bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular mRNA expression of interferon-gamma (IFN-γ), IL-4 and transforming growth factor-beta (TGF-β) in rats nasally tolerized against experimental autoimmune myasthenia gravis (EAMG)
Authors: Ma C-G , Zhang G-X , Xiao B-G , Link H
Published: November 2003

Nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR plus Freund’s complete adjuvant (FCA) resulted in prevention or marked decrease of the severity of EAMG, suppression of AChR-specific B cell responses and of AChR-reactive T cell functions. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radiolabelled synthetic oligonucleotide probes was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine IFN-γ, the B cell stimulating IL-4 and the immune response-down-regulating TGF-β. Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-γ, IL-4 and TGF-β mRNA-expressing cells compared with control rats receiving PBS nasally and injected with FCA only. Nasal tolerance to EAMG was accompanied by decreased numbers of AChR-reactive IFN-γ and IL-4 mRNA-expressing cells, and strong up-regulation of TGF-β mRNA-positive cells in lymphoid organs compared with non-tolerized EAMG control rats. The relative affinity of anti-AChR antibodies was lower, but muscle AChR amounts were higher in nasally tolerized rats compared with non-tolerized EAMG control rats. The results suggest that IFN-γ and IL-4 are central effector molecules in the development of EAMG, and that TGF-β plays an important role in tolerance induction to EAMG.
Interferon-gamma (IFN-γ) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats
Authors: Johansen H K , Hougen H P , Rygaard J , Høiby N
Published: November 2003

In a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF), we studied whether the inflammatory response could be altered by intraperitoneal treatment with recombinant rat interferon-gamma (rrIFN-γ). Rats were treated either before or after intratracheal challenge with P. aeruginosa embedded in alginate beads. Rats treated after challenge had a significant reduction in the severity of macroscopic lung inflammation compared with rats treated before challenge (P=0.004) and controls (P=0.003). The histopathology in controls was dominated by numerous polymorphonuclear leucocytes (PMN) (≥90%) surrounding the alginate beads like in CF. This could be caused by a Th2-like response. In contrast, a complete shift to a chronic-type inflammation dominated by mononuclear leucocytes (≥90% lymphocytes and plasma cells) and granulomas was observed in both rrIFN-γ-treated groups of rats. This could be caused by a Th1-like response. There was no significant difference in lethality between the groups, and the antibody titres against P. aeruginosa sonicate and alginate were similar in the treated rats and controls. Since the ongoing lung tissue damage in CF patients has been shown to be caused by elastase secreted by PMN, which dominate the P. aeruginosa lung infection, our findings offer a possible new strategy of modifying the inflammatory response in CF patients.
Subclasses of IgA antibodies in serum and saliva samples of newborns and infants immunized against rotavirus
Authors: Friedman M G , Entin N , Zedaka R , Dagan R
Published: November 2003

Little is known about subclass levels of IgA in serum or saliva of infants in the perinatal period. We have previously shown that very young infants are capable of responding to an experimental rotavirus vaccine with both serum and salivary IgA, and that small amounts of IgA are already detectable in cord blood of these infants. In the present study, total IgA1 and IgA2 antibodies in serum and saliva samples of some of these infants at birth, at 6 weeks of age, and at 12 weeks of age, were determined by a quantitative ELISA. Also, subclass-specific IgA antibodies to the rotavirus group A common antigen were determined by ELISA. The ratio of average serum concentrations of IgA1 to IgA2 for 14 infants at 6 weeks of age was 19:1, while in saliva it was 5:1. Between 6 and 12 weeks of age levels of serum IgA1 increased by 25%, while levels of IgA2 did not increase perceptibly. Concentrations of IgA1 were higher in infant sera than in saliva, while concentrations of IgA2 were slightly higher in saliva than in serum. When calculated as specific ELISA units per mg IgA1, more salivary IgA1 was specific for rotavirus than serum IgA1. Further studies are needed to determine when infant IgA2 levels rise to values more characteristic of children and adults. This may be of significance for infant mucosal immunizations if secretory IgA2, more resistant to bacterial proteases than IgA1, is required for efficient defence of the respiratory and intestinal tracts.
Antibodies to proteinase 3 mediate expression of vascular cell adhesion molecule-1 (VCAM-1)
Authors: Mayet W-J , Schwarting A , Orth T , Duchmann R , Meyer Zum Büschenfelde K-H
Published: November 2003

VCAM-1 was first identified as an adhesion molecule induced on human endothelial cells (HEC) by inflammatory cytokines such as IL-1, tumour necrosis factor (TNF), and lipopolysaccharide (LPS). The molecule binds to a variety of leucocytes, including B cells, T cells, basophils, eosinophils and monocytes. Vascular expression of VCAM-1 has been associated with a number of disease states, including rheumatoid arthritis and vasculitis. The detection of antineutrophil cytoplasmic antibodies (ANCA), especially to proteinase 3 (PR3), has become important in the diagnosis of Wegener’s granulomatosis (WG) and related vasculitides. Recently we were able to demonstrate a direct effect of anti-PR3 antibodies on neutrophil–endothelial interactions (Blood 1993; 82:1221). Binding of anti-PR3 antibodies to their antigen translocated into the membrane of HEC leads to an enhanced adhesion of neutrophils via induction of E-selectin (Clin Exp Immunol 1993; 94:440). The aim of this study was to investigate the effect of anti-PR3 antibodies on the expression of VCAM-1. HEC was isolated from umbilical vein and cultured on microtitre plates. After preincubatiion with purified anti-PR3 antibody, purified control antibodies (SS-A, SS-B, RNP) (IgG and F(ab′)2 fragments) or different cytokines (controls), VCAM-1 was detected on the surface of unfixed HEC by cyto-ELISA and polymerase chain reaction analysis. Incubation of HEC with anti-PR3 antibodies led to a marked increase of endothelial VCAM-1 expression with a peak after 8 h. Incubation with TNF-α also led to maximal VCAM-1 expression after 4–6 h (control). Increased adhesion of T lymphocytes to HEC after binding of anti-PR3 antibodies to their antigen could be confirmed by performing adherence assays. This effect could be inhibited by antibodies to VLA-4. In conclusion, we have been able to show that cytokine-like effects of anti-PR3 antibodies on HEC are not limited to induction of neutrophil adhesion. Anti-PR3 antibodies may thus contribute to the regulation of T lymphocyte migration from the blood by HEC in ANCA-related vasculitides.
The catabolism of intact, reactive centre-cleaved and proteinase-complexed C1 inhibitor in the guinea pig
Authors: Malek R , Aulak K S , Davis III A E
Published: November 2003

