bluuebunny/crossref_metadata_2025 huggingface dataset:
Extensive characterization of the immunophenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage−  cells
Authors: Almeida J , Bueno C , Alguero M C , Sanchez M L , Cañizo M C , Fernandez M E , Vaquero J M , Laso F J , Escribano L , San Miguel J F , Orfao A
Published: March 2003

Dendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II+/lineage− were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16 ± 0.06% of the PB nucleated cells and 55.9 ± 11.9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12 ± 0.04% of PB nucleated cells and 44.53 ± 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor α-chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1β (97 ± 5% of positive cells), IL-6 (96 ± 1.1% of positive cells), IL-12 (81.5 ± 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-α) (84 ± 22.1% of positive cells) as well as chemokines such as IL-8 (99 ± 1% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production.
Cytomegalovirus immune globulin intravenous (human) administration modulates immune response to alloantigens in sensitized renal transplant candidates
Authors: Sivasai K S R , Mohanakumar T , Phelan D , Martin S , Anstey M E , Brennan D C
Published: March 2003

One of the important parameters for prolonged waiting time for potential renal transplant recipients is the presence of preformed antibodies to human leucocyte antigen (HLA) antigens, which is often caused by previous transplants, pregnancy or transfusions. In vivo administration of specific and unselected polyclonal intravenous immunoglobulin (IVIGs) preparations have been shown to inhibit anti-HLA alloantibodies in highly sensitized patients. We sought to determine whether Cytogam (Medimmune Inc., Gaithersburg, MD, USA), a hyperimmune anticytomegalovirus immunoglobulin would (1) effect either in vitro or in vivo alloreactivity, and (2) whether Cytogam therapy could reduce the titre of preformed anti-HLA antibodies in highly sensitized patients. Alloreactivity was assessed by mixed lymphocyte reaction (MLR) and cytotoxic T lymphocyte (CTL) assay. A complement dependent microlymphocytotoxicity assay was done to assess for panel reactive antibody (PRA) status and the presence of anti-idiotypic antibodies in the Cytogam preparation. The MLR was inhibited by Cytogam in vitro in a dose-dependent fashion ranging from 31–92%. Significant inhibition of the MLR responses was not observed in recipients who received Cytogam in vivo (50 mg/kg). This could be a result of adminstration of a low dose of IVIG. However, CTL activity against the alloantigens in all individuals assessed was significantly inhibited after in vivo administration of Cytogam. Three of five individuals experienced a decrease of 5–32% in the PRA status at 4 weeks post administration of Cytogam. Cytogam also blocked the anti-HLA antibody titres in a microlymphocytotoxicity assay, suggesting the presence of anti-idiotypic antibodies. Our study was based on a single prophylactic dose of Cytogam (50 mg/kg), however, higher dose administration could be feasible by removing more fluid at dialysis, but should be given intradialytically to avoid volume overload. Overall, our results suggest that Cytogam can modulate the in vivo and in vitro T cell responses against the alloantigens.
Clonal expansion of T cells infiltrating in the airways of non-atopic asthmatics
Authors: Umibe T , Kita Y , Nakao A , Nakajima H , Fukuda T , Yoshida S , Sakamaki T , Saito Y , Iwamoto I
Published: March 2003

CD4+ T cells are thought to play an important role in airway inflammation in both atopic and non-atopic asthma. However, the mechanism by which T cells are activated in non-atopic asthma, where there is no causative antigen identified, is unknown. To elucidate this issue, we analysed T cell receptor (TCR) Vβ gene clonotypes of T cells in the bronchoalveolar lavage fluids (BALF) of non-atopic asthmatics using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and a sequencing method. We found that the numbers of TCR Vβ gene clonotypes of T cells in the BALF of non-atopic asthmatics were significantly increased compared with those of peripheral blood lymphocytes (PBL). We also found that there were several shared amino acid motifs in complementarity-determining region 3 (CDR3) of TCR Vβ genes from those T cell clones in BALF of non-atopic asthmatics, whereas these shared motifs were not found in the same Vβ family genes from PBL in the patients. Moreover, a conserved amino acid sequence was detected in two patients who shared a common HLA-DR allele. These results indicate that the infiltrating T cells in the airways of non-atopic asthmatics recognize relatively limited epitopes of antigens and are clonally expanded by antigen-driven stimulation.
In the rheumatoid pannus, anti-filaggrin autoantibodies are produced by local plasma cells and constitute a higher proportion of IgG than in synovial fluid and serum
Authors: Masson-Bessière C , Sebbag M , Durieux J-J , Nogueira L , Vincent C , Girbal-Neuhauser E , Durroux R , Cantagrel A , Serre G
Published: March 2003

IgG anti-filaggrin autoantibodies (AFA) are the most specific serological markers of rheumatoid arthritis (RA). They include the so-called ‘anti-keratin antibodies’ (AKA) and anti-perinuclear factor (APF), and recognize human epidermal filaggrin and other (pro)filaggrin-related proteins of various epithelial tissues. In this study we demonstrate that AFA are produced in rheumatoid synovial joints. In 31 RA patients, AFA levels were assayed at equal IgG concentrations in paired synovial fluids (SF) and sera. AFA titre-like values determined by indirect immunofluorescence and immunoblotting and AFA concentrations determined by ELISA were non-significantly different in serum and SF, clearly indicating that AFA are not concentrated in SF. In contrast, we demonstrated that AFA are enriched in RA synovial membranes, since the ELISA-determined AFA in low ionic-strength extracts of synovial tissue from four RA patients represented a 7·5-fold higher proportion of total IgG than in paired sera. When small synovial tissue explants from RA patients were cultured for a period of 5 weeks, the profile of IgG and AFA released in the culture supernatants was first consistent with passive diffusion of the tissue-infiltrating IgG (including AFA) over the first day of culture, then with a de novo synthesis of IgG and AFA. Therefore, AFA-secreting plasma cells are present in the synovial tissue of RA patients and AFA can represent a significant proportion of the IgG secreted within the rheumatoid pannus.
Increased synovial fluid levels of soluble CD23 are associated with an erosive status in rheumatoid arthritis (RA)
Authors: Ribbens C , Bonnet V , Kaiser M J , Andre B , Kaye O , Franchimont N , De Groote D , Beguin Y , Malaise M G
Published: March 2003

