baseline, patients exhibited significantly enhanced proportions and counts of CD40L+ and OX40+ cells within the CD4 subset which were corrected by weeks 8–16 of HAART. Interestingly, in the five patients showing viral load rebound during therapy in spite of increasing CD4 counts, the reduction of the levels of these costimulatory molecules was similarly maintained. Therapy induced a decrease in the over-expression of Fas, particularly in the CD4 subset where normal levels were reached at week 8. This reduction occurred in parallel with the major recovery of lymphoproliferative responses. Higher basal levels and lower reduction of Fas were associated with suboptimal suppression of viraemia. In conclusion, this previously undescribed increased expression of CD40L and OX40 may play a role in the HIV-associated pan-immune activation and represent a possible target for immunointervention, as suggested for several immunologically mediated diseases. Moreover, HAART induced an early correction of the over-expression of Fas, CD40L and OX40 in CD4 T cells which could be involved in the recovery of the cell traffic disturbances and in the T cell renewal capacity.
Studies of the mechanism of cytolysis by tumour-infiltrating lymphocytes
Authors: HISHII M , KURNICK J T , RAMIREZ-MONTAGUT T , PANDOLFI F
Published: March 2003

In order to determine the mechanism of tumour destruction by tumour-infiltrating lymphocytes (TIL), we examined the ability of both CD4+ and CD8+ effector TIL, and TIL clones, to manifest granzyme-mediated and Fas-mediated destruction of tumour targets. In many in vitro studies TIL have been shown to manifest anti-tumour reactivity, yet many tumours escape immunological destruction. To investigate the role of Fas expression and the concomitant sensitivity to the inducibility of apoptotic death, we derived TIL from four melanomas and one glioma. The glioma, and all but one of the melanomas, expressed Fas, but Fas-mediated apoptosis could only be detected if the targets were treated with cyclohexamide. The melanomas and the glioma all expressed detectable cytoplasmic Bcl-2 protein, known to exert anti-apoptotic activity. Lysis of tumours by CD8-enriched cultures and CD8+ clones was Ca2+-dependent and could not be modified by an anti-Fas MoAb. In CD4-enriched cultures or CD4+ clones with cytotoxic potential against tumour cells, cytotoxicity was also Ca2+-dependent. As Ca2+-dependent cytotoxicity is usually the result of secretion of perforin/granzyme-B, we investigated the presence of perforin in cytotoxic CD4+ clones and demonstrated the presence of granular deposits of this enzyme in some of the CD4+clones. Although an anti-Fas MoAb did not block the lysis of melanoma targets by CD4+ clones, the examination of Fas-dependent targets demonstrated that these clones also had the potential to kill by the Fas/Fas ligand system. These data suggest that the predominant mechanism in tumour killing by TIL appears to be perforin–granzyme-dependent, and that the solid tumour cell lines we studied are less susceptible to Fas-mediated apoptosis. As non-apoptotic pathways may enhance tumour immunogenicity, exploitation of the perforin–granzyme-dependent cytotoxic T lymphocyte (CTL) pathways may be important for achieving successful anti-tumour responses.
Elevated serum levels of soluble membrane cofactor protein (CD46, MCP) in patients with systemic lupus erythematosus (SLE)
Authors: KAWANO M , SEYA T , KONI I , MABUCHI H
Published: March 2003

Membrane cofactor protein (MCP, CD46) is a cell surface complement regulatory protein which acts as a cofactor for the factor I-mediated cleavage of the activated complement components C3b/C4b. To evaluate the clinical usefulness of serum soluble CD46 as a marker of disease activity in patients with SLE, serum levels of sCD46 were measured by ELISA, using two MoAbs (M160 and M177), each of which recognized two different epitopes on CD46 molecule in SLE, other autoimmune diseases and healthy controls. Serum sCD46 levels in active SLE patients (30.5 ± 14.1 ng/ml) were significantly higher than those of inactive SLE (5.8 ± 7.1 ng/ml; P = 0.0003), rheumatoid arthritis (14.9 ± 11.6 ng/ml; P = 0.0218), primary Sjögren's syndrome (12.3 ± 11.6 ng/ml; P = 0.0039) and normal controls (7.3 ± 3.6 ng/ml; P = 0.0005). The elevated serum sCD46 levels in active SLE patients significantly decreased from 30.5 ± 14.1 ng/ml to 8.0 ± 6.3 ng/ml after effective corticosteroid and immunosuppressant therapy (P = 0.018). Additionally, we found a significant negative association between increasing concentration of sCD46 and decreasing levels of CH50 in SLE (r = –0.598, P = 0.0009). These results suggest that sCD46 reflects in vivo activation of complement system and provides an additional useful serum parameter of active SLE.
Oligoclonal T cell expansions in patients with Behçet's disease
Authors: DIRESKENELI H , EKSIOGLU-DEMIRALP E , KIBAROGLU A , YAVUZ S , ERGUN T , AKOGLU T
Published: March 2003

