bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential humoral immune response against hepatitis C virus antigenic synthetic peptides in infected patients with and without mixed cryoglobulinaemia
Authors: Gabrielli A , Zhang Z-X , Cherubini G , Candela M , Savoldi S , Manzin A , Clementi M , Amoroso A , Sallberg M
Published: November 2003

In this study we have evaluated the prevalence of antibodies against core region peptides (residues 1–28, 21–38 and 51–68), the envelope 1, the non-structural (NS) 4 and 5 proteins of hepatitis C virus (HCV) in sera from 65 chronically HCV-infected patients, 47 with mixed cryoglobulinaemia (MC+) and 18 without (MC−). The major binding sites were located within the core region. Regions 1–28 and 51–68 were recognized by a similar proportion of MC+ and MC− patients, while peptide 21–38 was less frequently detected by samples from MC+ patients (65.5% versus 100%; P=0.011). The patterns of the reactions showed a minimum of three binding sites: one, located within region 51–68, was shared by both groups; a second determinant was identified at residues 1–21 for MC+ patients and at residues 28–38 for MC− patients; a third, not exactly localized, lay between residues 1 and 38. Recognition of NS5 peptides was not significantly different between MC+ and MC− patients, but while the former mostly reacted either with peptide 1 (residues 2294–2309) (five of 15 sera) or with peptide 2 (residues 2304–2319) (nine of 15 sera), the latter group showed a more scattered reaction. Antibodies to HCV peptides prevalently belonged to IgG1 subclass. However, whereas IgG1 antibodies against peptide 21–38 and peptide 1 of NS5 were more frequently found in MC− rather than in MC+ patients (100% versus 63.8%, P=0.003, and 22.2% versus 4.2%, P=0.025, respectively), IgG3 antibodies against region 1–28 were more frequent in MC+ patients (53.19% versus 16.6%, P=0.0078). Overall, the data suggest that a differential humoral immune response to HCV antigens occurs in patients with and without cryoglobulinaemia.
Enhanced binding of lymphocytes from patients with multiple sclerosis to tumour necrosis factor-alpha (TNF-α)-treated endothelial monolayers: associations with clinical relapse and adhesion molecule expression
Authors: Vora A J , Perkin G D , McCoy T , Dumonde D C , Brown K A
Published: November 2003

This study investigated the adherent properties and adhesion molecule expression of blood mononuclear cells (MNC) from a total of 84 patients with multiple sclerosis (MS). The MNC from MS patients were significantly more adherent than cells from normal healthy subjects to endothelial monolayers pretreated with 0.01 U/ml TNF-α (103% increase; P = 0.002), 0.1 U/ml TNF-α (80% increase; P < 0.01) and 1.0 U/ml TNF-α (41% increase; P < 0.02), and to endothelium pretreated with 10 U/ml IL-1β (44% increase; P < 0.05) and 100 U/ml interferon-gamma (IFN-γ) (100% increase; P < 0.05). This augmented adhesion was a property of the lymphocytes, in particular CD4+ cells, and was inversely related to the time of onset of clinical relapse. The percentage of lymphocytes bearing the adhesion molecules CD49d, CD29 and CD62L was increased in MS blood, but the level of CD29 and CD62L expression was reduced. We infer that circulating lymphocytes in MS are predisposed to cross endothelial barriers at sites where inflammation has already commenced.
Expression of tumour necrosis factor-alpha (TNF-α) mRNA and protein in pathological thyroid tissue and carcinoma cell lines
Authors: Aust G , Heuer M , Laue S , Lehmann I , Hofmann A , Heldin N-E , Scherbaum W A
Published: November 2003

There has been much controversy about the presence of TNF-α within thyroid tissue. We therefore conducted a study to determine if TNF-α mRNA is present in thyroid tissue and thyroid-derived cells. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed with a heterologous competitor fragment. Significantly lower levels of TNF-α mRNA were found in the autonomous nodules from patients with thyroid autonomy (TA; n = 4; 5.7 ± 1.3 arbitrary units (AU) (mean ± s.e.m.); P < 0.03) and in normal thyroid tissue (n = 2, 7.0 ± 3.1 AU) compared with tissue from patients with Graves’ disease (GD; n = 13; 27.9 ± 10.3 AU), non-toxic multinodular goitre (NTG; n = 5; 20.9 ± 5.8 AU) and perinodular tissue from TA patients (20.3 ± 4.0 AU). Higher levels were detected in tissues from patients with Hashimoto’s thyroiditis (HT; n = 2; 51.3 ± 10.3 AU). Cultures of pure thyroid-derived fibroblasts (46 ± 18 AU), thyrocytes (33 ± 8 AU), and the anaplastic thyroid carcinoma cell lines 8505 C (39 ± 11 AU), SW 1736 (214 ± 16 AU) and C 643 (3 ± 1 AU) showed significantly lower TNF-α mRNA levels than thyroid-derived lymphocytes (1650 ± 32 AU). TNF-α was detected in the supernatants of unstimulated lymphocytes (22.1 ± 1.1 pg/ml) and SW 1736 cells (3.5 ± 0.9 pg/ml), but not in unstimulated fibroblasts and thyrocytes. Using an intracellular labelling technique in flow cytometry, the immunophenotype of stimulated TNF-α-positive lymphocytes was determined as predominantly CD3+CD45RO+. Our results suggest that TNF-α is present in the thyroid tissue of different thyroid disorders. Thyroid-derived lymphocytes are potential TNF-α producers and may thus locally influence thyroid function.
Soluble adhesion molecules in sera of patients with leprosy: levels of soluble intercellular adhesion molecule-1 (sICAM-1) rapidly decrease during multi-drug therapy
Authors: Rieckmann P , Scholze G , Weichselbraun I , Ganapati R , Prange H W
Published: November 2003