Clearance rates in the guinea pig were determined for intact guinea pig and human C1 inhibitor, the complexes of both inhibitors with human C1s, β factor XIIa and kallikrein, and for each inhibitor cleaved at its reactive centre with trypsin. Intact human and guinea pig C1 inhibitor were cleared from the circulation more slowly (t½s of 9.7 h and 12.1 h and fractional catabolic rates (FCRs) of 0.09 and 0.117) than any of their cleaved or complexed forms. The reactive centre-cleaved inhibitors were cleared with half-lives of 6.75 h for humans and 10.1 h for the guinea pig. The complexes with target proteases were catabolized much more rapidly, with half-lives ranging from 3.08 h to 4.3 h. The complexes with kallikrein were cleared more slowly than those with C1s and β factor XIIa. Complexes prepared with the guinea pig and human inhibitors were cleared at equivalent rates. The free inactivated proteases were cleared at rates similar to the equivalent complexes, except for kallikrein, which was cleared more rapidly than its complex. The fact that the complexes with different target proteases differed in their catabolism and that protease and complex catabolism were similar suggests that protease may play a direct role in clearance.
Elevated levels of soluble CD14 in serum of patients with acute Plasmodium falciparum malaria
Authors: Wenisch C , Wenisch H , Parschalk B , Vanijanonta S , Burgmann H , Exner M , Zedwitz-Liebenstein K , Thalhammer F , Georgopoulos A , Graninger W , Looareesuwan S
Published: November 2003

Serum sCD14, tumour necrosis factor-alpha (TNF-α), IL-6, and endotoxin were analysed in 45 patients with complicated malaria, in 14 patients with Gram-negative septicaemia and in 24 healthy subjects by ELISA. Malaria patients with renal failure (n=16) had higher levels than patients without renal failure (n=29) (8116+1440 μg/lversus 9453+1017 μg/lP<0.05) and both had higher levels than patients with septicaemia (6155+1635μg/l) and normal subjects (2776+747 μg/l). A significant correlation between sCD14 and IL-6 (r=0.756) and TNF (r=0.822) existed. However, no relation between sCD14 and serum endotoxin or indices of clinical disease severity (parasitaemia, fever, parasite or fever clearance time) was seen. Although the role of sCD14 in malaria remains to be determined, elevated levels may participate in the inflammatory response in complicated malaria.
Anti-neutrophil cytoplasmic antibodies (ANCA) in ulcerative colitis: anti-cathepsin G and a novel antibody correlate with a refractory type
Authors: Sobajima J , Ozaki S , Okazaki T , Osakada F , Sumita S , Mori K , Nakao K
Published: November 2003

We analysed the clinical significance of ANCA in patients with ulcerative colitis. On either an indirect immunofluorescence assay or an ELISA with fixed neutrophils, 71% (25/35) of the patients were positive for ANCA. However, only half of them reacted with either cathepsin G or lactoferrin. Western blot assays revealed positive bands in 40% (10/25) of the antibody-positive patients. The sizes of the bands detected were ≈58, 47, 44, 40, and 28 kD. No significant correlation was found between the ANCA positivity and various variables, i.e. disease activity, extent of lesion, or treatment of the disease. The anti-cathepsin G and 28-kD positivity, however, significantly correlated with a refractory type of ulcerative colitis.
Human antibody response to a pneumococcal vaccine in SCID-PBL-hu mice and simultaneously vaccinated human cell donors
Authors: Aaberge I S , Steinsvik T E , Groeng E-C , Leikvold R-B , Løvik M
Published: November 2003

Severe combined immunodeficient (SCID) mice were transplanted intraperitoneally with human peripheral blood lymphocytes (PBL) from nine healthy human donors (SCID-PBL-hu mice). None of the donors had ever received pneumococcal vaccine. Ten days after transplantation, 62 out of 111 transplanted mice and six of the nine donors were vaccinated with a 23-valent pneumococcal polysaccharide vaccine. For each donor, human IgG was detected in 91.7–100% of the SCID-PBL-hu mice, whereas specific human IgG antipneumococcal antibodies were demonstrated in 16.7–100% of the vaccinated SCID-PBL-hu mice. Most of the mice transplanted with cells from the same donor showed similar antibody response patterns in terms of kinetics and antibody levels. A significant antibody response was only obtained in mice that received cells from donors with relatively high antipneumococcal antibody levels at the time of transplantation, or donors that showed a substantial increase in antibody levels after vaccination. The immune response in the SCID-PBL-hu mice did not always reflect the ability of the respective donor to produce antipneumococcal antibodies. The donor dependency of the antipneumococcal antibody response has great practical importance for the use of the SCID-PBL-hu model. Donors should not be chosen randomly. By selecting donors whose cells have been found to result in successful engraftment, functional SCID-PBL-hu mice can be obtained for the study of human immune responses and function in an in vivo experimental model.
Reversal of the inhibitory effects of HIV-gp120 on CD4− T cells by stimulation through the CD28 pathway
Authors: Faith A , Yssel H , O’Hehir R E , Lamb J R
Published: November 2003