Behçet's disease (BD) is a multisystem disorder with oral and genital ulcers, mucocutaneous, ocular, joint, vascular and central nervous system involvement. In this study, the peripheral T cell repertoire was analysed in patients with BD with MoAbs against T cell receptor (TCR) Vβ gene products in CD4+ and CD8+ T cell compartments, and these were compared with rheumatoid arthritis (RA) patients and healthy controls (HC). In the CD4+ T cell compartment, oligoclonal TCR Vβ expression was observed in 56% of BD (10/18), 71% of RA (5/7) patients and 21% (3/14) of HC. In the CD8+ T cell group 50% of BD (9/18), 57% of RA patients and 28% of HC (4/14) had an oligoclonal TCR repertoire. An increase of TCR Vβ5.1 subset was observed in five BD patients among CD8+ T cells. Other elevations of TCR Vβ subsets were heterogeneously distributed with one to three different Vβ subsets. Our results suggest an antigen-driven oligoclonal increase of T cells in BD. There was no overall increase in any Vβ group to suggest a superantigen effect. Analysis of the responsible antigens causing the increase in T cell subsets may give insights into the aetiopathogenesis of BD and immunomodulation of these T cells may lead to new treatments.
Prevention of B220+ T cell expansion and prolongation of lifespan induced byLactobacillus caseiin MRL/lpr mice
Authors: Mike A , Nagaoka N , Tagami Y , Miyashita M , Shimada S , Uchida K , Nanno M , Ohwaki M
Published: March 2003

We examined the therapeutic effect of heat-killed Lactobacillus casei (LC) on MRL/lpr mice. Ingestion of a diet containing 0.05% (w/w) LC from the weaning period prolonged the lifespan and tended to reduce the proportion of B220+ T cells in the spleen and mesenteric lymph nodes (MLN) of MRL/lpr mice. When LC was intraperitoneally injected once a week after the age of 8 weeks, I-A− macrophages accumulated in the spleen as well as the peritoneum and macrophage progenitors increased in the bone marrow. Moreover, the amount of IL-6 mRNA in peritoneal macrophages was reduced by LC injection. Splenocytes from LC-injected MRL/lpr mice exhibited lower proliferative responses to mitogens than those from control MRL/lpr mice and the increase in number of B220+ T cells in the spleen and MLN was prevented by LC injection. However, LC injection affected neither expression of interferon-gamma (IFN-γ) and IL-4 mRNAs nor proliferative capacities of splenic T cells. Our findings demonstrate that LC injection accelerates macrophage recruitment and prevents the expansion of B220+ T cells without affecting the functions of T cells in MRL/lpr mice. These immunological modulations induced by LC may lead to prolongation of the lifespan of MRL/lpr mice.
Increased levelsin vivoof mRNAs for IL-8 and macrophage inflammatory protein-1α (MIP-1α), but not of RANTES mRNA in peripheral blood mononuclear cells of patients with atopic dermatitis (AD)
Authors: Hatano Y , Katagiri K , Takayasu S
Published: March 2003

Chemokines play an important role in the selective movement of leucocytes into inflammatory areas and they also activate various cells in inflamed tissues. However, it is unclear which cells are the main sources of chemokines in actual inflammatory diseases, even though both mononuclear cells and non-inflammatory resident cells are able to produce chemokines in vitro and the former cells are also the main target of chemokines. To clarify the roles of chemokines that are produced by mononuclear cells in AD, we measured levels in vivo of mRNA for IL-8 and MIP-1α, as well as the level of regulated upon activation normal T cell expressed and secreted (RANTES) mRNA in freshly isolated peripheral blood mononuclear cells from patients with AD. We compared the results with those from psoriatic patients, and patients without AD who were suffering from other cutaneous diseases and eosinophilia. Levels of mRNAs were determined by semiquantitative reverse transcriptase-polymerase chain reactions. Levels of IL-8 and MIP-1α mRNA were elevated not only in atopic patients but also in non-atopic patients with inflammatory skin disease associated with eosinophilia, compared with levels in psoriatic patients and healthy controls. Levels of RANTES mRNA were similar in atopic patients but they were lower in the other two groups of patients when compared with levels in healthy controls. In atopic patients, the levels of both IL-8 and MIP-1α mRNAs but not of RANTES mRNA decreased with improvements in symptom scores after therapy. These findings suggest that mononuclear cells are not only the target of chemokines but might also play an important role in the pathogenesis of AD by producing IL-8 and MIP-1α.
Interferon-alpha (IFN-α) enhances cytotoxicity in healthy volunteers and chronic hepatitis C infection mainly by the perforin pathway
Authors: Kaser A , Enrich B , Ludwiczek O , Vogel W , Tilg H
Published: March 2003