The clinicopathological spectrum of leprosy is associated with an altered immunological reaction. The expression of adhesion molecules on endothelial cells directs the cellular traffic to sites of local skin and nerve inflammation. Soluble forms of adhesion molecules, which are released upon cytokine activation, can be detected in the circulation and may reflect ongoing tissue inflammation. We determined the serum levels of sICAM-1, sE-selectin and sL-selectin in 74 patients with leprosy (tuberculoid form, n = 23; lepromatous form, n = 36; acute leprous reaction, n = 16) and 15 healthy age- and sex-matched control donors. Patients with lepromatous leprosy had significantly higher levels of sICAM-1 (564 ± 174 versus 450 ± 92 versus 334 ± 57 ng/ml) and E-selectin (90 ± 31 versus 74 ± 29 versus 50 ± 10 ng/ml) than patients with tuberculoid leprosy and normal donors (P<0.01). No differences between groups were detected for L-selectin. Patients with leprous reactions had similar high levels to lepromatous patients. Twenty lepromatous patients were re-examined after 4 weeks of therapy. A significant decrease in sICAM-1 serum levels was observed after 1 month of anti-mycobacterial treatment, which was accompanied by a reduction of mycobacteria in skin biopsies (P<0.01). Patients with leprous reactions (n = 13) also demonstrated a drop in sICAM-1 after anti-inflammatory therapy. sE-selectin and sL-selectin serum values decreased only in lepromatous patients after therapy. It can be concluded that soluble adhesion molecules like sICAM-1 and sE-selectin are promising activity markers in patients with leprosy, which may be useful for treatment monitoring.
Regulation of the neutralizing anti-hepatitis B surface (HBs) antibody response in vitro in HBs vaccine recipients and patients with acute or chronic hepatitis B virus (HBV) infection
Authors: Böcher W O , Herzog-Hauff S , Herr W , Heermann K , Gerken G , Meyer Zum BüSchenfelde K-H , Löhr H F
Published: November 2003

Antibodies directed to the HBs antigen indicate viral clearance and the development of life-long immunity in patients that recovered from HBV infection. In HBs antigen vaccine recipients anti-HBs antibodies provide protective immunity. However, little is known about the regulation of this HBs-specific antibody response. The existence of anti-HBs-secreting B cells was demonstrated using the highly sensitive ELISPOT technique compared with conventional ELISA in serum and cell culture supernatants. In the peripheral blood of patients with acute self-limited hepatitis B, HBs-specific B cells were demonstrated with a high frequency despite undetectable anti-HBs serum antibodies. HBV-immunized patients that had recovered from infection and vaccine recipients had significantly lower frequencies, whereas chronic HBV carriers and negative controls showed no anti-HBs-secreting B cells. Coculture experiments of isolated B and T cells revealed that the anti-HBs antibody response was restricted to the presence of T helper cells, but not to identical HLA class II molecules. Allogeneic T cells derived from vaccine recipients or chronic HBV carriers stimulated the HBs-specific B cell response in HBs vaccine recipients. Otherwise, isolated T helper cells could never provide sufficient help to induce the HBs-specific B cell response in chronic HBV carriers. Furthermore, peripheral blood mononuclear cells (PBMC) of six out of 10 vaccine recipients, one out of five HBV-immunized patients, but of no chronic HBV carrier showed a proliferative response to different HBs antigen preparations. This study demonstrated a high frequency of circulating anti-HBs-producing B cells in the early phase of acute HBV infection, but a lower frequency of HBs-specific B cells years after resolution of HBV infection. In chronic HBV carriers, however, deficient HBs-specific T and B cell responses were observed.
Rapid cytotoxicity of human B lymphocytes induced by VH4-34 (VH4.21) gene-encoded monoclonal antibodies
Authors: Bhat N M , Bieber M M , Stevenson F K , Teng N N H
Published: November 2003

We have previously described two human cold agglutinin MoAbs 216 and A6(H4C5), that are derived from the VH4-34 (VH4.21) gene that bind specifically to a cell surface ligand on human B lymphocytes. In this study, we report that binding of 216 and A6(H4C5) leads to rapid killing of target B cells. This complement-independent cytotoxicity was measured by three independent assays, cell viability dye uptake on FACS, 3H-thymidine uptake, and the 3(4,5)-dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxicity was specific for CD20+ mononuclear cells in human spleen and peripheral blood. The MoAbs were also cytotoxic to human B cell lines Nalm-6, OCI-LY8, Arent and SUP-B8, but not to T cell lines HuT 78 and PEER. As observed by scanning electron microscopy, membrane pores were formed within 15 min of exposure to the MoAbs. Cytotoxic activity was dependent on MoAb concentration and temperature of exposure. Killing was greater at 4°C than 37°C. Sodium azide and EDTA did not block the cytotoxic activity. No DNA fragmentation typical of apoptosis was observed. This rapid cytotoxic activity, independent of physiologic cellular processes and independent of complement, suggests a novel mechanism of cell death via membrane perturbations.
Experimental hypersensitivity pneumonitis: cellular requirements
Authors: Schuyler M , Gott K , Edwards B
Published: November 2003