The effects of exposure to HIV-gp120 on proliferation and cytokine production by T cell lines were investigated. T cell lines were generated by stimulation of peripheral blood mononuclear cells from several healthy donors with cross-linked anti-CD3 antibodies and IL-2. These T cell lines exhibited the characteristics of Th1 cells, producing IL-2 and interferon-gamma (IFN-γ), but not IL-4, on stimulation with anti-CD3 antibodies. In the presence of gp120, stimulation with anti-CD3 antibodies was inhibited in terms of both proliferative responses and the secretion of IL-2 and IFN-γ. Similar effects were observed when a T cell line was stimulated in the presence of a synthetic peptide representing the CD4-binding region of gp120. Neither gp120 nor the CD4-binding region peptide had any effect on IL-4 production by the T cell lines. Stimulation through the CD28 pathway partially restored both the proliferative effect and cytokine production by T cell lines in response to anti-CD3 antibodies in the presence of gp120. Anti-CD28 antibodies also partially restored cytokine production when purified CD4+ T cells from a T cell line were stimulated with anti-CD3 antibodies in the presence of gp120. Anti-gp120 antibodies partially or completely reversed the inhibitory effects of gp120 on T cell proliferation. These results indicate that stimulation through the CD28 pathway may restore defective CD4+ T cell responses in HIV-infected individuals.
Autoantibodies that stabilize the molecular interaction of Ku antigen with DNA-dependent protein kinase catalytic subunit
Authors: Satoh M , Ajmani A K , Stojanov L , Langdon J J , Ogasawara T , Wang J , Dooley M A , Richards H B , Winfield J B , Carter T H , Reeves W H
Published: November 2003

DNA-dependent protein kinase (DNA-PK) consists of a DNA binding subunit (Ku autoantigen), and a catalytic subunit (DNA-PKcs). In the present study, human autoantibodies that recognize novel antigenic determinants of DNA-PK were identified. One type of autoantibody stabilized the interaction of DNA-PKcs with Ku and recognized the DNA-PKcs–Ku complex, but not biochemically purified DNA-PKcs. Another type recognized purified DNA-PKcs. Autoantibodies to Ku (p70/p80 heterodimer), ‘stabilizing’ antibodies, and antibodies to DNA-PKcs comprise a linked autoantibody set, since antibodies recognizing purified DNA-PKcs were strongly associated with stabilizing antibodies, whereas stabilizing antibodies were strongly associated with anti-Ku. This hierarchical pattern of autoantibodies specific for components of DNA-PK (anti-Ku>stabilizing antibodies>anti-DNA-PKcs) may have implications for the pathogenesis of autoimmunity to DNA-PK and other chromatin particles. The data raise the possibility that altered antigen processing and/or stabilization of the DNA-PKcs–Ku complex due to autoantibody binding could play a role in spreading autoimmunity from Ku to the weakly associated antigen DNA-PKcs.
Heterozygous and homozygous factor H deficiency states in a Dutch family
Authors: Fijen C A P , Kuijper E J , Te Bulte M T , Van De Heuvel M M , Holdrinet A C J M , Sim R B , Daha M R , Dankert J
Published: November 2003

Factor H, a 150-kD protein, is an important down-regulating protein of the alternative pathway of the complement system. Presently, only 15 persons, representing seven families, have been described with homozygous factor H deficiency. Deficiency of this protein, inherited as an autosomal recessive trait and resulting in uncontrolled breakdown of C3, results in depletion of components of the alternative pathway (factor B, properdin) and of the terminal pathway (C5), and is associated with the onset of bacterial infections, glomerulonephritis and systemic lupus erythematosus (SLE). The proband of the family in this study suffered from subacute cutaneous lupus erythematosus and had had meningococcal meningitis due to serogroup X. She had a complete factor H deficiency at the protein level as determined by Western blotting. Among 21 relatives of the proband studied, encompassing three generations, 10 had low factor H levels, including the two children of the proband, indicating a heterozygous factor H deficiency state. In serum samples of the proband and 11 relatives prospectively studied, a strong correlation of factor H levels with C3, C3 haemolytic activity, factor B and properdin levels (P<0.0001) was found. Alternative pathway protein levels were significantly lower (Mann–Whitney test; Z values 3.6–2.7) in sera from the four heterozygous relatives studied than in sera from the seven non-deficient relatives. In addition, a defect of the 37/42-kD H-related protein was found in the proband and two of 21 relatives, compared with four of 40 controls. A defect of the 24/29-kD H-related protein was present in one of 21 relatives studied and in none of the 40 controls.
Cytotoxicity in patients with different clinical forms of Chagas’ disease
Authors: Brodskyn C I , Barral A , Bulhões M A , Souto T , Machado W C , Barral-Netto M
Published: November 2003