Cell-mediated cytotoxicity is exerted via perforin and Fas ligand (FasL). We have recently shown that IFN-α up-regulates FasL expression in T cells isolated from healthy volunteers and augments activation-induced T cell death. Since the Fas/FasL system is implicated in the pathogenesis of hepatic failure and both molecules have been shown to be up-regulated in hepatitis C virus (HCV) infection, we studied whether cytotoxicity via the FasL system is enhanced by IFN-α and therefore could contribute to hepatic injury. We investigated FasL and perforin expression in peripheral blood mononuclear cells (PBMC) derived from HCV+ donors by Northern analysis and soluble FasL synthesis by ELISA. Natural killer (NK) cell and cytotoxic T lymphocyte (CTL) cytotoxicity was studied by 51Cr-release assays. IFN-α up-regulates FasL mRNA and protein synthesis in mitogen-activated PBMC of HCV+ individuals, as previously found in healthy subjects. Stimulation with IFN-α increases perforin mRNA levels in PBMC. In NK cytotoxicity assays, the enhancement of cytotoxicity by IFN-α is mainly due to the perforin pathway, while the FasL pathway plays only a minor role. In CTL cytotoxicity experiments neither the FasL nor the perforin pathway is further enhanced by IFN-α. Our data suggest that up-regulation of perforin by IFN-α results in elevated cytotoxicity, suggesting that IFN-α might support elimination of virally infected cells via this pathway. In contrast, the major effect of IFN-α on the Fas/FasL system might be the enhanced elimination of activated T cells as a means of finally limiting a T cell response.
Characterization of immune function and analysis of RAG gene mutations in Omenn syndrome and related disorders
Authors: Wada T , Takei K , Kudo M , Shimura S , Kasahara Y , Koizumi S , Kawa-Ha K , Ishida Y , Imashuku S , Seki H , Yachie A
Published: March 2003

Omenn syndrome was recently found to be caused by missense mutations in RAG1 or RAG2 gene that result in partial V(D)J recombination activity. Although the clinical hallmarks of the disease are well defined, there have been several cases with clinical findings similar to, but distinct from Omenn syndrome. The data on immune functions and RAG gene mutations of such cases are limited. We described five Japanese infants from four unrelated families, including two cases of Omenn syndrome and three cases of related disorders. Sibling cases with typical Omenn phenotype were found to be compound heterozygotes of R396C and L885R mutations in RAG1. The former has been reported in European cases and may constitute a hot spot. The latter is a novel missense mutation. Infants with related disorders exhibited erythroderma, eosinophilia, hypogammaglobulinaemia, decreased number of B cells and skewing to Th2, and their lymph node specimens showed architectural effacement, lymphocyte depletion and histiocytic hyperplasia, each of which is seen characteristically in Omenn syndrome. However, in these cases serum IgE levels were low or undetectable. We found no mutation in RAG genes except for a K820R substitution in RAG1, which was regarded to be a functional polymorphism, in two of these cases. Our study suggests that RAG missense mutation may be a genetic abnormality unique to Omenn syndrome with characteristic clinical and laboratory findings. Variations of Omenn syndrome, or related disorders, may represent a different type of immunodeficiency, distinct from abnormalities in lymphoid-specific recombinase activity.
Targeted Fc2′-3-PE40 chimeric protein abolishes passive cutaneous anaphylaxis in mice
Authors: Fishman A , Prus D , Belostotsky R , Lorberboum-Galski H
Published: March 2003

The alarming increase in the incidence of allergic diseases in the past decade has led to a clear call for more effective treatment. Recently, we reported on the construction of a chimeric protein for targeted elimination of cells expressing FcεRI receptors. This chimeric protein, designated Fc2′-3-PE40, is composed of a Fc fragment of mouse IgE attached to a truncated form of Pseudomonas exotoxin. The Fc2′-3-PE40 chimeric protein was found to be highly cytotoxic to mouse mast cell lines and primary mouse mast cells. We now demonstrate that Fc2′-3-PE40 successfully prevents the development of passive cutaneous anaphylaxis reaction (PCA) in mice. Treatment with Fc2′-3-PE40 for 7 days prevented the PCA reaction in mice by 80% compared with that in control mice given only PBS. Fc2′-3-PE40M, the mutated, enzymatically inactive analogue of Fc2′-3-PE40, did not display this activity. Fc2′-3-PE40 was also effective when given as a single dose 16 h before antigen exposure, resulting in complete inhibition of the PCA reaction. Moreover, treatment with Fc2′-3-PE40 did not cause mast cell degranulation, as the serum histamine values of mice treated with Fc2′-3-PE40 were within the range obtained for control, untreated mice. Thus, the Fc2′-3-PE40 chimeric protein offers a novel approach to the treatment of allergic disorders.
Mechanisms of the in vitro fungicidal effects of human neutrophils against Penicillium marneffei induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)
Authors: Kudeken N , Kawakami K , Saito A
Published: March 2003

