bluuebunny/crossref_metadata_2025 huggingface dataset:
Early decrease in surface expression of HLA-DQ predicts the development of infection in trauma patients
Authors: Taylor J V , Gordon L E , Polk H C
Published: March 2003

The behaviour of human leucocyte antigen-DR (HLA-DR) following injury has been extensively studied. However, the behaviour of other class II antigens following trauma has not been characterized as well, despite evidence that HLA-DQ genotype influences the response to several bacterial antigens. Our study attempts to characterize and analyse the behaviour of HLA-DQ after trauma in patients with and without infection. Twenty-five patients were studied following major injury. Fifteen of the 25 patients developed infection (men = 11, women = 4); 10 patients developed no infection (men = 9, women = 1). The mean age was 34 ± 12 years for patients with no infection and 52 ± 20 years for those with infection. Monocyte HLA-DQ surface expression was determined using FITC-labelled antibodies and flow cytometry. Expression was compared with a control population of 11 healthy volunteers. The percentage of monocytes expressing HLA-DQ following trauma was reduced in patients with infection and in those without infection, but returned to normal (days 8–14) only in those patients who did not develop infection. Monocyte HLA-DQ mean channel fluorescence was reduced on day 1, but quickly returned to normal in those patients who subsequently developed infection. Stimulated with lipopolysaccharide, the initial samples of 13 patients who developed infection showed that surface expression on these monocytes could be elevated into the normal range. We conclude that HLA-DQ is an additional early marker of outcome that may not function merely as an immune suppressor. The maintained ability of HLA-DQ to present self-antigens may be important in the initial stages of the host response to injury.
IL-1β- and IL-4-induced down-regulation of autotaxin mRNA and PC-1 in fibroblast-like synoviocytes of patients with rheumatoid arthritis (RA)
Authors: Kehlen A , Lauterbach R , Santos A N , Thiele K , Kabisch U , Weber E , Riemann D , Langner J
Published: March 2003

Autotaxin (ATX) is a 125-kD ectonucleotide pyrophosphate/phosphodiesterase, which was initially isolated and cloned from human melanoma cells as a potent stimulator of tumour cell motility. ATX shows 44% identity to the plasma cell membrane marker PC-1. Recently, we described the decreased expression of ATX mRNA in cultured fibroblast-like synoviocytes (SFC) of patients with RA by interferon-gamma. In this study using a competitive reverse transcriptase-polymerase chain reaction, we show an increased ATX mRNA expression in SFC from patients with RA in comparison with synoviocytes from non-RA patients. The median ATX mRNA amount in SFC of RA patients (440 pg/μg total RNA) was five-fold higher than the expression in synoviocytes from non-RA patients (80 pg/μg total RNA) or foreskin fibroblasts (MRHF cells, 90 pg/μg total RNA). In contrast to the elevated ATX mRNA expression in SFC of patients with RA, we did not measure increased mRNA amounts of PC-1 in these cells. Both the ATX mRNA amount and the 5′-nucleotide phosphodiesterase (PDE) activity of SFC lysate were reduced after treatment of SFC with the cytokines IL-1β or IL-4. IL-1β and IL-4 induced a down-regulation of PC-1 mRNA and protein expression in SFC. In SFC treated with transforming growth factor-beta the expression of PC-1 mRNA and protein was increased, whereas no significant effect on ATX mRNA expression was detectable. Pharmacological drugs used in therapy for RA, such as dexamethasone, cyclosporin, methotrexate and indomethacin, did not show a statistically significant effect on either ATX mRNA or PC-1 mRNA expression. Only pentoxifylline suppressed ATX mRNA as well as PC-1 mRNA expression. In conclusion, we show a tight regulation of ATX and PC-1 gene expression by cytokines detectable in the inflamed tissue of RA. Further investigations will deal with the regulation of ATX protein expression as well as with the function of ATX in RA.
Activation of signal-transducer and activator of transcription 1 (STAT1) in pouchitis
Authors: Kühbacher T , Gionchetti P , Hampe J , Helwig U , Rosenstiel P , Campieri M , Buhr H J , Schreiber S
Published: March 2003

Activation of signal transducer and activator of transcription 1 (STAT1) is a hallmark of IFN-γ receptor signal transduction but is also part of the signalling pathway of other cytokines/growth factor receptors. In ulcerative colitis, high levels of activation and expression of STAT1 have been observed in comparison with both Crohn's Disease and normal controls. Pouchitis develops in some patients after Ileal-Pouch-Anal-Anastomosis (IPAA). The pathophysiology and aetiology of pouchitis is still unclear. Recent studies have shown an increased production of proinflammatory cytokines including IFN-γ. To investigate the expression and activation of STAT1 in pouchitis and the influence of treatment, patients were followed longitudinally from pouch operation. Diagnosis of pouchitis was made by clinical, endoscopic and histological criteria. Biopsies were obtained during routine endoscopy and snap frozen in liquid nitrogen. Nuclear and cytosolic extracts were prepared and the expression and activation of specific transcription factors were assessed by Western blot, electrophoretic mobility shift assay and immunofluorescence. Patients who develop pouchitis show highly increased levels of STAT1α as well as STAT1β expression and activation in comparison with both normal pouch and normal ileal mucosa. Improvement of pouchitis during antibiotic therapy relates to a normalization of STAT1 expression and activation. We conclude that activation of STAT1 correlates to clinical disease activity and therefore STAT1 could play an important role in the pathophysiology of pouchitis. Similarities in the pattern of activation of STAT1 in pouchitis and ulcerative colitis may suggest a common pathway in the immunopathophysiology of both diseases.
Inhibition of HIV-1-mediated syncytium formation and virus replication by the lipophosphoglycan fromLeishmania donovaniis due to an effect on early events in the virus life cycle
Authors: Genois N , Barbeau B , Olivier M , Tremblay M J
Published: March 2003