There are few studies on cell-mediated cytotoxicity in human Chagas' disease. In the present study, we evaluated peripheral blood mononuclear cell (PBMC) cytotoxicity activity from chagasic patients with different clinical forms of disease. To verify the cytotoxic response, we performed cell lysis assays using 51Cr-labelled K562 cells as targets. Results are reported as lytic units (LU=number of cells required for 30% lysis) per million PBMC. Exposure of patients’ PBMC to Trypanosoma cruzi antigen led to an increase in cytotoxic activity compared with unstimulated patient cells against K562. Asymptomatic cardiomyopathy patients had higher responses (37.8±5.0 LU/106 PBMC; mean±s.d. than indeterminate (11.5±3.6 LU/106) and symptomatic cardiomyopathy (7.8±2.5 LU/106). Normal control PBMC stimulated with T. cruzi antigen had 4.36±1.31 LU/106 PBMC against K562. Addition of recombinant interferon-gamma (IFN-γ) did not lead to significant increase in cytotoxicity in any group of patients. On the other hand, recombinant human IL-12 significantly increased cytotoxic responses from symptomatic cardiomyopathy patients and normal controls who presented low levels of cytotoxicity induced by T. cruzi antigen. The combined use of IL-12 and a neutralizing anti-IFN-γ antibody did not change IL-12-induced cytotoxic responses, showing the direct role of this cytokine on natural killer (NK) cells. NK cells were the main cells responsible for the lysis of K562 target cells as evidenced by testing cell subsets following magnetic cell sorting. These data demonstrate that chagasic patients with different clinical forms of disease have PBMC which respond to T. cruzi antigen with a cytotoxic response, and this response is up-regulated by IL-12.
Generation of biologically active anti-Cryptococcus neoformans IgG, IgE and IgA isotype switch variant antibodies by acridine orange mutagenesis
Authors: Spira G , Paizi M , Mazar S , Nussbaum G , Mukherjee S , Casadevall A
Published: November 2003

Administration of MoAbs to Cryptococcus neoformans capsular glucuronoxylomannan (GXM) can alter the course of infection in mouse models. However, the effectiveness of these antibodies appears to depend on isotype and specificity. Comparison of isotype protection efficacy requires families of MoAbs with identical fine specificity and different constant region domain. The generation of such families by hybridoma technology is not always possible because the immune response produces MoAbs of limited classes or subclasses. In these instances isotype switch variants can be isolated in vitro. Unfortunately, standard methods of recovering spontaneous switch variants are often unsuccessful, mainly because of the low frequency of switching. In this study we demonstrate that acridine orange stimulation of an IgG3 anti-C. neoformans-producing hybridoma can be used to recover the entire set of isotype switch variants: IgG1, IgG2b, IgG2a, IgE and IgA. All isotype switch variants bind to GXM; fine specificity mapping, using an 11 amino acid peptide polysaccharide mimetope, revealed conservation of binding site specificity. Furthermore, all isotype switch variants reacted with an anti-idiotopic MoAb. The functional activity of this set of MoAbs was demonstrated by their ability to enhance phagocytosis and anti-fungal efficacy of human macrophage-like THP-1 cells, with IgG3 being the most effective and IgE being the least effective.
Methylprednisolone differentially regulates IL-10 and tumour necrosis factor (TNF) production during murine endotoxaemia
Authors: Marchant A , Amraoui Z , Gueydan C , Bruyns C , Le Moine O , Vandenabeele P , Fiers W , Buurman W A , Goldman M
Published: November 2003

IL-10 is an endogenous antiinflammatory cytokine that inhibits TNF biosynthesis and protects mice  from lipopolysaccharide (LPS)-induced lethality. As synthetic glucocorticoids are widely used as antiinflammatory agents, we analysed the effects of methylprednisolone administration on IL-10 biosynthesis during murine endotoxaemia. We found that low doses of methylprednisolone (2–10 mg/kg) markedly inhibited TNF production but did not affect serum levels of IL-10, while a high methylprednisolone dose (50 mg/kg) increased LPS-induced IL-10 levels. In parallel, we observed that LPS-induced IL-10 production is TNF-independent in this experimental setting. Experiments conducted in vitro indicated that methylprednisolone (from 0.01 to 100 μg/ml) also increased the biosynthesis of IL-10 by LPS-activated mouse peritoneal macrophages. We conclude that methylprednisolone differentially regulates IL-10 and TNF production induced by LPS both in vivo and in vitro at the macrophage level.
Modulation of Langerhans cell phenotype, migration and maturation by agents known to cause herpes simplex virus reactivation in a mouse model
Authors: Manickasingham S P , Hill T J , Williams N A
Published: November 2003

The factors which control whether an asymptomatic or symptomatic infection in the skin follows reactivation of herpes simplex virus (HSV) in the sensory ganglia remain unclear. Xylene, retinoic acid and dimethylsuphoxide (DMSO) all stimulate similar levels of virus reactivation in the ganglia of latently infected mice, yet give rise to high, moderate and very low incidences of clinical skin disease, respectively. This observation suggests that the chemicals may be capable of affecting the local microenvironment of the skin. In the present study we have investigated the effects of xylene, retinoic acid and DMSO on Langerhans cell (LC) phenotype and function. The results show that none of the chemicals inhibit the phenotypic maturation of Langerhans cells in vitro. However, DMSO induced a dramatic elevation in class II MHC expression on Langerhans cells. In addition, fewer antigen-bearing dendritic cells (DC) were evident in the lymph nodes if xylene was administered to the skin prior to challenge with the contact sensitizer, FITC. None of the chemicals inhibited the accumulation of DC in the lymph nodes after such an antigen-challenge, suggesting that xylene was affecting not migration, but antigen uptake. The inhibition of antigen uptake by xylene together with the activation of LC in terms of class II expression by DMSO may explain, in part, the relative abilities of these chemicals to allow the establishment of recurrent HSV disease in the skin.
The treatment of systemic lupus erythematosus (SLE) in NZB/W F1 hybrid mice; studies with recombinant murine DNase and with dexamethasone
Authors: Macanovic M , Sinicropi D , Shak S , Baughman S , Thiru S , Lachmann P J
Published: November 2003