We examined the in vitro fungicidal activity of human neutrophils against conidia and yeast cells of Penicillium marneffei. Neutrophils showed a small but significant anti-fungal effect against the yeast form of P. marneffei. Treatment of neutrophils with GM-CSF significantly augmented their anti-fungal activity. In contrast, the conidia form resisted killing even by stimulated neutrophils. Neutrophil fungicidal effect was not inhibited by superoxide dismutase (SOD), while the same treatment significantly suppressed the killing of Candida albicans by GM-CSF-stimulated neutrophils. For effective killing of P. marneffei yeast cells by GM-CSF-stimulated neutrophils, direct contact between the two was essential; interference in such interaction by separation using a 0·45-μm-pored membrane prevented such an effect. Addition of colchicine attenuated GM-CSF-stimulated neutrophil fungicidal activity in a dose-dependent manner. This effect did not appear to be mediated by interference with neutrophil mobility toward yeast cells, because similar results were obtained when the cultures were set in round-bottomed wells which facilitate their direct contact. Finally, granular extracts derived from unstimulated neutrophils significantly suppressed the growth of microorganisms. Pretreatment of neutrophils with GM-CSF markedly enhanced this effect. The fungicidal activity of granular lysates was strongly, but not completely, reduced by heat treatment. Considered together, our results indicate that GM-CSF-stimulated neutrophils killed the yeast form of P. marneffei present in close proximity, probably in a superoxide anion-independent mechanism, but through exocytosis of granular enzymes which were largely heat-labile.
The development of post-kala-azar dermal leishmaniasis (PKDL) is associated with acquisition ofLeishmaniareactivity by peripheral blood mononuclear cells (PBMC)
Authors: Gasim S , Elhassan A M , Kharazmi A , Khalil E A G , Ismail A , Theander T G
Published: March 2003

PKDL develops in about 50% of Sudanese patients treated for visceral leishmaniasis (kala-azar). Patients with kala-azar were entered into this study and followed for a period of up to 2 years. During follow up 12 patients developed PKDL and eight did not. Proliferative responses and cytokine production to Leishmania donovani and control antigens were measured in vitro using PBMC isolated at the time of diagnosis of kala-azar, after treatment of visceral leishmaniasis, during follow up, and at the time of diagnosis of PKDL. Proliferative responses and interferon-gamma (IFN-γ) production were low at diagnosis and increased after treatment of kala-azar in both patients who developed (group 1) and those who did not develop PKDL later (group 2). In group 1, development of PKDL was always associated by an increased PBMC response to Leishmania antigen in proliferation and IFN-γ production assays. There were no differences in Leishmania antigen-induced production of IL-4, IL-5 and IL-10 between or within the two groups. We have previously shown that Leishmania parasites spread to the skin during visceral leishmaniasis and proposed that PKDL was the result of an immunological attack on parasites, which have survived in the skin despite the drug treatment. The finding that PKDL develops after treatment of kala-azar as Leishmania-reactive T cells start to circulate in peripheral blood in sufficient numbers to be detected in in vitro assays supports this hypothesis.
Expression of HLA-DR, costimulatory molecules B7-1, B7-2, intercellular adhesion molecule-1 (ICAM-1) and Fas ligand (FasL) on gastric epithelial cells inHelicobacter pylorigastritis; influence ofH. pylorieradication
Authors: Archimandritis A , Sougioultzis S , Foukas P G , Tzivras M , Davaris P , Moutsopoulos H M
Published: March 2003