We previously demonstrated that Thy1.2+, CD4+, Ia− T cells are responsible for transfer of murine adoptive experimental hypersensitivity pneumonitis (adoptive EHP). To characterize the culture conditions necessary for development of these cells, we depleted cell cultures of Thy1.2+, CD4+, CD8+, or Ia+ cells using MoAbs and complement or magnetic beads, prior to culture of sensitized C3H/HeJ murine spleen cells (SC) with Micropolyspora faeni. After culture, cells were transferred to recipients which were later challenged intratracheally with M. faeni. The extent of pulmonary inflammatory changes in these animals was determined 4 days after intratracheal (i.t.) challenge with M. faeni. Cultured M. faeni-sensitized SC which had been treated before culture with media, complement only, anti-CD8 plus complement or magnetic beads alone could transfer EHP to naive animals. SC treated with anti-Thy1.2 or anti-CD4 plus complement could not transfer EHP. Treatment of SC with anti-Iak plus magnetic beads diminished the ability of cultured cells to transfer EHP. We conclude that the ability to produce cells able to adoptively transfer EHP is dependent on the presence of Thy1.2+, CD4+ and Ia+ cells, but not CD8+ cells, at the onset of culture.
A vascular smooth muscle-specific CD4+ T cell line that induces pulmonary vasculitis in MRL-+/+ mice
Authors: Sumita S , Ozaki S , Okazaki T , Sobajima J , Nakao K
Published: November 2003

We established a T cell line, MV1, specific for rat vascular smooth muscle antigen from the regional lymph nodes of immunized MRL/Mp-+/+ mice. Adoptive transfer of MV1 T cells induced vasculitis lesions in the lungs of the syngeneic recipient mice pre-treated with cyclophosphamide. Flow cytometric analysis showed that MV1 was a CD4+ T cell line. The T cells proliferated in the presence of the vascular smooth muscle antigen and mitomycin C-treated syngeneic spleen cells. The cross experiments using an ovalbumin-specific T cell line demonstrated that MV1 was specific for vascular smooth muscle antigen. The antigen-specific proliferation of MV1 was CD4-dependent, which was consistent with the flow cytometric analysis. In addition, MV1 T cells, upon activation with anti-CD3 antibody or antigen-specific activation, killed A20.2J mouse B lymphoma cells. MV1 T cells also killed a CD95 (Fas)-transfected T lymphoma line, but not its parental Fas-negative cell line. These findings indicate that MV1 T cells killed target cells via a Fas ligand (FasL)/Fas pathway. The cytotoxicity of MV1 T cells may play an important role in the development of vasculitis in this model. Although the antigenic epitopes of MV1 and the lung specificity of vasculitis remain to be clarified, MV1-induced vasculitis should serve as an experimental model of human pulmonary vasculitis.
Frequency of anti-bactericidal/permeability-increasing protein (BPI) and anti-azurocidin in patients with renal disease
Authors: Yang J J , Tuttle R , Falk R J , Jennette J C
Published: November 2003

The major subtypes of anti-neutrophil cytoplasmic antibodies (ANCA) detected by indirect immunofluorescence assay (IFA) are P-ANCA and C-ANCA. In patients with vasculitis, myeloperoxidase (MPO) is the major P-ANCA antigen and proteinase 3 (PR3) is the major C-ANCA antigen. BPI and azurocidin, which are also called 57-kD cationic antimicrobial protein (CAP 57) and 37-kD cationic antimicrobial protein (CAP 37), respectively, have been proposed as less frequent target antigens for C-ANCA and P-ANCA. In patients with renal disease, we determined the frequency of antibodies against BPI and azurocidin. By IFA on alcohol-fixed neutrophils, monoclonal and polyclonal anti-BPI antibodies produced a C-ANCA pattern, whereas rabbit anti-azurocidin antibody produced a P-ANCA pattern. By ELISA, sera from 229 P-ANCA-positive patients, 99 C-ANCA-positive patients and 48 ANCA-negative (by IFA) patients with renal biopsies were tested for reactivity with recombinant human BPI and purified human azurocidin. Of these sera, 17.5% of P-ANCA, 30.3% of C-ANCA and 20.8% of IFA-ANCA-negative sera were positive for anti-BPI; and 8.3% of P-ANCA, 3.0% of C-ANCA and 8.3% of IFA-ANCA-negative sera were positive for anti-azurocidin. There was no statistical difference in frequency of anti-BPI between pauci-immune necrotizing and crescentic glomerulonephritis (NCGN) and other glomerular disease (OGD), and there was a lower frequency of anti-azurocidin in NCGN samples than in OGD samples. By Western blot, anti-BPI-positive sera reacted with a 57-kD BPI band and anti-azurocidin-positive sera with a 29-kD azurocidin band. In conclusion, there is a low frequency of anti-BPI and anti-azurocidin antibodies in ANCA-positive patient sera; however, this does not correlate with NCGN, which is a marker for ANCA-associated small vessel vasculitis, and a similar positivity is found in IFA-ANCA-negative patients with renal disease. Therefore, serologic detection of anti-BPI and anti-azurocidin is not diagnostically specific in patients with renal disease.
Natural killer (NK) lymphocytosis induced by Epstein–Barr virus (EBV) reactivation in monoclonal gammopathy of undertermined significance (MGUS)
Authors: Ishiyama T , Koike M , Watanabe K , Watanabe H , Fukuchi K , Tsuruoka N
Published: November 2003