The effects of recombinant mouse DNase on a well established murine model of spontaneous SLE have been evaluated. Daily intraperitoneal injections of DNase were given to female NZB/NZW F1 mice during the period of disease development from 4 to 7 months of age or at the height of disease activity from the age of 7 months for 3 weeks. This treatment was compared with the injections of diluent and with an immunosuppressive dose of dexamethasone. The effects of treatment were evaluated using the immunological parameters of disease activity (antinucleoprotein antibody, immune complexes, serum immunoglobulins, anti-cardiolipin antibodies), proteinuria, serum creatinine and renal histopathology (light microscopy, immunofluorescence and electron microscopy). The dose of dexamethasone used (1 mg/kg per day from the age of 4 months) was sufficient to suppress the development of lupus entirely. Treatment with DNase starting at the age of 4 months postponed the development of the disease by about 1 month and extended the period from the onset of disease to death by about 30%. Mice treated for 3 weeks during the most active phase of the disease at 7 months of age showed more dramatic effects. Proteinuria and serum creatinine were significantly reduced and renal histopathology was strikingly less severe than in the control group. Immune complexes involving DNA-containing antigens are believed to play a crucial role in the pathogenesis of SLE. DNA-nucleoprotein, even in immune complexes, can be destroyed by DNase. This enzyme therefore provides a rational way to interfere with the disease process. The results reported here encourage a trial of recombinant human DNase in human SLE and lupus nephritis.
In vitro and in vivo functional analysis of CD+ and CD5− B cells of autoimmune NZB ∨ NZW F1 mice
Authors: Ye Y-L , Chuang Y-H , Chiang B-L
Published: November 2003

Polyclonal B cell activation has been thought to play the critical role in production of autoantibodies, and possible activation of autoreactive T cells in murine lupus, especially abnormal expansion of CD5+ B cells, is one of the characteristic findings in these mice. The aim of this study was to investigate further the characteristics and function of CD5+ and CD5− B cells. Both CD5+ and CD5− B cells were isolated for in vitro autoantibody production, cytokine expression and in vivo anti-DNA antibody production with the reconstitution of severe combined immunodeficient (SCID) mice. The data showed: (i) both CD5+ and CD5− B cells produced a high level of anti-DNA antibody after stimulation with lipopolysaccharide (LPS) plus IL-5; (ii) both peritoneal CD5+ and CD5− B cells expressed a high level of IL-10 mRNA after stimulation with LPS, while in contrast CD5− B cells of non-autoimmune BALB/c mice did not express IL-10 mRNA after stimulation; (iii) SCID mice reconstituted with either CD5+ or CD5− B cells all produced significant levels of anti-DNA antibodies in vivo and manifested with proteinuria. These data suggest both CD5+ and CD5− B cells play important roles in polyclonal B cell activation and subsequent autoantibody production. Generalized polyclonal B cell activation, instead of expanding a certain subpopulation of B cells, contributed to the pathogenesis of autoimmunity in murine lupus.
Non-MHC genes determine the development of lupus nephritis in H-2 identical mouse strains
Authors: Sutmuller M , Ekstijn G L , Ouellette S , Heer E DE , Bruijn J A
Published: November 2003

Susceptibility to systemic lupus erythematosus (SLE) and, in particular, lupus nephritis is strongly influenced by genetic factors. Previous studies have shown that MHC-related antigens influence the development of SLE. In the current study, we set out to investigate how non-MHC genes influence the pathogenesis of glomerulonephritis in chronic graft-versus-host disease (GVHD) in mice, a model for lupus nephritis. For the induction of GVHD we used parent-to-F1 hybrid mouse strain combinations. DBA/2, BALB/c, BALB.D2 and C57Bl/10.D2 (BL10.D2) donor lymphocytes carrying an H-2d haplotype were injected into H-2b/d F1 hybrids of BL10 mice, which differed only at non-MHC loci. Within these hybrid strains the development of immune complex glomerulonephritis was investigated by monitoring the occurrence of autoantibodies in the circulation, deposition of immunoglobulins in the glomeruli, development of albuminuria, and glomerulosclerosis. In diseased DBA/2 mice albuminuria developed 6 weeks after induction of the disease. Mice with a BALB background developed a lupus-like syndrome characterized by albuminuria starting 8 weeks after induction of the GVHD. During the development of the GVHD, polyclonal B cell activation occurred in both the DBA/2 and BALB/c strains, resulting in the formation of autoantibodies. Only the strain combination using DBA/2 mice developed anti-GBM antibodies. In DBA/2 and BALB strain combinations immune complexes were detected in a granular pattern along the glomerular capillary walls. In the DBA/2 recipients a linear pattern of immunoglobulin depositions preceded the granular phase. This study demonstrates that: (i) non-MHC genes govern the pathogenesis of immune complex nephritis in this model by influencing the autoantibody profile; and (ii) the presence of anti-GBM antibodies in the early stages of the disease is a conditio sine qua non for the development of full-blown glomerulonephritis and glomerulosclerosis in this model.
Human T cell responses to recombinant mite antigens of Dermatophagoides farinae
Authors: FUJII S , ONO K , SHIGETA S , JYO T , YAMASHITA U
Published: November 2003