Synovial fluid (SF) levels of soluble CD23 (sCD23) were determined in 96 patients presenting with an inflammatory knee effusion (73 with RA and 23 with reactive arthritis (ReA) serving as a control inflammatory non-erosive group) and were correlated with the degree of joint destruction, with local immune parameters (IL-1β, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12 and sCD25) and with serum markers of inflammation, C-reactive protein and erythrocyte sedimentation rate. RA patients, classified as erosive or not according to Larsen’s grade, were separated as follows: (i) 13 patients with non-erosive RA; (ii) 16 RA patients with erosions in hands but not in knees, matched for disease duration with the first group; (iii) 44 RA patients with hand and knee erosions, matched with the second group for rheumatoid factor positivity but of longer disease duration. SF sCD23 levels were significantly increased in both erosive RA groups compared with non-erosive diseases, whether RA or ReA (P < 0·05), whose SF levels were not different. SF IL-10 showed a similar profile to that of SF sCD23 and was the only other parameter characteristic of erosive RA, but no direct correlation was found between the two. SF sCD23 was significantly correlated with IL-12 (r = 0·65, P = 0·0001) and sCD25 (r = 0·39, P = 0·0019) exclusively in the two erosive RA populations. In conclusion, these data showing that increased levels of sCD23 are not only found in the SF of erosive joints but also in knee SF of patients with erosive RA but without knee x-ray-diagnosed erosions suggest that this parameter might be of predictive value for joint destruction. Longitudinal studies are however needed to confirm its potential clinical interest.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) but not granulocyte colony-stimulating factor (G-CSF) induces plasma membrane expression of proteinase 3 (PR3) on neutrophilsin vitro
Authors: Hellmich B , Csernok E , Trabandt A , Gross W L , Ernst M
Published: March 2003

The theoretical risk of triggering vasculitis resulting from administration of G-CSF and GM-CSF to patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), such as Wegener’s granulomatosis (WG), who develop agranulocytosis due to cytotoxic therapy, is unknown. Since there is strong evidence that activation of polymorphonuclear neutrophils (PMN) induced by binding of ANCA to PR3 or myeloperoxidase (MPO) expressed on their plasma membrane is involved in the pathogenesis of systemic vasculitides (SV), we studied the surface expression of PR3 and MPO on PMN from healthy donors in response to G-CSF and GM-CSF in vitro by flow cytometric analysis. Increasing doses of G-CSF did not alter PR3 expression on either untreated or tumour necrosis factor-alpha (TNF-α)-primed donor PMN significantly. In contrast, GM-CSF significantly increased PR3 membrane expression on both intact PMN and neutrophils primed with TNF-α. MPO expression was not significantly altered by either G-CSF or GM-CSF. In summary, these data demonstrate that GM-CSF, but not G-CSF, induces plasma membrane expression of PR3 on PMN in vitro. Since in AAV accessibility of the antigen (PR3 or MPO) to the antibody (ANCA) on the plasma membrane of PMN is thought to be essential for neutrophil activation by ANCA, the results of the present study suggest that administration of GM-CSF to patients with WG with neutropenia implies a definite theoretical risk of deterioration of vasculitis via this mechanism.
Characterization of T cell clones derived from lymph nodes and lungs ofPseudomonas aeruginosa-susceptible and resistant mice following immunization with heat-killed bacteria
Authors: Kondratieva T K , Kobets N V , Khaidukov S V , Yeremeev V V , Lyadova I V , Apt A S , Tam M F , Stevenson M M
Published: March 2003

Pseudomonas aeruginosa-resistant BALB/c and susceptible C57Bl/6 (B6) mice were immunized with heat-killed Pseudomonas either in the foot pad or via the trachea, and panels of Pseudomonas-specific T cell clones were developed from lymph nodes and lungs. All clones from either strain, whether of lymph node or lung origin, were CD3+CD4+CD8−TCRαβ+. The efficacy of cloning from lymph node cells was comparable between BALB/c and B6 mice. All lymph node BALB/c clones proliferated in response to Pseudomonas antigen in a dose-dependent manner, and this response was MHC class II-restricted. Vigorous proliferation by a considerable proportion of B6 T cell clones occurred in the absence of specific antigen. Lymph node clones from either strain could be categorized as either Th1 or Th0 on the basis of interferon-gamma (IFN-γ)/IL-4 production. In either mouse strain the efficacy of cloning from lung tissue was substantially lower than from lymph nodes, but the efficacy of cloning from BALB/c compared with B6 lungs was higher. Four lung T cell clones from BALB/c and two from B6 mice were expanded for further analyses, and an interstrain difference was observed in cytokine production. Both B6 lung T cell clones were Th1-like and produced IFN-γ but not IL-4 and IL-10, whereas four BALB/c lung T cell clones were Th2-like and produced IL-4 and IL-10 but not IFN-γ. These observations suggest that differences in the CD4+ Th response in the lung may contribute to differences among inbred mouse strains in the level of resistance to bronchopulmonary Pseudomonas infection.
Influence of fluticasone and salmeterol on airway effects of inhaled organic dust;anin vivoandex vivostudy
Authors: Ek A , Palmberg L , Larsson K
Published: March 2003