Previous findings have indicated that the major surface molecule of Leishmania, lipophosphoglycan (LPG), could abrogate HIV-1-induced syncytium formation and virus replication. In the present work, we were interested in characterizing this inhibitory process. Data from a new luciferase-based semiquantitative assay for syncytium formation, relying on the coincubation of a T-cell line containing an HIV-1 LTR-driven luciferase construct with a cell line chronically infected with HIV-1, confirmed that LPG was indeed a strong inhibitor of HIV-1-dependent syncytium formation and that this inhibition was dose-dependent. As determined by flow cytometric analyses, this inhibition was not apparently due to downregulation of CD4, CXCR4 or LFA-1, three distinct surface glycoproteins known to be important in HIV-1 mediated syncytium formation. Furthermore, LPG did not seem to affect signal transduction pathways in T cells as judged by measurement of HIV-1 LTR-driven reporter gene activity upon treatment with different stimuli. However, pretreatment of either of the cell lines used in the assay with LPG led to a significant decrease of virus-mediated syncytium formation, which was further accentuated when both cell lines were pretreated. LPG inhibition of HIV-1 replication was next assessed. When measuring either infection with luciferase-encoding recombinant HIV-1 particles or multinucleated giant cell formation following an acute virus infection, we again observed that LPG was efficient at blocking HIV-1 replication. Specific assays probing different steps of viral entry demonstrated that attachment was not hindered by LPG but that viral entry was modulated, suggesting that LPG targets a postbinding step. Hence, incorporation of LPG into a target cell membrane could influence its fluidity and diminish both the virus-cell and cell-to-cell fusion processes initiated by HIV-1.
Activation of human monocytes/macrophages by hypo-osmotic shock
Authors: Frenkel O , Shani E , Ben-Bassat I , Brok-Simoni F , Shinar E , Danon D
Published: March 2003

Phagocytosis and secretion of interleukins and growth factors put the macrophage in the centre of the wound healing process. For the last four years over 400 human ulcers have been treated in elderly and paraplegic patients by local application of monocytes prepared from a blood unit, in a unique, closed, sterile system. The process of preparation includes a step of hypo-osmotic shock, which induces monocyte/macrophage activation. This is different from any other known method of activation. In the present study we evaluated the efficacy of the hypo-osmotic shock. We found enhanced levels of IL-1 (P = 0·004) and IL-6 (P = 0·001) in the incubation medium (100% autologous serum) of the activated cells, as compared with controls, prepared in the same system. The IL-1 reached a plateau after 6 and 12 h incubation at 37°C, in both experimental and control incubation medium. The level of IL-6 was further elevated after 12 and 24 h incubation in experimental and control incubation mediums (P = 0·001). The phagocytosis of fluorescent beads was markedly enhanced after hypo-osmotic shock (P = 0·005).

The osmotic shock induced macrophages were compared to those stimulated with LPS, and osmotic shock was proved to be at least as efficient method of stimulation as LPS.
Imbalance between interleukin-6 and adrenomedullin mRNA levels in peripheral blood mononuclear cells of patients with lupus nephritis
Authors: Nishitani Y , Kubo A , Iwano M , Minamino N , Hamano K , Fujimoto T , Nishino T , Shiiki H , Yonemasu K , Dohi K
Published: March 2003

In this study, we measured the mRNA levels of adrenomedullin (AM), C-type natriuretic peptide, vascular endothelial growth factor, interleukin-1β (IL-1β) and interleukin-6 (IL-6) in peripheral blood mononuclear cells (PBMC) of 34 patients with lupus nephritis (LN) (15 active and 19 inactive) and 30 healthy volunteers. mRNA levels were measured using a real-time quantitative PCR method. Compared with healthy volunteers, IL-6 mRNA levels were elevated in LN patients (P < 0.005), while AM mRNA levels were decreased (P < 0.05). Also, IL-6 mRNA levels were higher and AM mRNA levels lower in active LN patients compared with inactive LN patients. In addition, IL-6 mRNA levels positively correlated and AM mRNA levels negatively correlated with SLE disease activity index and laboratory findings, such as blood urea nitrogen, serum creatinine, 50% haemolytic unit of complement and urinary excretion of protein over 24 h. Furthermore, IL-6 mRNA levels were negatively correlated with AM mRNA levels within the same LN patients. With regard to pathological findings, our results showed that IL-6 mRNA levels were higher, and AM mRNA levels significantly lower in patients with a high activity index compared to those with a low activity index. Following treatment with prednisolone, IL-6 mRNA levels in active LN patients decreased and AM mRNA levels increased to levels comparable to those in inactive LN and healthy volunteers. In vitro studies further demonstrated that elevated IL-6 mRNA levels in active LN patient PBMC were suppressed by the addition of adrenomedullin. Our results suggest that an imbalance between IL-6 and AM levels may play an important role in the progression of SLE, and that the mRNA levels of these genes in PBMC may be used as a disease activity index for SLE.
Escherichia coli-induced expression of IL-1α, IL-1β, IL-6 and IL-8 in normal human renal tubular epithelial cells
Authors: Brauner A , Söderhäll M , Jacobson S H , Lundahl J , Andersson U , Andersson J
Published: March 2003