We investigated the relationship between NK lymphocytosis and EBV infection in MGUS. We found that two out of 10 patients showed an increase of NK cells compared with normal controls. In addition, these two patients had far higher levels of CD5LOW+ NK cells (activated NK cells) than controls. EBV DNA was detected by polymerase chain reaction in peripheral blood mononuclear cells from the two patients, although the other eight patients with MGUS and all 20 normal controls had no detectable EBV DNA. Furtheremore, EBV DNA was detected in sorted CD5LOW+ NK cells. These results suggest that reactivation of EBV might be related to the increase of NK cells, particularly CD5LOW+ NK cells in some patients with MGUS.
Structural polymorphisms of complement receptor 1 (CR1) in systemic lupus erythematosus (SLE) patients and normal controls of three ethnic groups
Authors: Moulds J M , Reveille J D , Arnett F C
Published: November 2003

CR1 exhibits a molecular weight polymorphism and variability in the number of C3b-binding sites. Because this may affect immune complex clearance, we used erythrocytes to investigate the CR1 size polymorphism in SLE patients from three ethnic groups. The CR1-C allele was found more frequently in African–Americans, but the frequency did not differ between controls (10%, n = 63) and SLE patients (9%, n = 79). A 160-kD band similar to CR1-C was noted in a number of patients and was shown to be a proteolytic cleavage fragment. The study shows that the smallest form of CR1, i.e. CR1-C, is not a genetic risk factor for SLE and that the frequencies of the CR1 structural alleles do not differ from race-matched healthy controls.
Elastase, but not proteinase 3 (PR3), induces proteinuria associated with loss of glomerular basement membrane heparan sulphate after in vivo renal perfusion in rats
Authors: Heeringa P , Van Den Born J , Brouwer E , Dolman K M , Klok P A , Huitema M G , Limburg P C , Bakker M A H , Berden J H M , Daha M R , Kallenberg C G M
Published: November 2003

Elastase, but not PR3, induces proteinuria associated with loss of glomerular basement membrane (GBM) heparan sulphate after in vivo renal perfusion in rats. PR3 and elastase are cationic neutral serine proteinases present in the azurophilic granules of polymorphonuclear leucocytes. Release of these proteolytic enzymes along the glomerular capillary wall may induce glomerular injury. Here, we investigated the effects of PR3 and elastase on the induction of proteinuria and glomerular injury after renal perfusion of these enzymes in Brown–Norway rats. Perfusion of active elastase induced a dose-dependent proteinuria 24 h after perfusion, while inactivated elastase did not. Perfusion of comparable amounts of active PR3 did not induce proteinuria. Light and electron microscopy showed no morphological abnormalities in any experimental group. However, immunohistology revealed that proteinuria occurring after perfusion of active elastase was associated with a strong reduction in intraglomerular expression of the heparan sulphate side chain and, to a lesser extent, of the protein core of heparan sulphate proteoglycans (HSPG). In vitro, both elastase and PR3 digested HSPG. However, PR3 bound to a lesser extent to HSPG than elastase. We conclude that elastase, but not PR3, induces proteinuria after in vivo renal perfusion. This differential effect probably relates to different binding to the GBM of those enzymes due to differences in their isoelectric points. Degradation of heparan sulfate proteoglycans, leading to the disappearance of their side chains that contribute to the polyanionic structure of the GBM, appears to be involved in the induction of proteinuria after perfusion of elastase.
Histaminergic regulation of interferon-gamma (IFN-γ) production by human natural killer (NK) cells
Authors: Asea A , Hansson M , Czerkinsky C , Houze T , Hermodsson S , Strannegård Ö , Hellstrand K
Published: November 2003

Monocytes, recovered from human peripheral blood by counter-current centrifugal elutriation, effectively inhibit the production of IFN-γ by CD3−/56+ NK cells in response to IL-2. This study aimed at defining the nature of the inhibitory signal, particularly the importance of monocyte-derived reactive metabolites of oxygen. It was found that monocytes recovered from patients with chronic granulomatous disease (CGD), a condition characterized by deficient NADPH-oxidase activity of phagocytes, did not inhibit IFN-γ production by NK cells. Further, catalase, a scavenger of hydrogen peroxide, completely reversed the inhibitory signal, whereas scavengers of the superoxide anion, hypohalous acids, the hydroxyl radical, or nitric oxide synthesis inhibitors such as L-NMMA were ineffective. Inhibition of IFN-γ production was operating on a pre-translational level, as indicated by the inability of enriched NK cells to accumulate IFN-γ mRNA in the presence of elutriated monocytes. Hydrogen peroxide, at micromolar concentrations, reconstituted the inhibition of IFN-γ production when added to enriched NK cells. Histamine, a biogenic amine which inhibits the generation of reactive oxygen metabolites in monocytes, abrogated the inhibition of IFN-γ production in NK cells; by this mechanism, histamine strongly synergized with IL-2 to induce IFN-γ in mixtures of NK cells and monocytes. The synergizing effect of histamine was specifically mediated by H2-type histamine receptors. We conclude that: (i) the induction of IFN-γ mRNA in NK cells is effectively down-regulated by products of the oxidative metabolism of monocytes; and (ii) histamine effectively enhances IFN-γ production by preventing monocyte-induced oxidative damage to NK cells.
Circulating levels of IL-11 and leukaemia inhibitory factor (LIF) do not significantly participate in the production of acute-phase proteins by the liver
Authors: Gabay C , Singwe M , Genin B , Meyer O , Mentha G , Le Coultre C , Vischer T , Guerne P-A
Published: November 2003