Inhalation of dust from swine confinement buildings induces airway inflammation with an increase in both inflammatory cell numbers and secretion of proinflammatory cytokines in the lungs. It is not known whether anti-asthma drugs, which influence airway inflammation in asthma, also influence the airway reaction to inhaled organic dust. In the present study we examined the effects of a ß2-agonist (salmeterol) and an inhaled steroid (fluticasone) on the swine dust-induced cell and cytokine content of the lower airways, and cytokine release in cultured alveolar macrophages. Healthy volunteers were pretreated with inhaled salmeterol (n = 8), fluticasone propionate (n = 8) or placebo (n = 8) for about 2 weeks and exposed to dust in a pig house. Bronchoalveolar lavage was performed both before medication and after dust exposure. Cell differential counts and cytokine analyses in bronchoalveolar lavage fluid (BALF) were examined. Alveolar macrophages were cultured and cytokine release was studied, both in unstimulated cells and after lipopolysaccharide (LPS) stimulation. Unstimulated alveolar macrophages from swine dust-exposed individuals released less IL-6, IL-8 and tumour necrosis factor-alpha (TNF-α) after, than before, exposure (P < 0.01). Medication did not influence basal cytokine production. Fluticasone inhibited LPS-induced IL-6 and IL-8 release (P < 0.05). There was no significant difference between the groups. There was a large and significant increase (P < 0.05) in alveolar macrophage, granulocyte, lymphocyte numbers, and IL-6 and TNF-α content in BALF in all three groups following dust exposure, with no significant difference between the groups. These findings suggest that drugs which are known to influence and control airway inflammation in asthma do not have major effects on airway inflammation induced by the inhalation of organic dust.
The C5a receptor is expressed by human renal proximal tubular epithelial cells
Authors: Zahedi R , Braun M , Wetsel R A , Ault B H , Khan A , Welch T R , Frenzke M , Davis A E
Published: March 2003

The C5a receptor is expressed by a variety of cell types. These studies demonstrate by immunohistochemistry that the receptor is present on the surface of proximal and distal tubular epithelial cells from normal kidney. In addition, the receptor was detected on transitional epithelial cells of the ureter and bladder. Primary proximal tubular cultures and a proximal tubular cell line both also expressed the C5a receptor, as demonstrated by immunofluorescence and by FACS analysis. The presence of mRNA encoding the receptor was confirmed by reverse transcriptase-polymerase chain reaction analysis. As opposed to its effect on glomerular mesangial cells, the receptor did not mediate a proliferative response by the proximal tubular cells. C5a also did not enhance the synthesis/secretion of transforming growth factor-beta 1, monocyte chemoattractant protein-1, platelet-derived growth factor-AB or tumour necrosis factor-alpha by cultured proximal tubular cells. Therefore, although the C5a receptor clearly is expressed by proximal tubular cells, clarification of its functional relevance on this cell type awaits further studies.
Accumulation of CD4+CD7− T cells in inflammatory skin lesions: evidence for preferential adhesion to vascular endothelial cells
Authors: Liu L , Abken H , Pföhler C , Rappl G , Tilgen W , Reinhold U
Published: March 2003

The CD7− subset of CD4+ memory T cells reflects a stable differentiation state of post-thymic helper T cells and represents a small subpopulation in circulating blood. We here demonstrate that CD7− T cells preferentially accumulate in skin lesions under chronic inflammatory conditions irrespective of the particular disease. As adhesion to vascular endothelial cells (EC) is required for migration of circulating lymphocytes into tissues, we analysed the adherence of purified subsets of CD4+ memory T cells to endothelial cells in vitro. Compared with CD4+CD7+ T cells, cells of the CD4+CD7− subset preferentially adhere to EC, which is moreover increased after prestimulation of EC with tumour necrosis factor-alpha (TNF-α). Stimulated EC increase expression of intercellular adhesion molecule-1 (CD54) and E-selectin (CD62E), the ligand of which, cutaneous lymphocyte-related antigen (CLA), is highly expressed in CD4+CD7− T cells but not in CD4+CD7+ T cells. LFA-1 is expressed in a bimodal distribution on CD4+CD7− T cells in contrast to CD4+CD7+ cells, whereas VLA-1, VLA-3, and VLA-5 are nearly similarly expressed in both T cell subsets. Our results imply that the preferred adherence of CD4+CD7− memory T cells to vascular EC, which is increased after long-term EC stimulation with TNF-α, is likely to facilitate their accumulation in various inflammatory skin lesions.
Induction of necrosis and apoptosis of neutrophil granulocytes byStreptococcus pneumoniae
Authors: Zysk G , Bejo L , Schneider-Wald B K , Nau R , Heinz H-P
Published: March 2003