The aim of the present study was to investigate whether the IL-1 family cytokines, in addition to IL-6 and IL-8, could be induced in normal human cortical epithelial cells in response to bacterial stimuli. Human renal tissue was obtained from 9 patients undergoing elective tumour nephrectomy. Renal cortical epithelial cells of tubular origin were prepared from the unaffected tissue. The proximal tubular cells were stimulated for 2, 6 and 24 h with a heat-inactivated pyelonephritogenic Escherichia coli strain DS-17. Cultured unstimulated tubular cells served as controls. IL-1α, IL-1β, IL-1 receptor antagonist, IL-6, IL-8, IL-10, TNF-α, G-CSF and GM-CSF were analysed using immunohistochemistry at the single cell level. The nonstimulated cells were found to express low levels of IL-6 and IL-8 (mean value < 3% of total cells). In contrast, E. coli exposure resulted in significantly increased incidences of IL-6 and IL-8 expressing cells (mean values ≈18% of total cells) peaking within two hours of stimulation (P < 0·008 and P < 0·02 versus non-stimulated cells, respectively). A gradual decrease was thereafter observed at 6 and 24 h, respectively, although persistently higher compared to controls. A different kinetic response was found for IL-1α, IL-1β and IL-1 receptor antagonist-expressing cells, which peaked 24 h after E. coli stimulation (mean values 3–10%) (P < 0·008, P < 0·02, P < 0·02 versus non-stimulated cells, respectively). Low levels of TNF-α and GM-CSF were found in 3 of the 9 donated epithelial cells, peaking at 2 h, and IL-10 and G-CSF producing cells in 1 patient each. In conclusion we found that heat-inactivated pyelonephritic E. coli induced a proinflammatory cytokine response in the normal human proximal tubular cells including the IL-1 family, IL-6 and IL-8.
Activation of HIV-1-specific immune responses to an HIV-1 vaccine constructed from a replication-defective adenovirus vector using various combinations of immunization protocols
Authors: Yoshida T , Okuda K , Xin K-Q , Tadokoro K , Fukushima J , Toda S , Hagiwara E , Hamajima K , Koshino T , Saito T
Published: March 2003

We constructed a recombinant replication defective adenovirus vector containing the env gene (Ad-Bal) derived from macrophage-trophic HIV-1 (HIV-1 Bal). We then immunized mice with this vector using several administration routes and protocols, and examined the immune response. When the Ad-Bal viral vector (over 1 × 107 pfu) was injected subcutaneously, both humoral and cell-mediated immunities were induced. However, immune response induced by the Ad-Bal vector alone was weaker than that induced by the recombinant vaccinia viral vector. We then employed the following three immunization protocols: (l) DNA vaccination followed by immunization with the Ad-Bal; (2) vaccination using the Ad-Bal vector followed by DNA vaccination; and (3) DNA vaccination followed by Ad-Bal infection and passive transfer of dendritic cells (DCs) infected with the Ad-Bal. Among the three protocols, the last gave the strongest humoral and cell-mediated immunity. These results suggest that the combination of DNA vaccination, Ad-Bal vector infection and passive transfer of Ad-Bal-infected DCs can induce strong immunity against HIV-1 Bal.
Colonic epithelial cells induce endothelial cell expression of ICAM-1 and VCAM-1 by a NF-κB-dependent mechanism
Authors: Maaser C , Schoeppner S , Kucharzik T , Kraft M , Schoenherr E , Domschke W , Luegering N
Published: March 2003

Epithelial cells are positioned in close proximity to endothelial cells. A non-contact coculture system was used to investigate whether colonic epithelial cells activated with various cytokines are able to provide signals that can modulate ICAM-1 and VCAM-1 expression on endothelial cells. Coculture of human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC-1) with TNF-α/IFN-γ-stimulated human colon epithelial cell lines led to a significant up-regulation of endothelial ICAM-1 and VCAM-1 expression. Increased ICAM-1 and VCAM-1 expression by endothelial cells was accompanied by an increase in endothelial cell NF-κB p65 and NF-κB-DNA-binding activity. Inhibition of endothelial NF-κB activation using the proteosome inhibitors MG-132 and BAY 11–7082 resulted in a significant decrease of ICAM-1 expression, indicating an important role for NF-κB in this response. This cross-talk may represent a biological mechanism for the gut epithelium to control the colonic inflammatory response and the subsequent immune cell recruitment during inflammation.
Reactive oxygen intermediates, nitrite and IFN-γin Indian visceral leishmaniasis
Authors: Kumar R , Pai K , Sundar S
Published: March 2003

Reactive oxygen intermediates (ROI), nitrite and interferon-gamma (IFN-γ) production were investigated at different times during treatment in 10 patients with visceral leishmaniasis (VL). Hydrogen peroxide (H2O2), superoxide (O2−) and IFN-γ production by cultured monocytes from patients with active VL were significantly lower compared with the healthy controls. In contrast, nitrite levels in the supernatants from monocyte cultures of VL patients were comparable to healthy controls and increased significantly during antileishmanial therapy. On day 20 of treatment, a significant increase in the release of H2O2, O2− and IFN-γ was observed. However, at follow-up, 4 months after the end of treatment, the production of H2O2, O2−, IFN-γ and nitrite had declined significantly. Thus, the impairment in hydrogen peroxide and superoxide production suggests that down-regulation of these mediators may be involved in the reduced killing of parasites by monocytes of active VL patients. Furthermore, the monocytes regained respiratory burst activity as the antileishmanial therapy progressed, suggesting that an immune-based mechanism is involved in successful drug therapy.
Co-incubation of pig islet cells with spleen cells from non-obese diabetic mice causes decreased insulin release by non-T-cell- and T-cell-mediated mechanisms
Authors: You S , Rivereau A-S , Gouin E , Saï P
Published: March 2003