There is evidence that Helicobacter pylori infection up-regulates the expression of HLA class II molecules by gastric epithelial cells (GEC). In this study we evaluated whether GEC are capable of expression of costimulatory molecules in H. pylori gastritis. The expression of FasL by GEC, before and after eradication of H. pylori, was also studied. Thirty patients (23 men) aged 27–81 years (53·67 ± 13·99 years (mean ± s.d.)) with dyspepsia were studied. Upper gastrointestinal endoscopy was performed and six biopsies were obtained (antrum, n= 3; corpus, n= 3) for Campylobacter-Like Organisms (CLO) test and histology; 23 (16 men) were H. pylori+ and seven (all men) were H. pylori− by both methods and served as controls. Helicobacter pylori eradication therapy was given to H. pylori+ patients and all patients were re-endoscoped after 116 ± 9 days. Formalin-fixed paraffin-embedded tissue sections were stained by the ABC immunoalkaline phosphatase method. In H. pylori gastritis HLA-DR was expressed and correlated with disease activity (P < 0·01). No HLA-DR was observed in controls. In H. pylori-eradicated patients significant decrease of HLA-DR was found (antrum, P < 0·001). ICAM-1 was expressed by GEC in 80% of H. pylori+ patients; ICAM-1 expression did not correlate with gastritis parameters and decreased significantly after eradication (antrum, P < 0·01). B7-1 and B7-2 were expressed on H. pylori+ samples and their expression decreased after eradication, albeit not significantly. Weak epithelial expression of both B7 molecules was observed in all the controls. FasL was steadily expressed by GEC in both H. pylori+ and H. pylori− patients and remained almost unchanged after eradication. These findings suggest that GEC may acquire antigen-presenting cell properties in H. pylori infection through de novo expression of HLA-DR and costimulatory molecules. This seems to be attenuated after eradication and resolution of mucosal inflammation. The same cells exhibit the capacity to control the inflammatory process, probably by inducing apoptotic cell death to Fas-bearing infiltrating lymphocytes.
Prednisolone inhibits cytokine-induced adhesive and cytotoxic interactions between endothelial cells and neutrophilsin vitro
Authors: Heimbürger M , Lärfars G , Bratt J
Published: March 2003

We assessed whether prednisolone influenced the ability of human polymorphonuclear neutrophils (PMN) to adhere to and cause lysis of human umbilical vein endothelial cells (HUVEC) in vitro (as measured by the release of 51Cr). Pretreatment of the endothelium with IL-1β or tumour necrosis factor-alpha (TNF-α) caused prominent endothelial E-selectin expression and endothelial hyperadhesiveness for neutrophils, as well as PMN-mediated cytotoxicity. All these processes were dose-dependently reduced when prednisolone was added to the assay system. This protective effect remained when HUVEC alone were pretreated with the drug prior to washing and cytokine activation. Likewise, when HUVEC cytotoxicity was induced by the nitric oxide (NO) donor S-nitroso-acetyl-penicillamine (SNAP), prednisolone reduced cell injury significantly. In contrast, prednisolone did not interfere with signalling systems between TNF-α-stimulated HUVEC and quiescent PMN such as IL-8 generation and release of cytosolic Ca2 + in the PMN. Thus, in this in vitro model of vasculitis, prednisolone dose-dependently reduced cytokine-induced E-selectin expression and HUVEC hyperadhesiveness for neutrophils, as well as reducing neutrophil-dependent cytotoxicity against HUVEC via NO-dependent steps.
Change in perforin-positive peripheral blood lymphocyte (PBL) subpopulations following exercise
Authors: Staats R , Balkow S , Sorichter S , Northoff H , Matthys H , Luttmann W , Berg A , Virchow J C
Published: March 2003

Perforin, one of the cytotoxic proteins of the immune system, plays a prominent role in protection against viral and bacterial infections. We investigated its expression in PBL and their CD3+, CD4+, CD8+ and CD16+ and/or CD56+ subpopulations in endurance athletes before and after a triathlon. Lymphocyte subpopulations were analysed by flow cytometry following separation of peripheral blood mononuclear cells and staining with antibodies against specific membrane antigens and intracellular perforin. The number of total lymphocytes decreased from 2.1 × 103/μl before the triathlon to 1.0 × 103/μl 1 h after the triathlon (P < 0.01). Interestingly, there was already a significant spontaneous decline in the percentage of CD3+/perforin+, and in CD8+/perforin+ cells, in the week proceeding the triathlon, when subjects were instructed to refrain from strenuous exercise training. The percentage of CD3+/perforin+, CD8+/perforin+ and CD16+ and/or CD56+/perforin+ cells in each lymphocyte subpopulation decreased 1 h after exercise even further from 14.3% to 5.8% (P < 0.05), 18.5% to 6.5% (P < 0.05) and 77.3% to 67.3%, respectively. However, at 18 h and 48 h after exercise the percentage of perforin-expressing CD3+, CD8+ and CD16+/56+ cells increased again towards baseline levels. Compared with normal controls, baseline perforin co-expression in CD3+ and CD8+ lymphocytes was significantly higher in trained athletes. From our data we conclude that trained athletes have an increased percentage of perforin+ PBL and that following exercise the percentage of perforin+ and therefore potentially cytotoxic lymphocytes transiently decreases in peripheral blood.
T cell lines specific for a synthetic Heymann nephritis peptide derived from the receptor-associated protein
Authors: Wu H , Zhang G Y , Knight J F
Published: March 2003