To investigate the contribution of IL-11 and LIF to acute-phase protein (APP) production, we first analysed the effects of IL-11 and LIF on production of C-reactive protein (CRP), fibrinogen, and haptoglobin by human primary hepatocytes. We also measured the serum levels of IL-11, LIF, and CRP in serum from patients with inflammatory rheumatic diseases to assess the role of these cytokines in the APP response in vivo. We included patients with conditions associated with a high APP response such as rheumatoid arthritis (RA) or spondylarthropathy (SpA), and others usually associated with a weak APP response such as systemic lupus erythematosus (SLE), in order to investigate whether these cytokines could account for the differences in APP responses. Our results showed that IL-11 and LIF induced only minimal stimulation on production of APP by human primary hepatocytes compared with IL-6, known as the major inducer. Serum levels of CRP were elevated in RA and SpA, and significantly higher than in SLE patients. Despite the presence of a high APP response in some of our patients and despite the fact that we used sensitive assays to measure IL-11 and LIF, serum levels of both cytokines were not detected in any of the tested sera. In conclusion, our results show that circulating levels of IL-11 or LIF do not contribute significantly to the production of APP in vivo, and that they do not account for the difference in APP response between SLE and other inflammatory rheumatic diseases.
Dendritic cells and tolerance induction
Authors: Steptoe R J , Thomson A W
Published: November 2003

Dendritic cells (DC) are widely accepted as the most potent antigen-presenting cells (APC), and considerable interest has been generated in their potential for the immunological therapy of cancer and infectious disease. Recently, however, a broader understanding of the phenotypic diversity and functional heterogeneity of DC has been acquired. Thus, in addition to having a role in central tolerance, DC are now regarded as potential modulators of peripheral immune responses. Harnessing this potential may offer a new approach to the immunosuppressive therapy of allograft rejection or autoimmunity. Here, the concept of ‘tolerogenic’ DC is placed in the context of rapidly accumulating new evidence of the diverse properties of these important APC.
IL-4 down-regulates the responsiveness of human intraepithelial lymphocytes
Authors: Ebert E C , Roberts A I
Published: November 2003

IL-4 is an important regulator of intestinal inflammation, yet little is known regarding its action on intestinal lymphocytes. Intestinal lymphocytes were isolated from jejunal mucosa of patients undergoing gastric bypass operations for morbid obesity. The impact of IL-4 was measured by its effects on proliferation using 3H-thymidine incorporation, IL-2 production using the CTLL assay, and IL-2 receptor generation using immunofluorescence. The production of IL-4 was measured by ELISA. IL-4 significantly inhibited the proliferation of intraepithelial lymphocytes (IEL) to a variety of stimuli as well as the development of lymphokine-activated killer (LAK) cells. In contrast, it had no effect on the proliferation of CD8+ T cells from peripheral blood. The inhibitory effect of IL-4 on IEL did not occur during activation, since IL-2 production and receptor expression were not altered. Rather, it occurred during cell cycling, since over 50% inhibition resulted whether IL-4 was added at the initiation of an IL-2-stimulated culture or after 24 or 48 h incubation. IL-4 was secreted by activated lamina propria lymphocytes (LPL) but not by IEL. IL-4,produced by activated LPL, may enter the epithelial compartment and down-regulate responsiveness of IEL.
Intrathyroidal cytokine gene expression in Hashimoto's thyroiditis
Authors: Ajjan R A , Watson P F , Mcintosh R S , Weetman A P
Published: November 2003

Hashimoto’s thyroiditis (HT) is an organ-specific autoimmune disease in which cytokines are likely to have a role in the initiation and perpetuation of the disease. Using the reverse transcription-polymerase chain reaction (RT-PCR) we analysed the cytokine profile in four HT tissue samples. Furthermore, cell fractionation was carried out on two tissue samples and cytokine profile was studied in CD4+ and CD8+ T cells, in addition to the residual cellular infiltrate composed of CD4− and CD8− cells. Our results showed IL-1β, IL-4 and IL-6 mRNA expression in three out of four tissue samples, whereas IL-1α, IL-2, IL-8, IL-10, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) were expressed in all tissue samples studied. Expression of IL-1α and IL-1β was absent in both CD4+ and CD8+ subsets. However, IL-2, IL-4, IL-6, IL-8, IFN-γ and TNF-α mRNA were detected in both CD4+ and CD8+ subsets. IL-10 was expressed in the CD4+ subset in one sample, whereas it was negative in both CD8+ fractions. All the cytokines studied were expressed in the residual infiltrate. These results suggest a mixed Th1 and Th2 response in HT, both of which may have an important role in the pathophysiology of the thyroid destructive process through cell-mediated cytotoxicity, and/or humoral autoimmunity manifested by autoantibody production.
Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry
Authors: North M E , Ivory K , Funauchi M , Webster A D B , Lane A C , Farrant J
Published: November 2003

Using three-colour flow cytometry, we have measured intracellular IL-2, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) induced in human CD4+ and CD8+ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC-conjugated anti-cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4+ cells can make IL-2 than the other two cytokines, and that there are more TNF-α-positive CD4+ cells than cells with IFN-γ. For normal CD8+ cells the highest production of cytokine is of IFN-γ (up to 50% of the cells) especially at longer times (10–20h) of stimulation. For CD8+ cells, IL-2-positive cells exceed those with TNF-α. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20h) increased the detection of IL-2-positive cells without apparently reducing the percentage of cytokine-positive CD4+ or CD8+ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL-2 and TNF-α but significantly raised levels of production of IFN-γ in both CD4+ and CD8+ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1-type response.
Thy-1 molecule associates with protein tyrosine kinase(s) in rat mesangial cells
Authors: Narisawa-Saito M , Yamanashi Y , Morioka T , Oite T , Shimizu F
Published: November 2003