We studied T cell responses to four glutathione S transferase (GST)-fused mite antigens prepared in our laboratory using peripheral blood lymphocytes from mite-sensitive patients with bronchial asthma. Of the four recombinant antigens, purified GST-Mag3 had the strongest ability to cause patients' lymphocytes to proliferate, and its potency was almost comparable to that of crude mite bodies (Dfb) and faeces (Dff) antigens and a purified major antigen, Der f 2. The responder lymphocytes were mainly T cells, because the proliferative response was depleted by the treatment of lymphocytes with anti-CD3 antibody and complement, but not with anti-CD20 antibody and complement. The responsiveness of lymphocytes to GST-Mag3 correlated with that to Der f 2, but GST-Mag3 displayed slightly higher activity to stimulate lymphocytes than Der f 2. Simultaneously, the levels of Dff- and GST-Mag3-specific IgE antibodies correlated with the responsiveness of lymphocytes to GST-Mag3. These results suggest that Mag3 is a new valuable antigen for the response of T cell proliferation in mite-sensitive patients.
The systemic lupus erythematosus (SLE) disease autoantigen—calreticulin can inhibit C1q association with immune complexes
Authors: KISHORE U , SONTHEIMER R D , SASTRY K N , ZAPPI E G , HUGHES G R V , KHAMASHTA M A , REID K B M , EGGLETON P
Published: November 2003

Following its release from cells during infection and inflammation, calreticulin (CRT) can act as an autoantigen in diseases such as SLE. Why CRT is a target of protective immunity and whether it may interfere with innate immunity once released from cells during inflammation is unclear. In the present study, we found that CRT was detected more frequently in SLE sera and in higher amounts than found in control sera. Approximately 40% of SLE sera tested contained autoantibodies against CRT as detected by ELISA and immunoblotting. CRT was found to be predominantly in the sera of SLE patients associated with immune complexes and C1q, and only bound to the surfaces of neutrophils in the presence of low levels of calcium and magnesium. In order to further investigate the C1q–CRT interaction, recombinant CRT and its discrete domains (N-, P-, and C-domains) were produced in Escherichia coli. CRT binds to globular head region of C1q primarily via its N- and P-domains. The N-domain was shown to be the most autoantigenic region of CRT, as the anti-CRT autoantibodies from most patients reacted against this region. CRT also altered C1q-mediated immune functions. The P-domain of CRT bound to C1q and reduced the binding of immune complexes in SLE sera to immobilized C1q. Full length CRT and its N- and P-domains were able to reduce the C1q-dependent binding of immune complexes to neutrophils and solid-phase bound C1q. We conclude that CRT, once released from leucocytes during inflammation, may not only induce an antigenic reaction, but also interfere with C1q-mediated inflammatory processes.
Accessory cell function of a human colonic epithelial cell line HT-29 for bacterial superantigens
Authors: LIU Z X , SUGAWARA S , HIWATASHI N , NOGUCHI M , RIKIISHI H , KUMAGAI K , TOYOTA T
Published: November 2003

The expression and up-regulation of cell adhesion molecules on a human colonic epithelial cell line HT-29, and the peripheral blood T lymphocyte proliferation responses to bacterial superantigens presented by this cell line were investigated, compared with peripheral blood monocytes. In HT-29 cells, there was constitutive expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) at a low level, but no constitutive expression of HLA-DR, LFA-1, B7-1 and B7-2 molecules. After stimulation with the supernatants of staphylococcal enterotoxin B (SEB)-stimulated peripheral blood mononuclear cells for 48 h, there was significant up-regulation of HLA-DR and ICAM-1 molecules (both >90% positive). However, this stimulation had no effect on the expression of LFA-1, B7-1, B7-2 and LFA-3 molecules. In the presence of all tested superantigens SEB, toxic shock syndrome toxin-1, and streptococcal pyogenic exotoxin A, stimulated HT-29 cells caused significant T cell proliferation. When monocytes were used as antigen-presenting cells (APC), the MoAbs against HLA-DR, B7-2 and LFA-3 showed a significant inhibition of SEB-induced T cell proliferation. Anti-ICAM-1 MoAb had no effect on this response. On the other hand, when stimulated HT-29 cells were used as APC, the MoAbs against HLA-DR and ICAM-1 significantly inhibited SEB-induced T cell proliferation. In contrast to monocytes, anti-B7-2 and anti-LFA-3 had no effect on this response. SEB could not induce HT-29 cells to produce IL-8 directly; however, SEB significantly induced the stimulated HT-29 cells to produce IL-8 in the presence of T cells. Thus these data demonstrate that the products of superantigen-stimulated T cell activation can increase the expression of HLA-DR and ICAM-1 molecules on HT-29 cells significantly. Stimulated HT-29 cells can serve as APC to bacterial superantigens. This response is an HLA-DR- and ICAM-1-dependent, but B7-2- and LFA-3-independent process, which was different from professional APC monocytes.
Expression of molecules involved in B lymphocyte survival and differentiation by synovial fibroblasts
Authors: EDWARDS J C W , LEIGH R D , CAMBRIDGE G
Published: November 2003