In vitro studies were conducted in the non-obese diabetic (NOD) mouse, prone to Type 1 autoimmune diabetes, to investigate the mechanisms involved in cell-mediated rejection of pig islet xenografts. Our previous work concerning the mechanisms of proliferation of xenogeneic lymphocytes to pig islet cells (PIC) was not indicative of PIC impairment. Consequently, a test was developed based on perifusion analysis of the alteration of basal and stimulated insulin release from adult PIC incubated with mouse splenocytes or subsets. Compared with PIC incubation alone or with syngeneic pig splenocytes, co-incubation with mouse whole spleen cells resulted in a decrease of basal and stimulated insulin release (P < 0·001). Two components of this alteration were detected separately: PIC impairment was decreased (P < 0·01) after removal of plastic-adherent cells from spleen cells, but maintained (P < 0·01) when plastic-adherent cells alone were co-incubated with PIC. The increase of murine interleukin-1β when mouse plastic-adherent spleen cells were cultured with PIC (P < 0·04) was indicative of macrophage activation. Soluble factors produced during co-incubation of mouse splenocytes or plastic-adherent cells with PIC were involved in the impairment process, since supernatant fluids collected during previous PIC–mouse cell co-incubations directly altered (P < 0·01) insulin release from PIC. Moreover, impairment of PIC by mouse spleen cells was abolished (P < 0·01) by gadolinium chloride (which inhibits macrophages), but not by cyclosporin A. Another mechanism was apparent, since co-incubation of PIC with purified mouse T cells or CD4+ T cells, re-mixed with antigen-presenting cells, led to a decrease (P < 0·01) of insulin release. This model, based on the alteration of dynamic basal and stimulated insulin release, is indicative of in vitro cell-mediated alteration of PIC in the NOD mouse. The effect of whole spleen cells was rapid, and a crucial role was played by plastic-adherent cells. Two mechanisms were responsible for the behaviour of these cells: an early direct effect (at least in part via soluble products); and the indirect presentation of PIC xenoantigens (leading to impairment by CD4+ T lymphocytes).
Antigen-specific suppression of cultured lymphocytes from patients with neurocysticercosis
Authors: Bueno E C , Vaz A J , Machado L R , Livramento J A , Ávila S L M , Ferreira A W
Published: March 2003

The biological parasite–host interactions involved in neurocysticercosis (NC) are of a complex nature. A lymphoproliferation assay was performed using mononuclear cells from 11 patients with NC, who were classified according to the alterations obtained by imaging examinations. Antigen extracts from the membrane and/or scolex of Taenia solium and from the vesicular fluid of Taenia crassiceps were used. Mononuclear cells from patients with NC showed antigen-specific suppression when compared with a control group. The patients presenting calcified cysts showed higher suppression when compared with patients in the active phase of disease. The antigen in the vesicular fluid of T. crassiceps seems to play a suppressor role in vitro, completely inhibiting cell proliferation induced by the mitogens phytohaemagglutinin, concanavalin A and pokeweed mitogen.
De-novo humoral immune responses to cancer-associated autoantigens during transition from chronic liver disease to hepatocellular carcinoma
Authors: Zhang J Y , Zhu W , Imai H , Kiyosawa K , Chan E K L , Tan E M
Published: March 2003

A feature of hepatocellular carcinoma (HCC) is that antecedent liver cirrhosis and chronic hepatitis are common precursor conditions and during transition to malignancy some patients develop autoantibodies which were not present during the preceding chronic liver disease phase. Serum samples from such patients can be used to immunoscreen cDNA expression libraries to identify genes encoding the new autoantigens. We demonstrate here the de novo appearance of antibodies to p62, a cytoplasmic protein which has been shown to bind to a developmentally regulated fetal species of insulin-like growth factor II (IGF-II) mRNA. Another antibody appearing during the transition period was against CENP-F, a cell cycle-related nuclear protein with maximum expression in the G2 and M phases of the cell cycle and previously shown to have a high association with malignancy. In three additional patients in whom serial serum samples were examined, new appearance of anti-p62 was detected in two patients and anti-CENP-F in one patient. This study demonstrates that transition to malignancy can be associated with autoantibody responses to certain cellular proteins which might have some role in tumorigenesis.
Flow cytometric measurement of intracellular migration inhibition factor and tumour necrosis factor alpha in the mucosa of patients with coeliac disease
Authors: O'Keeffe J , Lynch S , Whelan A , Jackson J , Kennedy N P , Weir D G , Feighery C
Published: March 2003

There is increasing evidence that proinflammatory cytokines contribute to many of the small intestinal features in coeliac disease. The aim of the study was to investigate the expression of two proinflammatory cytokines, migration inhibition factor (MIF) and tumour necrosis factor alpha (TNF-α) in duodenal biopsy specimens from patients with coeliac disease on a gluten-free diet and normal control subjects. A flow cytometric system was used to analyse intracellular protein levels of MIF and TNF-α in freshly isolated cells from duodenal biopsies taken from 12 patients with treated coeliac disease and 10 healthy control subjects. From the biopsy specimens, single cell suspensions of the epithelium and lamina propria were prepared using EDTA/DTT and enzymes. Intracellular cytokine expression was studied in intraepithelial lymphocytes (IELs), lamina propria T cells (LP T) and intestinal epithelial cells using different surface labelling antibodies. MIF protein was constitutively expressed in IELs, LP T cells and epithelial cells from normal intestinal mucosa. In contrast, although TNF-α was found in LP T cells, this cytokine was virtually undetectable in either IELs or epithelial cells. In coeliac disease, intracellular levels of MIF were significantly higher in epithelial cells compared with control subjects (P = 0·005). Raised levels of TNF-α were found in epithelial cells (P = 0·03) as well as IELs (P = 0·045) from coeliac patients compared with controls. The findings from this study show up-regulated expression of MIF and TNF-α in IELs and epithelial cells of histologically normal mucosa in patients with coeliac disease. Increased expression of proinflammatory cytokines in cells occupying the epithelial layer could help explain the rapidity with which the coeliac mucosa may respond to gluten challenge.
Localization of pro-inflammatory (IL-12, IL-15) and anti-inflammatory (IL-11, IL-13) cytokines at the foetomaternal interface during murine pregnancy
Authors: Zourbas S , Dubanchet S , Martal J , Chaouat G
Published: March 2003