Pathogenic antigens involved in the induction of Heymann nephritis (HN), an experimental rat model of human membranous nephritis, have been identified in megalin (gp330) and the receptor-associated protein (RAP) [1,2]. A pathogenic epitope has been identified in RAP (amino acid 1–86) that plays a significant role in the formation of immune deposits in glomeruli in HN. A synthetic peptide (P31−53) derived from RAP1−86 contains a pathogenic epitope recognized by antibodies eluted from glomerular immune deposits and includes two putative RT-1B1 MHC class II-binding motifs. We have investigated whether RAP P31−53 can be recognized by T cells. Five peptide-specific T cell lines were generated from regional lymph node (LN) T cells from Lewis rats immunized with P31−53. The T cell lines were characterized by using a T cell proliferation assay for their specificity, FACS and MHC restriction assay for the phenotype, reverse transcription-polymerase chain reaction for TCR Vβ repertoire and cytokine expression, and cloning and sequencing for the analysis of the CDR3 sequence of TCR. The helper function of the T cell line was confirmed by autoantibody production in vitro. In this study, we clearly identify that the synthetic pathogenic peptide P31−53 contains a T cell epitope recognized by CD4+ Th2 cells in Lewis rats. This recognition was restricted by MHC class II RT1.B1. These CD4+ Th2 cells were able to promote B cells to produce specific antibodies and used a restricted set of TCR Vβ genes with preferential usage of Vβ18. A charged amino acid motif at the CDR3 region of predominant TCR Vβ subfamilies may contribute to the specific ability of these cells to recognize the immunogenic T cell epitope within RAP peptide P31−53.
Predominant type-2 response in infants with respiratory syncytial virus (RSV) infection demonstrated by cytokine flow cytometry
Authors: Bendelja K , Gagro A , Baće A , Lokar-Kolbas R , Kršulović-Hrešić V , Drazenović V , Mlinaric-Galinović G , Rabatić S
Published: March 2003

Acute RSV infection in infancy may produce some asthma-like symptoms and may be followed by a recurrent wheeze later in childhood. It has been proposed that RSV infection stimulates type-2 cytokine responses, resembling those found in atopy and asthma. Peripheral blood cells were obtained from RSV-infected infants (n = 30) and healthy controls (n = 10). After in vitro restimulation of the cells, intracellular IL-4 and interferon-gamma (IFN-γ) were measured by flow cytometry. The cells from RSV-infected infants produced more IL-4 and less IFN-γ than those from healthy controls. IL-4 production was more frequent in CD8 than in CD4 cells, and the bias toward IL-4 production was greatest in infants with mild infections, whereas IFN-γ production increased with disease severity. Our conclusions are that RSV infection is associated with IL-4 production in peripheral T cells, and that peripheral blood in infants with severe disease may be depleted of cytokine-producing cells.
Deficiency in circulating natural killer (NK) cell subsets in common variable immunodeficiency and X-linked agammaglobulinaemia
Authors: Aspalter R M , Sewell W A C , Dolman K , Farrant J , Webster A D B
Published: March 2003

Absolute and relative NK cell numbers were determined in peripheral whole blood by flow cytometry in patients with common variable immunodeficiency (CVID) (n = 55) and X-linked agammaglobulinaemia (XLA) (n = 19) on regular immunoglobulin (IVIG) therapy. Absolute CD3−CD16+ NK cell numbers were significantly reduced in CVID patients (median 108/μl, range 23–815), compared with normal subjects (n = 60) (289/μl, range 56–640, P < 0·001). Total lymphocyte concentrations were significantly lower in CVID (median 1587/μl, range 523–7519) compared with normal subjects (median 2019/μl, range 1124–3149, P = 0·004), with the percentage of NK cells also being significantly decreased (median 7·5%, range 3·0–33·0%, compared with 14·2%, range 2·6–30·8%, P < 0·001). In XLA, absolute NK cell numbers (median 140/μl, range 32–551, P < 0·001) but not relative numbers were significantly reduced compared with normal controls. We excluded the possibility that IVIG interferes with in vitro binding of CD16 MoAbs. Further analysis of NK cell subsets showed a deficiency of both CD16+ and CD56+ cells in CVID, most marked in the CD3−CD8dim subpopulation, which may be due to increased homing of these cells to the gut. Serial studies on a small number of patients suggest that IVIG therapy has no short-term effect on NK cells, although we cannot exclude an effect with prolonged use. Although there are no obvious clinical effects of the NK depletion in CVID and XLA, this may be a factor in their predisposition to cancer.
Kinetic studies of the production of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-α) in macrophages stimulated withBurkholderia pseudomalleiendotoxin
Authors: Utaisincharoen P , Tangthawornchaikul N , Kespichayawattana W , Anuntagool N , Chaisuriya P , Sirisinha S
Published: March 2003