The synovitis of rheumatoid arthritis (RA) is one of few pathological lesions in which B lymphocyte accumulation progresses to the extent of germinal centre formation. The present study was designed to assess the ability of synovial fibroblasts to express molecules implicated in B lymphocyte survival and differentiation, both in vivo, and in response to cytokines in vitro. Normal and diseased synovia were examined by indirect immunofluorescence. In all tissues synovial intimal fibroblasts showed co-expression of vascular cell adhesion molecule-1 (VCAM-1) and complement decay-accelerating factor (DAF) comparable to that of follicular dendritic cells (FDC), but not complement receptor 2 (CR2). In rheumatoid synovia, subintimal cells showed variable expression of VCAM-1 and DAF, with bright co-expression of VCAM-1, DAF and CR2 in lymphoid follicle centres. B lymphocytes, some of which were proliferating cell nuclear antigen-positive, were present in contact with subintimal cells expressing VCAM-1 with or without DAF or CR2. B lymphocytes were rarely present in the intimal layer, and, where present, showed fragmentation. In vitro, synovial fibroblasts exposed to tumour necrosis factor-alpha (TNF-α) in combination with interferon-gamma (IFN-γ) showed enhanced expression of VCAM-1, in comparison with fibroblasts from skin and lung and, unlike skin and lung fibroblasts, also expressed DAF and CR2. These findings support the hypothesis that synovial targeting in RA involves an enhanced ability of synovial fibroblasts to support B lymphocyte survival. This appears to be dependent, not on the constitutive expression of VCAM-1 and DAF on intimal cells, but on the increased ability of subintimal cells to respond to proinflammatory cytokines, perhaps critically in the expression of VCAM-1.
Migration of human intestinal lamina propria lymphocytes, macrophages and eosinophils following the loss of surface epithelial cells
Authors: MAHIDA Y R , GALVIN A M , GRAY T , MAKH S , McALINDON M E , SEWELL H F , PODOLSKY D K
Published: November 2003

Lymphocytes and macrophages are present in the normal intestinal lamina propria, separated from the epithelial monolayer by the basement membrane. There is evidence for movement of mononuclear cells through the lamina propria, entering from the systemic circulation and exiting via lymphatic channels. The goal of our studies was to investigate the capacity of cells to migrate out from the lamina propria into the lumen following the loss of surface epithelial cells. An in vitro model was therefore established in which normal human intestinal mucosal samples, denuded of the surface epithelium, were maintained in culture. Electron microscopy showed that during culture, large numbers (> 2 × 106/g tissue per 24 h) of cells migrated out of the lamina propria via discrete ‘tunnels’ which were in continuity with pores (diameter < 4 μm) in the basement membrane. The emigrating cells were T cells (68.5 ± 5.1%), macrophages (10.5 ± 1.3%) and eosinophils (7.1 ± 1.3%). Our studies have therefore demonstrated, for the first time, the capacity for large numbers of lymphocytes, macrophages and eosinophils to migrate out of the lamina propria, via basement membrane pores. We postulate that such emigration of cells occurs in vivo following the loss of surface epithelial cells due to injury, and could represent an important form of host defence against luminal microorganisms and also facilitate wound repair by enhancing restitution by neighbouring epithelial cells, via peptide factors.
Hypogammaglobulinaemia occurs in Fas-deficient MRL-lpr mice following deletion of MHC class II molecules
Authors: ANDERSON C C , MUKHERJEE R , SINCLAIR N R StC , JEVNIKAR A M
Published: November 2003

Fas (CD95)-mediated apoptosis in B and T cells is deficient in both human autoimmune lymphoproliferative syndrome and in MRL-lpr mice, a model for systemic lupus erythematosis (SLE). Autoimmune disease in these mice is associated with polyclonal B cell activation, increased serum immunoglobulin and autoantibodies. In non-autoimmune mice MHC class II is not required for normal serum immunoglobulin expression, and previously we have shown using MHC class II-deficient MRL-lpr mice (MRL-lpr Ab−/−) that generation of specific antibodies to DNA requires MHC class II-directed T cell help. In contrast, in the present study we demonstrate that MRL-lpr Ab−/−mice also have a profound reduction of total serum immunoglobulin levels, suggesting abnormal polyclonal regulation of B cells by MHC class II-directed T cells occurs in the autoimmune MRL-lpr strain. This abrogation of immunoglobulin production does not occur in MHC class II-deficient non-obese diabetic (NOD) mice, nor in MHC class I-deficient NOD or MRL-lpr mice. Reduced immunoglobulin levels in MRL-lpr Ab−/− mice were not due to a lack of B cells or to an increased loss of circulating immunoglobulin, but were associated with reduced numbers of surface IgG-positive B cells. These results define a general abnormal regulation of B cells in MRL-lpr mice through a process requiring MHC class II, and suggest that Fas deficiency may allow expansion of totally T-dependent B cells.
Identification and characterization of a DR4-restricted T cell epitope within chlamydia heat shock protein 60
Authors: DEANE K H O , JECOCK R M , PEARCE J H , GASTON J S H
Published: November 2003

An epitope within the 60 kD Chlamydia trachomatis heat shock protein (hsp) 60, recognized by a HLA-DRB1*0401-restricted T cell clone from a reactive arthritis patient, has been characterized. Stimulatory peptides contained a nine amino acid sequence (residues 38–46) predicted by algorithm to confer strong binding to DRB1*0401, with valine in the P1 position. The overall length of the peptide was critical for efficient recognition; peptides with at least one residue N-terminal to the putative P1 position were markedly more stimulatory than a peptide whose N-terminal is the P1 valine. Optimal responses were seen with 14mer peptides having two to three amino acids N- and C-terminal to the core 9mer. The sequence of the defined epitope is identical in hsp60 from both C. trachomatis and C. pneumoniae. Since the latter is a common respiratory pathogen, patients infected with C. trachomatis may already be primed for responses to hsp60 by prior infection with C. pneumoniae. Such secondary responses are important in the pathogenesis of chlamydia-induced inflammatory diseases such as trachoma. Priming by infection with enteric organisms was considered because of the similarity of the epitope sequence in Escherichia coli hsp60. However, although an E. coli-related peptide was recognized, intact E. coli hsp60 was not, suggesting that the epitope is cryptic in E. coli hsp60. Human hsp60 has six amino acid differences from chlamydial hsp60 in the epitope sequence and was not recognized. Thus cross-reactive recognition of self hsp60 could not be implicated in the pathogenesis of chlamydia-induced reactive arthritis in this patient.
Pituitary responsiveness to GH‐releasing hormone, GH‐releasing peptide‐2 and thyrotrophin‐releasing hormone in critical illness
Authors: Van den Berghe Greet , De Zegher Francis , Bowers Cyril Y. , Wouters Pieter , Muller Peter , Soetens Filip , Vlasselaers Dirk , Schetz Miet , Verwaest Charles , Lauwers Peter , Bouillon Roger
Published: November 2003