The involvement of some interleukins (ILs) in early and established pregnancy has been convincingly demonstrated, but little is known about the potential role of the more recently discovered ones. However, since many of these have positive or negative regulatory effects on both NK and T cells, it is highly probable that they also have regulatory functions in both implantation and placental development. Therefore, as a first step in tackling this problem, we have investigated whether several recently described pro- (IL-12, IL-15) and anti-inflammatory (IL-11, IL-13) cytokines were expressed at the uteroplacental interface by use of immunohistochemistry at different stages of gestation in mice.

Each of these molecules was found at the foetomaternal interface, with specific distributions and patterns of expression depending on both the cytokine itself and the stage of pregnancy. The significance of these data is discussed.
Elevated serum levels of matrix metalloproteinase-9 (MMP-9) in Kawasaki disease
Authors: Takeshita S , Tokutomi T , Kawase H , Nakatani K , Tsujimoto H , Kawamura Y , Sekine I
Published: March 2003

Matrix metalloproteinases (MMPs) play an important role in the progression of tumour cells and the invasion of inflammatory cells by degrading the extracellular matrix. In the MMP family, MMP-9 gelatinase is thought to contribute to the pathogenesis of inflammatory arteritis by disrupting the elastic lamina. The aim of the present study is to investigate the potential role of MMP-9 in Kawasaki disease (KD), an acute type of systemic vasculitis in children. We studied the total levels of MMP-9 (free proMMP-9 and free MMP-9) in the sera using a new assay system and the expression of MMP-9 mRNA in the leucocytes using reverse transcription-polymerase chain reaction in 18 patients with KD, 10 patients with sepsis and 10 healthy children (HC). The serum MMP-9 levels were significantly higher (P < 0·01) in the acute phase of KD than in the acute phase of sepsis and HC. In the time course of KD, the serum MMP-9 levels decreased significantly (P < 0·01) from the subacute through the convalescent phases. In the acute phase of KD, the serum MMP-9 levels showed a significantly positive correlation (P < 0·05) with the circulating leucocyte counts, especially the neutrophil counts. Furthermore, the expression of MMP-9 mRNA in the circulating leucocytes increased in the acute phase of KD and decreased from the subacute through the convalescent phases. These findings indicate that an excessive amount of MMP-9 is present in the plasma during the acute phase of KD, thus suggesting that circulating leucocytes may be a source of the MMP-9 secreted into the circulation.
T-cell re-population in HIV-infected children on highly active anti-retroviral therapy (HAART)
Authors: King D J S , Gotch F M , Larsson-Sciard E-L
Published: March 2003

In this pilot study, we address the nature of the re-population of the T-cell compartment in HIV-1+ (Human Immunodeficiency Virus 1), vertically infected children placed on successful regimens of HAART (highly active anti-retroviral therapy) incorporating 2 NRTI and a protease inhibitor. The clonality of the T-cell compartment and the abundance of RTEs (Recent Thymic Emigrants) were determined 2 weeks before and 20 weeks after initiation of HAART in a subgroup of children taking part in the PENTA (Paediatric European Network for the Treatment of AIDS) 5 trial. Analysis of the clonality of the circulating T-cell compartment was assessed using CDR3 spectratyping and analysed using the Kolmogorov–Smirnov two sample test. This revealed that a high degree of T-cell clonal restriction still exists 5 months into therapy, despite the appearance of previously undetectable T-cell clones within the periphery. We detected no increase in RTE abundance in this 5 month period, as determined by PCR detection of TRECs (T-Cell Receptor Excision Circles). We conclude that the observed re-population of T cells within the periphery of treated children is heavily reliant upon the maintenance/expansion of pre-existing cells during the 5 month period immediately following the initiation of therapy.
Generationin vitroof B-cell chronic lymphocytic leukaemia-proliferative and specific HLA class-II-restricted cytotoxic T-cell responses using autologous dendritic cells pulsed with tumour cell lysate
Authors: Goddard R V , Prentice A G , Copplestone J A , Kaminski E R
Published: March 2003

Immunotherapy using dendritic cells has shown encouraging results in both haematological and non-haematological malignancies. In this study, monocyte-derived dendritic cells from patients with B-CLL were cultured for 6 days in the presence of IL-4 and GM-CSF. Autologous B-CLL T-cells were cultured alone or with B-CLL lysate-pulsed and unpulsed autologous dendritic cells. IFN-γ secretion was assessed using ELISA. Cytotoxicity was assessed, after 21 days in culture and re-stimulation, using flow cytometry with and without blockade by anti-HLA class I, anti-HLA class II, anti-CD4, anti-CD8 and anti-TCRαβ monoclonal antibodies. B-CLL T cells stimulated with B-CLL lysate-pulsed autologous dendritic cells showed a significant (P = 0·0004) increase in IFN-γ secretion and a significant (P = 0·0008) increase in specific cytotoxicity to autologous B-cell targets, but none to autologous T cell or B cell targets from healthy individuals. B-CLL T cells cultured with (non-B-CLL) B-cell lysate-pulsed B-CLL dendritic cells showed no significant response. Pulsing dendritic cells from healthy volunteers with an autologous (non-B-CLL) B-cell lysate did not stimulate proliferation, cytokine production or cytotoxicity by autologous T cells. Pulsing B-CLL dendritic cells with allogeneic B-CLL lysates and culturing with autologous T-cells elicited cytotoxicity against autologous B-CLL targets in some cases, but not in others. Cytotoxicity was significantly reduced by blocking with anti-HLA class II (P = 0·001), anti-TCRαβ (P = 0·03) and anti-CD4 (P = 0·046) antibodies. Phenotyping of the responding T-cell population demonstrated the majority to be CD4 positive. Our data demonstrate that HLA class II-restricted proliferative and cytotoxic T-cell responses to B-CLL can be generated using autologous dendritic cells pulsed with tumour cell lysate.
Children with atopic dermatitis who carry toxin-positiveStaphylococcus aureusstrains have an expansion of blood CD5− B lymphocytes without an increase in disease severity
Authors: Arkwright P D , Cookson B D , Haeney M R , Sanyal D , Potter M R , David T J
Published: March 2003