Apoptosis followed by macrophage phagocytosis is the principal mechanism by which neutrophil granulocytes (PMN) are removed from the site of inflammation. To investigate whether Streptococcus pneumoniae causes apoptosis of PMN, we exposed PMN to viable and heat-killed pneumococci and purified pneumococcal cell walls (PCW). The occurrence of PMN cell death was quantified by flow cytometry using annexin V/propidium iodide labelling of the cells. Intracellular histone-associated DNA fragments were quantified by ELISA. The presence of apoptosis was confirmed by in situ tailing. Exposure of PMN to viable pneumococci caused necrosis of the cells. The pneumococcal cytotoxin pneumolysin, the bacterial production of hydrogen peroxide, and PCW contributed to necrosis. Heat-killed pneumococci accelerated the process of apoptosis observed in cultivated non-stimulated PMN in vitro. These results demonstrated that pneumococci induce PMN cell death. Depending on the intensity of the stimulus, PMN necrosis and apoptosis were observed.
Humoral hyporesponsiveness to a conjugate contraceptive vaccine and its bypass by diverse carriers using permissible adjuvant
Authors: Mandokhot A , Pal R , Nagpal S , Chauhan V S , Ahlawat S , Singh Om
Published: March 2003

A contraceptive vaccine directed against human chorionic gonadotropin (hCG) has previously undergone clinical testing that demonstrated the feasibility of the approach in preventing pregnancy in women. Some immunized volunteers however, did not respond with an adequate anti-hCG antibody response despite employing highly immunogenic bacterial toxoids as carriers. Since there is some evidence that T cell responses to a complex protein typically focus on a few immunodominant epitopes, we investigated the responsiveness to hCG in mice of different haplotypes using the protein carrier diphtheria toxoid (DT). Our data showed a differential carrier effect of DT. With the aim of making a more potent immunogen employing promiscuous pathogen-derived Th peptides as carriers, peptide:antigen stoichiometric ratios were optimized. When tested individually using alum as the adjuvant, three such peptide conjugates improved the anti-hCG response, though not consistently to levels higher than the DT conjugate. Immunization with a combination of these synthetic epitopes generated anti-hCG responses higher than those achieved with DT or with the individual peptides. Antibodies were of high affinity and capable of neutralizing the bioactivity of hCG, but were devoid of anti-peptide reactivity. These results support our view that differential hyporesponsiveness in a diverse population may arise from inadequate carrier effect and that it can be overcome by use of pathogen-derived broadly reactive non-B Th epitopes employing only alum, a permissible adjuvant.
Renal cytokine responses in acuteEscherichia colipyelonephritis in IL-6-deficient mice
Authors: Khalil A , Tullus K , Bartfai T , Bakhiet M , Jaremko G , Brauner A
Published: March 2003

The aim of this study was to investigate the influence of IL-6 on mortality, bacterial growth and cytokine expression in experimental acute pyelonephritis. Female IL-6-deficient mice and their wild-type counterparts, 8–10 weeks old, were infected with Escherichia coli CFT 073 or injected with NaCl 0·9% (w/v) via the urethra and thereafter obstructed for 6 h. Animals were killed at 48 h, 6 days or 8 weeks and cytokine and bacterial renal levels were assessed at each time point. We found that IL-6-deficient mice had increased mortality and extensive renal bacterial growth on day 6, compared with wild-type mice (P < 0·05) and the histopathological changes were generally more severe and widespread in the IL-6-deficient mice. Peak mRNA expression of IL-1β, IL-4, IL-10, IL-12 and interferon-gamma (IFN-γ) occurred 48 h after infection in both IL-6 knock out and wild-type mice. Transforming growth factor-beta (TGF-β) levels also peaked at 48 h in E. coli-infected wild-type mice, while in the IL-6-deficient strain both TGF-β mRNA and protein levels were significantly lower at 48 h than wild-type levels (P < 0·0008 and P < 0·03, respectively) and remained stationary throughout the study period. Animals injected with NaCl 0·9% (w/v) displayed a similar decrease in TGF-β expression (P < 0·02). When splenocytes from the IL-6-deficient mice were incubated with murine recombinant IL-6, TGF-β levels increased to those of wild-type mice. No increase was observed when splenocytes from wild-type mice were incubated with the same doses of rIL-6. We therefore conclude that IL-6 plays an important role in bacterial clearance and directly influences the TGF-β levels in experimental acute pyelonephritis. We also demonstrate that urethral obstruction per se induces an increase in TGF-β the magnitude of which is decreased in IL-6-deficient mice.
Induction of lymphocyte apoptosis in rat liver allograft with ongoing rejection by FTY720
Authors: Li X-K , Tamura A , Fujino M , Guo L , Kakefuda T , Funeshima N , Enosawa S , Amari M , Naoe S , Amemiya H , Suzuki S
Published: March 2003

The action mechanism of FTY720, a novel immunosuppressant, is completely different from conventional immunosuppressants. The drug, which triggers apoptosis in murine and human lymphocytes, has a potent immunosuppressive activity to prevent allograft rejection without any severe side-effect. The present study was designed to determine whether FTY720 induces apoptotic cell death in activated lymphocytes infiltrated into liver grafts with ongoing rejection. FTY720 was orally administered at 5 mg/kg to the recipients on day 3 and day 4 after grafting, when the graft rejection was histologically confirmed. The intragraft patterns of IL-2, interferon-gamma (IFN-γ), perforin, and granzyme B gene expression were detected by reverse transcriptase-polymerase chain reaction. The treatment reversed ongoing rejection and significantly prolonged recipient survival time compared with the control group. Light microscopic observation of the graft sections stained with the DNA nick-end labelling method showed that the apoptosis in the control allografts was mainly induced in hepatocytes, while that in the FTY720-treated allografts was in infiltrated lymphocytes. The rejection therapy with FTY720 did not alter the expression of IL-2, IFN-γ, and perforin mRNAs, but slightly decreased granzyme B expression. Our results suggest that FTY720 does not alter the intrinsic lymphocyte function to produce the rejection-related cytokines, but strongly induces apoptotic cell death in the activated lymphocytes. Thus, FTY720 affords new insight into the mechanisms underlying improvements in immunosuppressive treatments.
The impact of platelet-activating factor (PAF)-like mediators on the functional activity of neutrophils: anti-inflammatory effects of human PAF-acetylhydrolase
Authors: Kuijpers T W , Van Den BERG1 J M , Tool A T J , Roos D
Published: March 2003