Glycophosphatidylinositol (GPI)-linked Thy-1 molecules, well known cell surface markers of murine T cells, are present on the glomerular mesangial cells of the rat kidney. The administration of anti-Thy-1.1 MoAbs 1-22-3 and OX-7 to rats induces severe and mild complement-dependent mesangial proliferative glomerulonephritis, respectively. In order to determine whether protein-tyrosine kinase (PTK) activity is associated with Thy-1 molecules on rat mesangial cell surface, we performed an immune complex kinase assay, using anti-Thy-1 MoAbs 1-22-3 and OX-7, followed by reimmunoprecipitation with anti-phosphotyrosine, anti-fyn, anti-lck and anti-lyn antibodies. Physical association of PTK, p59fyn and p56/53lyn with Thy-1 molecules was demonstated in cultured rat mesangial cells. The activities of these kinases detected in MoAb 1-22-3 precipitates were higher than those in MoAb OX-7 precipitates. These results suggest that Thy-1 molecule transduces some signals also in rat mesangial cells.
Evidence for a selected humoral immune response encoded by VH4 family genes in the synovial membrane of a patient with rheumatoid arthritis (RA)
Authors: Voswinkel J , Trümper L , Carbon G , Hopf T , Pfreundschuh M , Gause A
Published: November 2003

The analysis of rearranged antibody-encoding genes from B cell foci in rheumatoid synovial tissue has characterized these cells as highly mutated memory B cells with a high proportion of members of the VH4 family. In order to characterize further the VH4 response in one patient, B cell-rich areas from different sections of synovial membrane (SM) were identified by CD20 staining, isolated by microdissection and pooled, in order to analyse highly enriched B cells without selection by in vitro culture procedures. From DNA of about 5 × 103 B cells rearranged VH genes were amplified by polymerase chain reaction (PCR) and cloned. Sequencing of 11 clones containing rearranged VH4 gene products revealed that seven were potentially functional, and all were mutated with 84–96% homology to known germ-line (gl) genes and VH4 gl genes amplified from the patient’s genomic DNA. Analysis of the complementarity determining region (CDR) 3 revealed that two products represented members of one B cell clone which differed by five nucleotide changes. Three of the five mutations encoded amino acid replacements in CDRs indicating antigen-driven expansion of one specific clone. Additional analyses of 25 members of three B cell clones from isolated aggregates showing intraclonal diversity in one of three clones provided further evidence that antigen selection takes place in the SM. Overall, the pattern of mutations and the replacement to silent (R:S) ratios were diverse, with six products indicating antigen selection by their high R:S ratios in CDRs. Although DNA analysis does not allow a characterization of antibody specificities, we can conclude from our analysis of antibody-encoding genes that selection by antigen and expansion of specific clones occur in the SM against the background of polyclonal activation.
Release of transforming growth factor-beta (TGF-β) and fibronectin by alveolar macrophages in airway diseases
Authors: Vignola A M , Chanez P , Chiappara G , Merendino A , Zinnanti E , Bousquet J , Bellia V , Bonsignore G
Published: November 2003

Asthma and chronic bronchitis are associated with airway remodelling, and airway macrophages are present in bronchial inflammation. TGF-β and fibronectin released by alveolar macrophages possess a fibrogenic potency. The potential role of alveolar macrophages in airway remodelling was studied in asthma and chronic bronchitis by the release of TGF-β and fibronectin. Alveolar macrophages were isolated by bronchoalveolar lavage in 14 control subjects, 14 asthmatics and 14 chronic bronchitics. The spontaneous and lipopolysaccharide (LPS)- or concanavalin A (Con A)-induced release of TGF-β and fibronectin was measured by ELISA. Alveolar macrophages from chronic bronchitics spontaneously release greater amounts of TGF-β and fibronectin than those from asthmatic and control subjects. Alveolar macrophages from asthmatics release greater amounts of TGF-β and fibronectin than those from control subjects. The spontaneous release of TGF-β is significantly correlated with that of fibronectin. Fibronectin release was significantly reduced after LPS stimulation, and TGF-β release was significantly increased after LPS stimulation, except in chronic bronchitis patients. Con A increased the release of TGF-β in cells from normal subjects. This study suggests that activated macrophages play a role in airway remodelling in chronic bronchitis and to a lesser extent in asthma.
IL-3 derived from CD4− T cells is essential for the in vitro expansion of mast cells from the normal adult mouse spleen
Authors: Harada M , Sumichika H , Hamano S , Ito O , Tamada K , Takenoyama M , Kimura G , Nomoto K
Published: November 2003

A null cell line (SCM1) was established by a culture of spleen cells (SC) from normal adult C57Bl/6 mice with complete medium alone for 10 days and followed by weekly cultures with a 25% WEHI-3 cell culture supernatant. Phenotype analysis showed that the SCM1 cells were negative for CD3, Thy1.2, B220, Mac-1, Gr-1, NK1.1 and MHC class II, but were positive for MHC class I, FcγRII/III, FcεRI, c-kit and the receptor against wheat germ agglutinin. These findings suggested that the SCM1 cells were mast cells. In an in vitro proliferation assay, SCM1 cells proliferated in the presence of either IL-3 or stem cell factor (SCF), but not in the presence of IL-4, whereas IL-4 showed an augmenting effect on their proliferation in the presence of either IL-3 or SCF. In analysing the mechanism by which such mast cells could be expanded from normal adult mouse SC, the addition of anti-IL-3 MoAb, but not anti-SCF MoAb, into the initial culture inhibited the subsequent expansion of either IL-3- or SCF-responding cells. The prior depletion of CD4+ T cells abrogated the capacity of the SC to enhance the expansion of SCF-responding cells, and this inability was restored by the addition of IL-3. Moreover, the culture supernatant of normal adult SC alone contained considerable levels of IL-3. Taken together, our findings suggest that, in an in vitro culture, CD4+ T cell-derived IL-3 therefore enhances the expansion of mast cells from the normal adult mouse spleen.
Site-directed serology of HIV-1 subtype B infection: relation between virus specific antibody levels and disease progression
Authors: Broström C , Sönnerborg A , Kim S , Sällberg M
Published: November 2003