Toxin-positive strains of Staphylococcus aureus (T + S. aureus) are present on the skin of some but not all patients with atopic dermatitis. Many staphylococcal toxins are superantigens, which can stimulate the immune response and thus may potentially lead to the very high levels of IgE characteristic of this condition, as well as exacerbating the clinical disease. The aim of this study was to determine whether the presence of T + S. aureus on the skin of children with atopic dermatitis was associated with in vivo evidence of a heightened humoral immune response, higher IgE levels and more severe clinical disease. Toxin gene expression in S. aureus isolated from the eczematous lesions of 28 children with atopic dermatitis was assessed by PCR. Clinical and immune data were also collected from this cohort. Thirteen of the 28 children (46%) were colonized with T + S. aureus strains. The presence of T + S. aureus was associated with a significant expansion in peripheral blood CD5− B cells (P = 0·01), and the more toxin types identified the greater the B-cell expansion (P = 0·002). However, in this cohort of children with atopic dermatitis, despite th in vivo expansion of B cells in children harbouring T + S. aureus, there was no associated increase in IgE levels or in clinical disease severity scores.
Intranasal BCG vaccination protects BALB/c mice against virulentMycobacterium bovisand accelerates production of IFN-γ in their lungs
Authors: Lyadova I V , Vordermeier H M , Eruslanov E B , Khaidukov S V , Apt A S , Hewinson R G
Published: March 2003

Local immune reactivity in the lungs of BALB/c mice was studied following (i) intranasal (i.n.) vaccination with Mycobacterium bovis BCG, (ii) intravenous (i.v.) challenge with a virulent M. bovis field isolate and (iii) i.n. vaccination with M. bovis BCG followed by i.v. challenge with an M. bovis field isolate. The results demonstrated that i.n. vaccination with BCG induced a high degree of protection against systemic M. bovis challenge, and that this protection correlated with a rapid production of IFN-γ after M. bovis challenge by lung T cells from vaccinated mice.
IL-15-induced conversion of monocytes to mature dendritic cells
Authors: Saikh K U , Khan A S , Kissner T , Ulrich R G
Published: March 2003

IL-15 is produced by a wide variety of tissues in response to inflammatory stimuli. We examined the effect of IL-15 in supporting the maturation of monocytes to dendritic cells in ex vivo culture. IL-15 transformed CD14+ monocytes to mature dendritic cells. These dendritic cells were similar to those obtained from monocyte cultures treated with a combination of the cytokines GM-CSF, IL-4 and TNF-α. The effects of IL-15 did not depend on endogenously produced GM-CSF. The IL-15-induced dendritic cells also expressed chemokines and stimulated strong allo-responses that were characteristic of mature dendritic cells. These data indicate that CD14+ monocytes respond to IL-15 by undergoing morphological transformation and acquiring characteristic dendritic cell features that facilitate antigen-specific responses of T cells. Thus, the release of IL-15 by inflammatory stimuli may induce the conversion of monocytes to immuno-stimulatory dendritic cells to support primary immune responses against pathogens.
Cytokine receptor signalling in neonatal macrophages: defective STAT-1 phosphorylation in response to stimulation with IFN-γ
Authors: Maródi L , Goda K , Palicz A , Szabó G
Published: March 2003

We reported earlier that neonatal monocyte-derived macrophages (MDM) could not be fully activated with IFN-γ, a finding that could not be attributed to lower expression of IFN-γ receptors on the neonatal cells. In this study we explored elements of IFN-γR-mediated signalling in cord monocytes and MDM. Intracellular expression of STAT-1 was analysed by flow cytometry. We have assessed phosphorylation of STAT-1 by using MoAbs that distinguish native and phosphorylated forms of STAT-1 on a discrete cell basis. Using MoAbs against the native form of STAT-1 revealed comparable expression of this protein in cord and adult cells (both monocytes and MDM). However, STAT-1 phosphorylation in response to IFN-γ was significantly decreased in neonatal monocytes (P < 0·05) and MDM (P < 0·01) compared to adult cells (n > 5 for each). These data suggest deficient cytokine-receptor signalling in neonatal mononuclear phagocytes exposed to IFN-γ. We propose that decreased STAT-1 phosphorylation and activation may represent developmental immaturity and may contribute to the unique susceptibility of neonates to infections by intracellular pathogens.
Comparative protective effects of recombinant DNA andMycobacterium bovisbacille Calmette–Guérin vaccines againstM. aviuminfection
Authors: Martin E , Triccas J A , Kamath A T , Winter N , Britton W J
Published: March 2003