Platelet-activating factor (PAF) is a proinflammatory agent in infectious and inflammatory diseases, partly due to the activation of infiltrating phagocytes. PAF exerts its actions after binding to a monospecific PAF receptor (PAFR). The potent bioactivity is reflected by its ability to activate neutrophils at picomolar concentrations, as defined by changes in levels of intracellular Ca2+ ([Ca2+]i), and induction of chemotaxis and actin polymerization at nanomolar concentration. The role of PAF in neutrophil survival is, however, less well appreciated.

In this study, the inhibitory effects of synthetic PAFR-antagonists on various neutrophil functions were compared with the effect of recombinant human plasma-derived PAF-acetylhydrolase (rPAF-AH), as an important enzyme for PAF degradation in blood and extracellular fluids. We found that endogenously produced PAF (–like) substances were involved in the spontaneous apoptosis of neutrophils. At concentrations of 8 µg/ml or higher than normal plasma levels, rPAF-AH prevented spontaneous neutrophil apoptosis (21 ± 4% of surviving cells (mean ± SD; control) versus 62 ± 12% of surviving cells (mean ± SD; rPAF-AH 20 µg/ml); P < 0·01), during overnight cultures of 15 h. This effect depended on intact enzymatic activity of rPAF-AH and was not due to the resulting product lyso-PAF. The anti-inflammatory activity of rPAF-AH toward neutrophils was substantiated by its inhibition of PAF-induced chemotaxis and changes in [Ca2+]i.

In conclusion, the efficient and stable enzymatic activity of rPAF-AH over so many hours of coculture with neutrophils demonstrates the potential for its use in the many inflammatory processes in which PAF (–like) substances are believed to be involved.
The release of leukotriene B4 from human skin in response to substance P: evidence for the functional heterogeneity of human skin mast cells among individuals
Authors: Okabe T , Hide M , Koro O , Nimi N , Yamamoto S
Published: March 2003

Substance P is located in cutaneous nerve fibres and induces wheal and flare responses, accompanied by granulocyte infiltration, upon intradermal injection. Studies with animal skin and rat peritoneal mast cells have suggested that substance P induces the release of histamine and leukotriene B4 (LTB4), a potent chemoattractant for granulocytes, from skin mast cells. However, the release of LTB4 has not been detected from mast cells enzymatically isolated from human skin. In order to investigate the mechanism of granulocyte infiltration induced by substance P in human skin, we studied the release of LTB4 and histamine in response to substance P, and the effect of dexamethasone using human skin obtained from 22 nonallergic individuals. Histamine was released from all skin tissue samples in a dose-dependent manner. However, the amount of LTB4 release, both constitutive and inducible, was variable among skin preparations. Substance P induced a large release of LTB4 from the skin of eight donors (twice to six times that of the spontaneous release), but no or only negligible release from the skin of 14 donors. The amount of constitutive release of LTB4 correlated with the amount of tissue histamine. Dexamethasone selectively abolished the inducible release of LTB4, without an effect on histamine release and the constitutive release of LTB4. These results suggest that substance P induces the release of LTB4 in a certain population of human individuals by a glucocorticosteroid-dependent mechanism, and plays an important role in neurogenic inflammation with granulocyte infiltration.
HLA-DRB1 gene transcripts in rheumatoid arthritis
Authors: Kerlan-Candon S , Combe B , Vincent R , Clot J , Pinet V , Eliaou J -F
Published: March 2003

Susceptibility to rheumatoid arthritis (RA) is associated with defined HLA-DRB1 alleles. However the molecular basis of this association is not known. Peculiarities in the expression of disease-linked DRB1 alleles could be involved since in healthy controls HLA-DRB1 gene expression varies according to the alleles in B cells. Peripheral blood B cells of healthy controls and RA patients were examined for their level of allelic DRB1 transcripts using a competitive PCR approach. Levels of DRB1 transcripts were greatly modified in RA and influenced by HLA-DRB1 genotype: patients with double dose of RA-associated alleles displayed up-regulated amounts of DRB1 gene transcripts whereas patients carrying either a single or no at risk allele had low levels of DRB1 transcripts, compared to control individuals. These differential levels of DRB1 gene expression were not influenced in any way by clinical, biological or therapeutic features of the patients. Various amounts of DRB1 mRNA may be related to variations of the density of DR molecules on B cells and consequently could influence the response of CD4 T cells. This particular regulation of DRB1 gene expression in RA patients could therefore represent one of the molecular mechanisms involved in the association of HLA DRB1 genes to RA.
Identification of ricin A-chain HLA class II-restricted epitopes by human T-cell clones
Authors: Tommasi M , Castelletti D , Pasti M , Fracasso G , Lorenzetti I , Sartoris S , Pera C , Ferrara G B , Tridente G , Colombatti M
Published: March 2003