The mechanism by which Burkholderia pseudomallei survives in macrophages is not clearly understood. In this study, we demonstrated that the mouse macrophage cell line (RAW 264.7) treated with lipopolysaccharide (LPS) from B. pseudomallei (BP-LPS) produced significantly less NO and TNF-α compared with those stimulated with the LPS from Escherichia coli and Salmonella typhi. The time required for the BP-LPS to trigger substantial NO and TNF-α release was at least 30 min, compared with < 5 min for the E. coli-LPS. A time course study of inducible nitric oxide synthase (iNOS) protein expression also indicated that the time required for macrophages stimulated with the BP-LPS to up-regulate iNOS was longer. The longer time lag for the BP-LPS to activate macrophages was probably due to the delay in up-regulation of iNOS and TNF-α mRNA transcription. These results indirectly suggest that the delay of the mediators' production may be due to a reduced rate of signal transduction initiated by the interaction of BP-LPS with the macrophage cell surface. The use of MoAb to phosphorylated p38 in a Western blot analysis provided data compatible with the notion that the maximum level of phosphorylated p38 from the cells activated with BP-LPS was attained at a slower rate. These results suggest that the unique structure of BP-LPS exhibits a property which may interfere with macrophage cell activation.
Effect of adhesion on inducible nitric oxide synthase (iNOS) production in purified human neutrophils
Authors: Webb J L , Polak J M , Evans T J
Published: March 2003

The production of nitric oxide (NO) within neutrophils is an important element of the innate immune response. We have previously shown that cytokines (IL-1α, tumour necrosis factor-alpha and interferon-gamma) induce human neutrophils in buffy coat preparations to produce iNOS. In order to define better the exact requirements for iNOS production within human neutrophils, we have studied the conditions needed for the production of iNOS in purified neutrophils. In contrast to buffy coat preparations, purified neutrophils in suspension did not produce an increase in iNOS following addition of cytokines. However, when purified neutrophils were allowed to adhere to glass surfaces either uncoated or coated with fetal calf serum (FCS), plasma, fibronectin or laminin, there was an increase in the percentage of iNOS-positive cells. The addition of cytokines during adhesion of these cells increased this proportion further. This was most marked for glass alone and FCS-coated glass on which the proportion of iNOS-positive cells increased to 22·7% and 35·5%, respectively, a significant increase compared with cytokine-treated neutrophils in suspension. Neither transmigration through activated endothelial monolayers nor the addition of soluble intercellular adhesion molecule-1 to purified neutrophil suspensions increased the percentage of iNOS-positive cells following cytokine stimulation. Adhesion of neutrophils to surfaces coated with IgG or complement also failed to increase cytokine-induced iNOS production. We conclude that iNOS production in human neutrophils requires not only cytokine stimulation, but also additional stimuli from adhesion to a surface.
Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells
Authors: Bengtsson Å , Lundberg M , Avila-Cariño J , Jacobsson G , Holmgren A , Scheynius A
Published: March 2003

The thiol antioxidant N-acetyl- l-cysteine (NAC), known as a precursor of glutathione (GSH), is used in AIDS treatment trials, as a chemoprotectant in cancer chemotherapy and in treatment of chronic bronchitis. In vitro, GSH and NAC are known to enhance T cell proliferation, production of IL-2 and up-regulation of the IL-2 receptor. The 120-kD CD30 surface antigen belongs to the tumour necrosis factor (TNF) receptor superfamily. It is expressed by activated T helper (Th) cells and its expression is sustained in Th2 cells. We have analysed the effect of GSH and NAC on the cytokine profile and CD30 expression on human allergen-specific T cell clones (TCC). TCC were stimulated with anti-CD3 antibodies in the presence of different concentrations of GSH and NAC. Both thiols caused a dose dependent down-regulation of IL-4, IL-5 and IFN-γ levels in Th0 and Th2 clones, with the most pronounced decrease of IL-4. Furthermore, they down-regulated the surface expression of CD30, and the levels of soluble CD30 (sCD30) in the culture supernatants were decreased. In contrast, the surface expression of CD28 or CD40 ligand (CD40L) was not significantly changed after treatment with 20 m m NAC. These results indicate that GSH and NAC favour a Th1 response by a preferential down-regulation of IL-4. In addition, the expression of CD30 was down regulated by GSH and NAC, suggesting that CD30 expression is dependent on IL-4, or modified by NAC. In the likely event that CD30 and its soluble counterpart prove to contribute to the pathogenesis in Th2 related diseases such as allergy, NAC may be considered as a future therapeutic agent in the treatment of these diseases.
Intradermal skin test with diabetes specific antigens in patients with type 1 diabetes
Authors: Crinò A , Cavallo M G , Corbi S , Mesturino C A , Ferrazzoli F , Coppolino G , Bizzarri C , Cervoni M , Monetini L , Pozzilli P ,   THE IMDIAB GROUP
Published: March 2003

Cell mediated immune response in vitro to a number of antigens has been reported in patients with Type 1 diabetes. The aim of the present study was to develop an in vivo intradermal (delayed type hypersensitivity) skin test using antigens known to be recognized by lymphocytes of patients with Type 1 diabetes and to compare, where possible, the in vivo response to the in vitro T cell proliferation to the same antigens.