A range of strategies are being explored to develop more effective vaccines against mycobacterial infection, including immunization with DNA plasmids encoding single mycobacterial bacterial genes and the use of recombinant live vectors based on the current vaccine, Mycobacterium bovis bacille Calmette–Guérin (BCG). We have compared these two approaches using a model of virulent M. avium infection, and the gene for the immunodominant 35 kDa protein which is shared by M. avium and M. leprae, but absent from BCG. Recombinant BCG over-expressing the M. avium 35 kDa protein (BCG-35) induced strong antigen-specific proliferative and interferon-γ (IFN-γ)-secreting T cell responses. These were comparable to those induced by a single immunization with a plasmid expressing the same antigen (DNA-35); however, repeat DNA-35 immunization evoked the strongest IFN-γ release. Immunization with BCG-35 significantly reduced the growth of virulent M. avium, although this effect was similar to that induced by wild-type BCG. Immunization with DNA-35 resulted in significantly greater (2 × log10) reduction in the growth of M. avium. Prime-boost strategies combining DNA-35 and BCG-35 increased the protective effect above that achieved by BCG-35, but they were not more protective than DNA-35 alone. Therefore, recombinant BCG-35 and BCG induced similar levels of protection in this model, and maximal protection against M. avium infection was attained by immunization with DNA encoding the 35 kDa protein.
Death-inducing receptors and apoptotic changes in lymphocytes of patients with heart transplant vasculopathy
Authors: Ankersmit H J , Moser B , Roedler S , Teufel I , Zuckermann A , Roth G , Lietz K , Back C , Gerlitz S , Wolner E , Boltz-Nitulescu G
Published: March 2003

The specific role of lymphocyte apoptosis and transplant-associated atherosclerosis is not well understood. The aim of our study was to investigate the impact of T cell apoptotic pathways in patients with heart transplant vasculopathy. Amongst 40 patients with cardiac heart failure class IV who have undergone heart transplantation, 20 recipients with transplant-associated coronary artery disease (TACAD) and 20 with non-TACAD were investigated one year postoperative. Expression of CD95 and CD45RO, and annexin V binding were measured by FACS. Soluble CD95, sCD95 ligand (sCD95L), tumour necrosis factor receptor type 1 (sTNFR1), and histones were measured in the sera by ELISA. The percentage of cells expressing CD3 and CD4 was significantly reduced in TACAD as well as in non-TACAD patients as compared with control volunteers. Interestingly, the proportion of CD19+ (B cells) and CD56+ (NK) cells was increased in TACAD groups (versus non-TACAD; P < 0·01, and P < 0·001, respectively). In contrast to sCD95, the expression of CD95 (APO-1/Fas) and CD45RO (memory T cells), and sCD95L were significantly increased in non-TACAD and TACAD patients. T cell activation via CD95 with consecutive apoptosis was increased in both groups. The concentration of sTNFR1, IL-10 and histones was significantly elevated in sera from TACAD than non-TACAD patients, and in both groups than in healthy controls. These observations indicate that the allograft may induce a pronounced susceptibility of CD4+ T cells to undergo apoptosis and antibody-driven activation-induced cell death. This data may suggest a paradox immune response similar to that seen in patients with autoimmune diseases.
IL-15 in human visceral leishmaniasis caused byLeishmania infantum
Authors: MILANO S , BELLA G DI , D’AGOSTINO P , BARBERA C , CARUSO R , ROSA M LA , FERLAZZO V , VITALE G , RUSSA C LA , GAMBINO G , CHIFARI N , MANSUETO S , CILLARI E
Published: March 2003

Interleukin (IL)-15 is a recently discovered cytokine with the ability to stimulate the proliferation activity of Th1 and/or Th2 lymphocytes. Here, we investigated the involvement of IL-15 in the immune response to Leishmania infantum infection by studying patients with visceral leishmaniasis (VL). We found that IL-15 is produced by leishmanial antigen (LAg)-stimulated peripheral blood mononuclear cells (PBMC) from active VL patients at a significantly higher level than those produced by cells from healed VL subjects or healthy controls. A significant increase in IL-15 serum blood levels was also observed in acute VL patients compared with healed ones. Furthermore, recombinant IL-15 had an appreciable effect in vitro in reducing IL-4 and increasing the production of IL-12 in response to LAg, but it was ineffective in altering the production of interferon-γ (IFN-γ). The production of endogenous IL-15 in acute VL patients appeared to be insufficient to activate both IFN-γ and IL-12, as attested by the absence of modification of these two cytokines by neutralization experiments in the presence of anti-IL-15 monoclonal antibodies (MoAB). On the contrary, the neutralization of IL-15 increased IL-4 production. Together, these results indicate that endogenous IL-15 plays a role in the suppression of Th2-type cytokines, even though it does not enhance the production of Th1 cytokines in acute VL patients. Since IL-15, in the presence of anti-IL-4 MoAb, caused a further increase in IL-12 production and led to a significant production of IFN-γ, one of its indirect effects on Th1 cell activation could be due to the latter’s effect on Th2 cytokines such as IL-4. Therefore, our observations indicate that there is a potential for IL-15 to augment the T-cell response to human intracellular pathogens.
Human T cell responses to peptides of theMycobacterium leprae45-kD serine-rich antigen
Authors: BRAHMBHATT S , HUSSAIN R , ZAFAR S , DAWOOD G , OTTENHOFF T H M , DRIJFHOUT J W , BOTHAMLEY G , SMITH S , LOPEZ F V , DOCKRELL H M
Published: March 2003