Activated T helper (Th) cell-dependent (TD) antibody responses were determined over an 8–10 year period in 28 patients infected with HIV-1 subtype B. Twelve patients remain asymptomatic with normal CD4+ cell counts for 101–114 months. These individuals were defined as long-term asymptomatic (LTA). Sixteen patients progressed to severe immunodeficiency within 58–120 months. In samples derived close to the diagnosis of HIV-1, CD4+ cell counts were higher among the LTAs (P < 0.01). Antibody production driven by activated Th cells was determined using peptides corresponding to HIV-1 V3US/Eur, gp41, and the hepatitis C virus (HCV) core proteins. The less Th cell-dependent B cell antibody response was represented by measles virus immunity. Close to HIV-1 diagnosis, variable third (V3), gp41, HCV core, and measles antibody titres were at similar levels among the LTAs and the progressors. With time the LTAs displayed unchanged levels of V3 and gp41 antibodies, and slightly decreasing levels of HCV core antibodies (P < 0.05). In contrast, the progressors showed a decrease in all these antibody responses (P < 0.05, for all). In both groups, the levels of measles antibody remained stable. Our data show that no significant change of the antibody responses of LTAs is seen, even after 101–114 months of known HIV-1 infection. Furthermore, the marked decrease of TD antibody production in the progressors suggests that activated Th cells may be excellent targets for HIV-1 infection.
Persistent reduction in IL-6 mRNA in peripheral blood mononuclear cells of patients with rheumatoid arthritis after treatment with a monoclonal antibody to CD54 (ICAM-1)
Authors: Schulze-Koops H , Lipsky P E , Kavanaugh A F , Davis L S
Published: November 2003

There is persistent excessive production of a number of pro-inflammatory cytokines in patients with rheumatoid arthritis (RA). A number of experimental therapies have been found to be effective in the treatment of RA, although the effects of these therapies on cytokine production have not been evaluated. One such experimental therapy involves administration of a MoAb to intercellular adhesion molecule-1 (ICAM-1) that has been shown to be clinically beneficial in approximately 60% of the patients, with some patients responding for up to 11 months. The current studies were carried out to determine whether the success of this therapy was associated with changes in mRNA levels for pro-inflammatory and other monocyte-derived cytokines in peripheral blood mononuclear cells (PBMC). Cytokine mRNA levels were assessed in freshly isolated unstimulated PBMC by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) after minimal in vitro manipulation of the cells. Anti-ICAM-1 MoAb administration was followed by an increase in IL-1β, IL-8, and tumour-necrosis factor-alpha (TNF-α) mRNA detected at 2 or 24 h after the initial infusion in the clinical responders, but no persistent change in these cytokine mRNA levels were observed that correlated with clinical benefit. By contrast, IL-6 mRNA levels declined immediately after the first MoAb infusion and reached pretreatment values variably after the 5 day treatment course. IL-6 mRNA levels remained significantly reduced in patients responding to therapy 1 month after treatment. Fluctuations in monocyte numbers within the PBMC after treatment did not account for the observed changes in cytokine mRNA levels. The results suggest that a decline in IL-6 mRNA levels is a pharmacological action of the MoAb to ICAM-1. Moreover, persistently diminished IL-6 mRNA levels induced by anti-ICAM-1 MoAb might be associated with the long-term benefit of this therapy. In addition, monitoring the activation status of monocytes in circulating PBMC may be useful in predicting response to therapy and warrants further investigation in a larger study population.
The behaviour of human vitronectin in vivo: effects of complement activation, conformation and phosphorylation
Authors: Peake P W , Greenstein J D , Pussell B A , Charlesworth J A
Published: November 2003

We examined the behaviour in vivo of native, specifically phosphorylated, and multimeric vitronectin to determine the effects of these modifications on its turnover, distribution and molecular behaviour. In normal rabbits, the plasma half-life (T1/2) of antigenically detected vitronectin was 8.00 ± 1.26 h (mean ± s.d.), with a fractional catabolic rate (FCR) of 18.77 ± 1.57%/h and extravascular/intravascular ratio (EV/IV) of 1.00 (0.48–1.60, median and range). For vitronectin selectively phosphorylated by protein kinase A, T1/2 was 8.87 ± 0.48 h, with a significantly smaller FCR of 10.85 ± 0.71%/h (P<0.005) and an EV/IV of 0.28 (0.15–0.36) (P<0.05 compared with antigenically detected vitronectin). In vitro, phosphorylation had no effect on the affinity of vitronectin for heparin–Sepharose, while complement activation with cobra venom factor (CVF) led to a two-fold enrichment of 32P-vitronectin within the SC5b-9 complex. In vivo CVF caused a rapid decrease in the circulating levels of 32P-vitronectin and was accompanied by the prompt appearance of a high mol. wt species consistent with SC5b-9. Despite specific incorporation of 32P-vitronectin into SC5b-9, both forms of the molecule had similar inhibitory effects on C9-mediated haemolysis of EAC1-7 cells. Urea-activated vitronectin was rapidly cleared from circulation with less than 15% remaining after 1 h while protein-bound label accumulated in the spleen, lung and liver. These results demonstrate that vitronectin is a rapidly metabolized protein whose in vivo behaviour is markedly altered when phosphorylated or activated to form multimers and SC5b-9.
Biased expression of T cell receptor Vβ genes in periodontitis patients
Authors: Yamazaki K , Nakajima T , Gemmell E , Kjeldsen M , Seymour G J , Hara K
Published: November 2003