The identification of ricin toxin A-chain (RTA) epitopes and the molecular context in which they are recognized will allow strategies to be devised that prevent/suppress an anti-RTA immune response in patients treated with RTA-based immunotoxins. RTA-specific human T-cell lines and T-cell clones were produced by in vitro priming of PBMC. The T-cell clones used a limited set of Vβ chains (Vβ1, Vβ2 and Vβ8) to recognize RTA epitopes. The use of RTA deletion mutants demonstrated that T-cell lines and T-cell clones from three out of four donors responded to RTA epitopes within the domain D124-Q223, whereas one donor recognized the region I1-D124. The response to RTA peptides of T-cell lines and T-cell clones from two donors allowed the identification of immunogenic segments (D124-G140 and L161-T190) recognized in the context of different HLA-DRB1 alleles (HLA-DRB1*0801, and HLA-DRB1*11011 and B1*03011, respectively). The response to L161-T190 was investigated in greater detail. We found that the HLA-DRB1*03011 allele presents a minimal epitope represented by the sequence I175-Y183 of RTA, whereas the HLA-DRB1*11011 allele presents the minimal epitope M174-I184. RTA peptides and an I175A RTA point mutant allowed us to identify I175 as a crucial residue for the epitope(s) recognized by the two HLA-DRB1 alleles. Failure of T-cell clones to recognize ribosome inactivating proteins (RIPs) showing sequences similar but not identical to RTA further confirmed the role of I175 as a key residue for the epitope recognized in the context of HLA-DRB1*11011/03011 alleles.
N-acetylcysteine induces shedding of selectins from liver and intestine during orthotopic liver transplantation
Authors: Taut F J H , Schmidt H , Zapletal C M , Thies J C , Grube C , Motsch J , Klar E , Martin E
Published: March 2003

In orthotopic liver transplantation (OLT), N-acetylcysteine (NAC) reduces ischaemia/reperfusion (I/R) injury, improves liver synthesis function and prevents primary nonfunction of the graft. To further elucidate the mechanisms of these beneficial effects of NAC, we investigated influence of high-dose NAC therapy on the pattern of adhesion molecule release from liver and intestine during OLT. Nine patients receiving allograft OLT were treated with 150 mg NAC/kg during the first hour after reperfusion; 10 patients received the carrier only. One hour after reperfusion, samples of arterial, portal venous and hepatic venous plasma were taken and blood flow in the hepatic artery and the portal vein was measured. Absolute concentrations of sICAM-1, sVCAM-1, sP-selectin and sE-selectin were not markedly different. However, balance calculations showed release of selectins from NAC-treated livers as opposed to net uptake in controls (P ≤ 0.02 for sP-selectin). This shedding of selectins might be a contributing factor to the decrease in leucocyte adherence and improved haemodynamics found experimentally with NAC-treatment.
Skin T cell proliferative response to M protein and other cell wall and membrane proteins of group A streptococci in chronic plaque psoriasis
Authors: Baker B S , Brown D W , Fischetti V A , Ovigne J-M , Porter W , Powles A , Fry L
Published: March 2003

To determine and compare the T cell response to M protein and other group A streptococcal (GAS) antigens, T cell lines (TCL) were cultured from the lesional skin of 33 psoriatic patients and 17 disease controls. GAS-reactive skin TCL were tested in proliferation assays with recombinant M6 protein, and extracts of cell wall and membrane from type M6 GAS and its corresponding M gene deletion mutant. Initially, GAS-reactive skin TCL were obtained from 16 of 25 (64%) psoriasis, and from seven of 17 (41%) control patients. Eleven psoriatic and four control GAS-reactive TCL proliferated to M6 cell wall extract, whereas all the TCL from both groups responded to the extract of M6 membrane proteins. This difference in response to the two extracts was significant for both groups of patients (psoriasis, P = 0·0335, controls, P = 0·0156). GAS-reactive TCL from a further eight psoriasis patients showed no difference in response to cell wall extract from M6 GAS (containing the M protein minus its C-terminus) compared to that of its corresponding M gene deletion mutant. Furthermore, GAS-reactive TCL did not proliferate to recombinant M6 protein. However, a small, but significant reduction in proliferation by the eight psoriatic GAS-reactive TCL to the M-negative (lacking the M protein C-terminus) compared to M6-positive membrane extract was observed (P = 0·01). These findings suggest that GAS-reactive T cells in skin lesions of chronic plaque psoriasis proliferate to streptococcal membrane and, to a lesser extent, cell wall proteins. However, psoriatic skin T cells do not recognize cell wall M protein.
Modulation of HIV transcription by CD8+ cells is mediated via multiple elements of the long terminal repeat
Authors: Maslove D M , Ni L W , Hawley-Foss N C , Badley A D , Copeland K F T
Published: March 2003