In order to identify T cell epitopes within the Mycobacterium leprae 45-kD serine-rich antigen, we analysed responses to overlapping 17-mer peptides encompassing the whole antigen in non-exposed UK controls, Pakistani leprosy patients and tuberculosis patients in both the United Kingdom and Pakistan. This antigen has been described as M. leprae-specific, although it has a hypothetical homologue in M. tuberculosis. Human peripheral blood mononuclear cells were stimulated with peptide for 5 days and IFN-γ measured in supernatants by ELISA. Some peptides were recognized more frequently by T cells from tuberculoid leprosy patients than those from UK controls, suggesting that such T cell epitopes might have diagnostic potential, while other peptides induced greater responses among UK control subjects. Short-term cell lines confirmed that these assays detected specific T cell recognition of these peptides. However, many tuberculosis patients also recognized these potentially specific peptides suggesting that there could be a true homologue present in M. tuberculosis.
Detection of antivimentin antibody in sera of patients with idiopathic pulmonary fibrosis and non-specific interstitial pneumonia
Authors: YANG Y , FUJITA J , BANDOH S , OHTSUKI Y , YAMADORI I , YOSHINOUCHI T , ISHIDA T
Published: March 2003

It has been suggested that the humoral immune system plays a role in the pathogenesis of non-specific interstitial pneumonia (NSIP). Although some circulating autoantibodies to cytoskeletal protein(s) have been suggested, the antimyofibroblast antibody has not been investigated in patients with idiopathic pulmonary fibrosis (IPF) and NSIP. The purpose of this study is to evaluate the existence of antimyofibroblast antibody in the sera of patients with IPF and NSIP. The MRC5 cell line was used as a model of myofibroblast. The anti-MRC5 cell antibody was characterized in a patient with NSIP using Western blotting. Since we found that one of the anti-MRC5 antibodies was an antivimentin antibody, we established an enzyme-linked immunosorbent assay (ELISA) to measure the levels of antivimentin antibody in the sera of patients with IPF (n = 12) and NSIP (n = 23). Initially, two anti-MRC5 cell antibodies were detected in the sera of patients with NSIP, one of which was characterized as the antivimentin antibody by Western blotting. The other was characterized as an antivimentin fragment antibody. We established an ELISA to measure the antivimentin antibody and found significantly higher levels in patients with IPF and NSIP than in normal volunteers. One of the anti-MRC5 cell antibodies in the serum of a patient with NSIP was against vimentin. The serum levels of antivimentin antibody were increased in patients with IPF and NSIP compared with that of normal volunteers. These results suggest that the antivimentin antibody may be involved in the process of lung injury in IPF and NSIP.
TCR usage and cytokine expression in peripheral blood and BAL T cells
Authors: BAKAKOS P , PICKARD C , SMITH J L , FREW A J
Published: March 2003

T cells are thought to play an important regulatory role in atopic asthma. We hypothesized that human blood and BAL T cell subsets bearing various TCR-Vβ genes might show selective differences in their cytokine profile. Peripheral blood (PB) and bronchoalveolar lavage (BAL) T cells from seven atopic asthmatic and six non-atopic non-asthmatic subjects were stimulated with PMA and ionomycin in the presence of monensin and analysed for TCR-Vβ expression and production of cytokines at the single cell level. The percentage of IFN-γ- and IL-2-producing BAL T cells was elevated compared with PB T cells from both the asthmatic subjects and the non-atopic, non-asthmatic controls. A small percentage of PB and BAL T cells produced IL-4 and IL-5, in asthmatic and normal subjects. In peripheral blood, the percentage of T cells expressing each cytokine was similar in the various TCR-Vβ subsets and in total CD3+ T cells in all normal and six of seven asthmatic subjects. However, there was a substantial degree of heterogeneity in the cytokine profile of BAL TCR-Vβ subsets compared with the total CD3+ T cells. This was more obvious in the asthmatic subjects with a reduction in the percentage of IFN-γ- and IL-2-expressing T cells (five of seven asthmatic subjects) and an increase in the percentage of IL-4- and IL-5-expressing T cells (two of seven asthmatic subjects). These data confirm previous findings of an elevated proportion of IFN-γ- and IL-2-producing BAL T cells while only a small proportion of PB and BAL T cells produce IL-4 and IL-5. Moreover, subsets of BAL T cells, defined by their TCR-Vβ usage, may differ in their cytokine profile compared with the total CD3+ T cells, implying that T cells expressing different Vβ elements may play different roles in regulating the airway inflammation in asthma.
Cerebrospinal fluid affects phenotype and functions of myeloid dendritic cells
Authors: PASHENKOV M , SÖDERSTRÖM M , HUANG Y-M , LINK H
Published: March 2003

Myeloid (CD11c+) dendritic cells (DC) are present in cerebrospinal fluid (CSF), as well as in the meninges and choroid plexus. Functional studies of these DC are hindered or impossible. To obviate this problem, we investigated the effects of CSF supernatants from patients with non-inflammatory neurological diseases (NIND), multiple sclerosis (MS), bacterial meningitis (BM) and Lyme meningoencephalitis (LM) on immature monocyte-derived DC (moDC) from healthy donors. CSF supernatants caused maturation of moDC (MS > LM > NIND > BM), as reflected by a decrease in CD1a, and an increase in HLA-DR, CD80 and CD86 expression. The maturation effect of MS CSF and LM CSF could be blocked by anti-TNF-α MoAb or recombinant human IL-10. moDC cultured with BM CSF either remained immature or turned into CD14+ macrophage-like cells and were relatively inefficient at inducing T cell responses in vitro. In contrast, moDC cultured with LM CSF induced strong Th1 responses. Both BM CSF and LM CSF contained IFN-γ, a cytokine that augments IL-12 production by moDC and hence should confer an ability to induce a Th1 response. However, BM CSF also contained high levels of IL-10, which could antagonize the effects of IFN-γ on moDC. moDC cultured with MS CSF induced a higher production of IFN-γ from T cells compared to moDC cultured with NIND CSF or BM CSF. In summary, soluble factors present in the CSF may influence the phenotype and functions of meningeal, choroid plexus and CSF DC which, in turn, may have an impact on the character of intrathecal T cell responses.