The skin test was performed in the following group of patients: 55 with recent onset Type 1 diabetes; 16 patients with Type 1 diabetes of longer duration; 10 patients with autoimmune thyroid disease and 20 patients with Latent Autoimmune Diabetes in Adults (LADA). Type 1 diabetes specific antigens for the skin test included glutamic acid decarboxilase (GAD65), insulin and beta casein, whereas diabetes non specific antigens included tetanus toxoid, diphteria, proteus, tubercolin, streptococcus, and glycerol as control. A multitest device consisting of heads delivering intradermally 10 µl of solution containing the antigens was applied to the forearms; the specific antigens were injected in one forearm whereas the non specific antigens were injected in the other forearm. Reading of the reaction, which was considered positive in the presence of a nodule of 2 mm diameter was performed 48 h after the multitest application. The in vitro T cell response to diabetes specific antigens used in the multitest was studied using conventional proliferation assays in patients with recent onset Type 1 diabetes and in age matched normal subjects.

Only recent onset Type 1 diabetes patients showed an in vivo positive response to GAD65, such response being detectable in 10 patients (18%). Two patients reacted also to beta casein and insulin, all other patient groups resulted negative but 2 patients with longer duration of Type 1 diabetes. There was no apparent link between the in vivo skin test and in vitro T cell proliferation to GAD65.

We conclude that in vivo cell mediated immune reaction to GAD65, insulin and beta casein can be visualized in a minority of patients with recent onset Type 1 diabetes. Further studies are required to determine specificity and whether altering the dose can improve the sensitivity of the test.
Prophylactic and therapeutic effects of a humanized monoclonal antibody against the IL-2 receptor (DACLIZUMAB) on collagen-induced arthritis (CIA) in rhesus monkeys
Authors: Brok H P M , Tekoppele J M , Hakimi J , Kerwin J A , Nijenhuis E M , De Groot C W , Bontrop R E , ‘t Hart B A
Published: March 2003

CIA in the rhesus monkey is an autoimmune-based polyarthritis with inflammation and erosion of synovial joints that shares various features with human rheumatoid arthritis (RA). The close phylogenetic relationship between man and rhesus monkey makes the model very suitable for preclinical safety and efficacy testing of new therapeutics with exclusive reactivity in primates. In this study we have investigated the prophylactic and therapeutic effects of a humanized monoclonal antibody (Daclizumab) against the α-chain of the IL-2 receptor (CD25). When Daclizumab treatment was started well after immunization but before the expected onset of CIA a significant reduction of joint-inflammation and joint-erosion was observed. A therapeutic treatment, initiated as soon as the first clinical signs of CIA were observed, proved also effective since joint-degradation was abrogated. The results of this study indicate that Daclizumab has clinical potential for the treatment of RA during periods of active inflammation and suppression of the destruction of the joint tissues.
Glomerular T cells in Heymann nephritis
Authors: Walters G , Wu H , Knight J F
Published: March 2003

Active Heymann nephritis (HN) is a rat model of human membranous nephropathy. The appearance of T cells within the glomeruli of HN rats suggests a role for these cells in the pathogenesis of the disease. The aims of this study were to investigate T cells infiltrating the glomerulus in HN in Lewis rats by polymerase chain reaction (PCR) of their Vβ chains, CDR3 spectratyping and sequencing. HN was induced in Lewis rats by immunization with renal tubular antigen (Fx1A) in CFA. Kidneys were collected between 4 and 10 weeks. The glomeruli were separated, homogenized and RNA extracted. RT-PCR, CDR3 spectratyping and sequencing were used to further characterize the infiltrating T cells. Multiple Vβ families showed restriction of their CDR3 spectratypes in each animal. Several TCR Vβ families had identical-sized restricted spectratypes across several different animals. Four Vβ families were sequenced. In three of those four families, the dominant clones showed identical sized CDR3 regions and a striking over-expression of Jβ2.6. Further analysis of the CDR3 regions of the Jβ2.6 clones showed a significant restriction of the amino acids at four of the six CDR3 positions. Glomerular T cells bearing similar CDR3 sequences, using Jβ2.6 and expressing at least two, and possibly more, Vβ genes are involved in the pathogenesis of HN.