HIV replication and LTR-mediated gene expression can be modulated by CD8+ cells in a cell type-dependent manner. We have previously shown that supernatant fluids of activated CD8+ cells of HIV-infected individuals suppress long terminal repeat (LTR)-mediated transcription of HIV in T cells while enhancing transcription in monocytic cells. Here, we have examined the effect of culture of T cells and monocytic cells with CD8+ supernatant fluids, and subsequent binding of transcription factors to the HIV-1 LTR. In transfections using constructs in which NFκB or NFAT-1 sites were mutated, the LTR retained the ability to respond positively to culture with CD8 supernatant fluid in monocytic cells. Nuclear extracts prepared from both Jurkat T cells and U38 monocytic cells cultured with CD8+ cell supernatant fluid demonstrated increased binding to the HIV-1 LTR at an AP-1 site which overlapped the chicken ovalbumin upstream promoter (COUP) site. In monocytic cells, increased binding activity was observed at the NFκB sites of the LTR. In contrast, an inhibition in binding at the NFκB sites was observed in Jurkat cells. Examination of two NFAT-1 sites revealed enhanced binding at − 260 to − 275 bp in U38 cells which was reduced by cellular activation. PMA and ionomycin-induced binding at a second NFAT-1 site (− 205 to − 216 bp) was abrogated by CD8+ cell supernatant fluid in T cells. These results, taken together, suggest that factors present in CD8+ supernatant fluids may act through several sites of the LTR to modulate transcription in a cell type-dependant manner.
HLA-DR expression on lymphocyte subsets as a marker of disease activity in patients with systemic lupus erythematosus
Authors: Viallard J F , Bloch-Michel C , Neau-Cransac M , Taupin J L , Garrigue S , Miossec V , Mercie P , Pellegrin J L , Moreau J F
Published: March 2003

A major problem in the management of SLE patients is to predict a flare or to distinguish between active and quiescent disease. Serological markers are widely used to assess disease activity, but many patients have close to or normal values for these parameters while exhibiting obvious disease-related signs and symptoms. This study aimed to determine which serological parameters, among ESR, ANA and anti-dsDNA antibody titres, CH50 and the HLA-DR expression on circulating T-lymphocyte subsets, best reflected the development of SLE flares. Sixty SLE patients were included, 34 with quiescent disease throughout the entire follow-up period and 26 who experienced an SLE flare defined as having active disease. According to univariate analysis, all parameters were significantly higher for patients with active disease, with the percentage of CD8+DR+ cells being the most significant parameter (P = 10−7). Multivariate logistic regression analysis identified three independent variables enabling the identification of a lupus flare: CH50, the CD8+DR+ and CD4+DR+ cell percentages among total lymphocytes. The CD8+DR+ cell percentage is the biological parameter most significantly associated with a flare (P < 0·001), even more powerful than CH50 (P < 0·01). HLA-DR expression on CD8+ lymphocytes clearly coincided with disease evolution in seven patients enrolled as having quiescent disease, but who experienced one flare during follow-up that subsequently resolved. The percentage of circulating CD8+DR+ lymphocytes appears to be a biological marker which accurately reflects disease activity. A larger prospective study is needed to demonstrate the real efficacy of this marker in predicting an exacerbation in SLE patients.
Effects of vitamin D on the growth of normal and malignant B-cell progenitors
Authors: Consolini R , Pala S , Legitimo A , Crimaldi G , Ferrari S , Ferrari S
Published: March 2003

As the effects of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) (VD, calcitriol) on the proliferation and differentiation potential of normal and leukaemic cells in vitro of myeloid lineage are known, we investigated the response to VD on the growth of both normal and malignant lymphoid progenitors. Effects of vitamin D on normal human lymphoid progenitors and B lineage acute lymphoblastic leukaemia (ALL) progenitors were assessed by using an in vitro cell colony assay specific for either B or T cell lineages. The expression of VDR on B untreated malignant progenitors at diagnosis was investigated by RT-PCR analysis. VD induced a significant inhibition of normal lymphoid cell progenitors growth of both T and B lineage. VD inhibited significantly also the growth of malignant B cell lineage lymphoid progenitors, without inducing cytotoxic effect. As it has been reported that VD effects on activated lymphocytes are mediated by 1,25-(OH)2-D3 nuclear receptor (VDR), we investigated VDR expression on malignant B cell progenitors. We did not detect VDR expression on these cells examined at diagnosis. We demonstrated that VD inhibited in vitro the clonogenic growth of both normal and malignant lymphoid B cell progenitors and that this inhibitory effect on malignant B cell progenitors was not related to VDR. Our work contributes to understanding of the mechanism of action of this hormone in promoting cellular inhibition of clonal growth of malignant lymphoid B cell progenitors, suggesting that the regulation of some critical growth and differentiation factor receptors could be a key physiological role of this hormone.
Influence of atopic heredity on IL-4-, IL-12- and IFN-γ-producing cells inin vitroactivated cord blood mononuclear cells
Authors: Gabrielsson S , Söderlund A , Nilsson C , Lilja G , Nordlund M , Troye-Blomberg M
Published: March 2003

Several reports have claimed that there is a greater risk for a child with an atopic mother to develop allergy as compared to a child with an atopic father. This suggests that the fetal environment during pregnancy might be of importance for the development of atopic disease. Both proliferative and cytokine responses have been detected in cord blood mononuclear cells (CBMC) after stimulation with allergens, suggesting allergen priming already in utero. The aim of this study was to investigate whether the atopic status of the mother influences cytokine production by CBMC. We compared interleukin (IL)-4, IL-12 and interferon (IFN)-γ-producing CBMC from children with double atopic heredity (dh), maternal atopic heredity only (mh) or no atopic heredity (nh). CBMC were stimulated in vitro with allergens (birch, ovalbumin and cat), phytohaemagglutinin (PHA) or purified protein derivative (PPD) and cytokine-producing cells were measured by the enzyme-linked immunospot assay. In response to PHA, the frequency of IL-4-producing cells, as well as the ratio of IL-4/IFN-γ-producing cells, were significantly higher in the dh group compared to the nh group. High numbers of IL-12-producing cells in response to allergens were detected, significantly highest in the nh group, followed by the dh and mh groups. Our results suggest that there is a stronger Th2 bias after in vitro stimulation of CBMC from children with atopic heredity, as reflected by higher IL-4/IFN-γ ratios in response to PHA, and lower numbers of IL-12-producing cells after allergen stimulation. Whether these differences influence later allergy development will be evaluated when the atopic status of the children is assessed at 2 years of age.
Serum macrophage-derived chemokine (MDC) levels are closely related with the disease activity of atopic dermatitis
Authors: KAKINUMA T , NAKAMURA K , WAKUGAWA M , MITSUI H , TADA Y , SAEKI H , TORII H , KOMINE M , ASAHINA A , TAMAKI K
Published: March 2003