The aim of the present study was to investigate the hypothesis that there is selective activation and expansion of a limited repertoire of T cell receptor (TCR)-bearing T cells in periodontitis tissue. We studied TCR Vβ gene expression in gingival tissues of periodontitis lesions and compared these with peripheral blood mononuclear cells (PBMC) from the same patients using polymerase chain reaction (PCR) amplification with 22 Vβ-specific sense primers in combination with a common antisense Cβ-specific primer. After initial therapy, gingival tissue specimens were obtained from 14 periodontitis patients at the time of periodontal surgery, and PBMC were also obtained from the same patients by density gradient centrifugation. Total RNA was extracted and analysed by reverse transcription PCR. The PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. For each product, gingival tissue and the respective peripheral blood were compared. The TCR repertoire identified in the PBMC in general overlapped with that of the gingival tissue. However, Vβ6 appeared to be over-expressed in the gingival tissues compared with the PBMC, but Vβ16 appeared to be under-expressed in the gingival tissues. Although it is not known whether this is due to antigen-specific activation or superantigen activation, the data suggest that there may be as yet uncharacterized T cell subsets in periodontal disease tissue.
The chemoattractant activity of rheumatoid synovial fluid for human lymphocytes is due to multiple cytokines
Authors: Al-Mughales J , Blyth T H , Hunter J A , Wilkinson PC
Published: November 2003

The majority of synovial fluids from 29 rheumatoid arthritis patients were strongly attractive for normal blood lymphocytes judged by assays of polarization and collagen gel invasion. While rheumatoid synovial fluids contained IL-15, IL-8, monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) at levels sufficient to attract lymphocytes, inhibition of the activity of any single cytokine using specific antibody did not abolish the activity of the fluid. However combinations of anti-cytokine antibodies used together (anti-IL-15+anti-MCP-1; anti-IL-8+anti-MCP-1 or +anti-MIP-1α) inhibited most of the activity, suggesting that attraction of lymphocytes by the fluids is due to a combination of attractants. Blood lymphocytes required activation by overnight culture to respond optimally, while rheumatoid synovial tissue lymphocytes responded to synovial fluids without a requirement for a period of culture. Lymphocytes derived from rheumatoid synovial fluids were poorly responsive to locomotor stimulants. Most of the responding cells from blood mononuclear cell fractions were T lymphocytes of the CD45RO isotype. Incubation in the presence of cyclosporin A or corticosteroids inhibited the response of lymphocytes to the fluids, but the presence of non-steroidal anti-inflammatory drugs (NSAIDs) and other agents used in therapy of the patients from whom the fluids were taken had no inhibitory effect.
Immune response and in vivo production of cytokines in patients with liver hydatidosis
Authors: Torcal J , Navarro-Zorraquino M , Lozano R , Larrad L , Salinas J C , Ferrer J , Roman J , Pastor C
Published: November 2003

Cytokines play an important role in the human immunological response, but the exact role of cytokines in the human immune response against parasites, especially against Echinococcus granulosus, remains unclear. IL-1, IL-2, IL-4 and tumour necrosis factor (TNF) levels in peripheral blood of 21 patients with liver hydatidosis were evaluated before surgical treatment, and the levels of IgA, IgM, IgG, IgE, specific IgE against E. granulosus, C3, C4 and BF complement fractions and CD20, CD3, CD4, CD8 and CD16 cell percentages were also determined, as was the relationship between these variables and cytokine levels. Data from hydatid patients were compared with data obtained from 21 healthy volunteers. Hydatid patients showed increases of IgG, IgE, IgEs and IL-2 (P < 0.01), and decreases of IL-1 and TNF levels (P < 0.001), but these variables (respectively) increased in patients showing cysts in the central area of the liver or with a wide opening of cysts in the biliary tract. The increase of IL-1, IL-2 and IL-4 showed a close relationship with the number, characteristics and above all the location of cysts within the liver itself. IgG and IL-4 levels and also IgG and IgE levels showed a significant correlation (P < 0.05).
Ligation of CR1 (C3b receptor, CD35) on CD4+ T lymphocytes enhances viral replication in HIV-infected cells
Authors: Mouhoub A , Delibrias C C , Fischer E , Boyer V , Kazatchkine M D
Published: November 2003

The present study provides evidence for a role of the C3b receptor, CR1 (CD35), in activation of HIV replication in CD4+ T lymphocytes. Ligation of CR1 with cross-linked anti-receptor MoAbs or with aggregated C3b enhanced transcription of viral genes and the release of p24 and RT activity from cultures of purified normal CD4+ T lymphocytes that had been infected with HIV-1 in vitro. No effect was observed upon ligation of CR2 (CD21), a C3 receptor that is also expressed on human CD4+ T cells. Cross-linking of CR1 also enhanced HIV replication in peripheral blood CD4+ lymphocytes isolated from HIV+ individuals. The enhancing effect of CR1 was not related to a mitogenic effect induced by stimulation of the receptor on T cells. The CR1 specificity of the enhancing effect was established by the observation that the addition of soluble recombinant CR1 to the cultures abolished the enhancement of p24 release induced by anti-CR1 MoAbs. Our results suggest that HIV replication may be triggered in resting HIV-infected CD4+ T lymphocytes through interaction with C3b-bearing immune complexes or particles.