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease characterized by the predominant infiltration of T cells, eosinophils and macrophages in lesional skin. Recently, macrophage-derived chemokine (MDC)/CCL22, a CC chemokine, was identified as a selective chemoattractant for CC chemokine receptor 4 (CCR4)-expressing cells, in addition to thymus and activation-regulated chemokine (TARC). We have previously reported that serum TARC levels correlate with the severity of AD. In this report, we investigated the participation of MDC in AD. First, we measured serum MDC levels in 45 patients with AD, 25 patients with psoriasis vulgaris and 25 healthy controls. Serum MDC levels in AD patients were significantly higher than those in healthy controls and psoriasis patients. Furthermore, the increases in serum MDC levels in AD patients were greater in the severely affected group than in the moderate or mild groups. We compared serum MDC levels in 11 AD patients, before and after treatment, and observed a significant decrease after treatment. Moreover, the serum MDC levels significantly correlated with the Scoring AD (SCORAD) index, serum soluble (s) E-selectin levels, serum soluble interleukin-2 receptor (sIL-2R) levels, serum TARC levels and eosinophil numbers in peripheral blood. Our study strongly suggests that serum MDC levels have a notable correlation with disease activity and that MDC, as well as the CC chemokine TARC, may be involved in the pathogenesis of AD.
Lidocaine inhibits secretion of IL-8 and IL-1βand stimulates secretion of IL-1 receptor antagonist by epithelial cells
Authors: LAHAV M , LEVITE M , BASSANI L , LANG A , FIDDER H , TAL R , BAR-MEIR S , MAYER L , CHOWERS Y
Published: March 2003

Lidocaine and related local anaesthetics have been shown to be effective in the treatment of ulcerative colitis (UC). However, the mechanisms underlying their therapeutic effect are poorly defined. Intestinal epithelial cells play an important role in the mucosal inflammatory response that leads to tissue damage in UC via the secretion of pro-inflammatory cytokines and chemokines. The aim of this study was to evaluate the direct immunoregulatory effect of lidocaine on pro-inflammatory cytokine and chemokine secretion from intestinal epithelial cells. HT-29 and Caco-2 cell lines were used as a model system and treated with lidocaine and related drugs. The expression of IL-8, IL-1β and the IL-1 receptor antagonist (RA) were assessed by ELISA and quantification of mRNA. In further experiments, the effect of lidocaine on the secretion of IL-8 from freshly isolated epithelial cells stimulated with TNFα was tested. Lidocaine, in therapeutic concentrations, inhibited the spontaneous and TNFα-stimulated secretion of IL-8 and IL-1β from HT-29 and Caco-2 cell lines in a dose-dependent manner. Similarly, suppression of IL-8 secretion was noted in the freshly isolated epithelial cells. Other local anaesthetics, bupivacaine and amethocaine, had comparable effects. Lidocaine stimulated the secretion of the anti-inflammatory molecule IL-1 RA. Both the inhibitory and the stimulatory effects of lidocaine involved regulation of transcription. The results imply that the therapeutic effect of lidocaine may be mediated, at least in part, by its direct effects on epithelial cells to inhibit the secretion of proinflammatory molecules on one hand while triggering the secretion of anti-inflammatory mediators on the other.
Adhesion to fibronectin viaα4 integrin (CD49d) protects B cells from apoptosis induced by serum deprivation but not via IgM or Fas/Apo-1 receptors
Authors: GARCIA-GILA M , LÓPEZ-MARTIN E M , GARCIA-PARDO A
Published: March 2003

Apoptosis is a regulated event crucial to the development and proliferation of normal and malignant B cells. We have studied the role of signals delivered via α4 integrin on apoptosis triggered by three different pathways on these cells. For apoptosis induced by serum deprivation, culturing B cells on the recombinant fibronectin fragment H89, a known ligand for α4β1 integrin, resulted in statistically significant (P < 0·005) higher viability values (68%, 65% and 67%) for Ramos, Nalm-6 and EHEB cells, respectively, than culturing cells on poly lysine (42%, 42% and 48%). An antiα4 MoAb reverted the protecting effect, thus confirming that it was due specifically to α4 engagement. Similarly, cells cultured on FN-III4-5, a recently identified fibronectin region which binds activated α4 integrin, also showed statistically significant higher viability than poly lysine cultures. α4 engagement however, did not prevent apoptosis induced on Ramos cells via surface IgM. Adhesion of IM-9 cells, a myeloma cell line carrying functional Fas receptors, to the H89 fragment neither increased cell viability upon triggering apoptosis via Fas when compared to poly lysine. These results indicate that α4 signalling may overcome B cell apoptosis induced by the lack of growth factors but does not seem to affect the IgM or Fas apoptotic pathways, thus suggesting different intracellular mechanisms for these processes.