OBJECTIVE Protein hypercatabolism and preservation of fat depots are hallmarks of critical illness, which is associated with blunted pulsatile GH secretion and low circulating IGF‐I, TSH, T4 and T3. Repetitive TRH administration is known to reactivate the pituitary‐thyroid axis and to evoke paradoxical GH release in critical illness. We further explored the hypothalamic‐pituitary function in critical illness by examining the effects of GH‐releasing hormone (GHRH) and/or GH‐releasing peptide‐2 (GHRP‐2) and TRH administration.

PATIENTS AND DESIGN Critically ill adults (n=40; mean age 55 years) received two i.v. boluses with a 6‐hour interval (0900 and 1500 h) within a cross‐over design. Patients were randomized to receive consecutively placebo and GHRP‐2 (n=10), GHRH and GHRP‐2 (n=10), GHRP‐2 and GHRH+GHRP‐2 (n=10), GHRH+GHRP‐2 and GHRH+GHRP‐2+TRH (n=10). The GHRH and GHRP‐2 doses were 1μg/kg and the TRH dose was 200μg. Blood samples were obtained before and 20, 40, 60 and 120 minutes after each injection.

MEASUREMENTS Serum concentrations of GH, T4, T3, rT3, thyroid hormone binding globulin (TBG), IGF‐I, insulin and cortisol were measured by RIA; PRL and TSH concentrations were determined by IRMA.

RESULTS Critically ill patients presented a striking GH response to GHRP‐2 (mean±SEM peak GH 51±9 μg/l in older patients and 102±2μg/l in younger patients; P=0.005 vs placebo). The mean GH response to GHRP‐2 was more than fourfold higher than to GHRH (P=0.007). In turn, the mean GH response to GHRH+GHRP‐2 was 2.5‐fold higher than to GHRP‐2 alone (P=0.01), indicating synergism. Adding TRH to the GHRH+GHRP‐2 combination slightly blunted this mean response by 18% (P=0.01).

GHRP‐2 had no effect on serum TSH concentrations whereas both GHRH and GHRH+GHRP‐2 evoked an increase in peak TSH levels of 53 and 32% respectively. The addition of TRH further increased this TSH response < ninefold (P=0.005), elicited a 60% rise in serum T3 (P=0.01) and an 18% increase in T4 (P=0.005) levels, without altering rT3 or TBG levels. GHRH and/or GHRP‐2 induced a small increase in serum PRL levels. The addition of TRH magnified the PRL response 2.4‐fold (P=0.007). GHRP‐2 increased basal serum cortisol levels (531±29nmol/l) by 35% (P=0.02); GHRH provoked no additional response, but adding TRH further increased the cortisol response by 20% (P=0.05).

CONCLUSIONS The specific character of hypothalamic‐pituitary function in critical illness is herewith extended to the responsiveness to GHRH and/or GHRP‐2 and TRH. The observation of striking bursts of GH secretion elicited by GHRP‐2 and particularly by GHRH+GHRP‐2 in patients with low spontaneous GH peaks opens the possibility of therapeutic perspectives for GH secretagogues in critical care medicine.
Bone turnover and bone mineral density in patients with congenital adrenal hyperplasia
Authors: Guo C‐Y. , Weetman A. P. , Eastell R.
Published: November 2003

BACKGROUND AND OBJECTIVE Glucocorticoid replacement is the most effective therapy for patients with congenital adrenal hyperplasia (CAH). It has been reported that excessive steroid therapy leads to bone loss and osteoporosis, but it is uncertain whether steroid replacement therapy affects bone turnover and bone mineral density (BMD) in adult patients with CAH.

DESIGN Case‐control study: patients with CAH were compared to normal subjects, individually matched for age and body weight.

PATIENTS Eleven patients, aged 19–65 years, were evaluated in this study. The age at diagnosis of CAH was 0–26 years. Nine patients (6 females and 3 males) were diagnosed with 21‐hydroxylase deficiency and 2 male patients with 11‐hydroxylase deficiency. 17‐Hydroxyprogesterone levels in patients had never been below the reference range in the previous 2 years. These patients were individually matched for sex, age and weight to 11 healthy controls.

MEASUREMENTS Total body, lumbar spine and femoral neck BMD was measured by dual‐energy X‐ray absorptiometry. Serum bone Gla‐protein (BGP, osteocalcin) and bone alkaline phosphatase (BAP) were measured to assess bone formation whereas serum tartrate‐resistant acid phosphatase (TRAP) and urinary cross‐linked N‐telopeptides of type I collagen (NTx) were measured to assess bone resorption. NTx was expressed as a fraction of urinary creatinine excretion (NTx/Cr). Serum dehydroepiandrosterone sulphate (DHEA‐S) and androstenedione levels were measured to assess adrenal androgen status.

RESULTS Serum DHEA‐S, androstenedione, BGP and BAP and urinary NTx/Cr were decreased in patients when compared with controls (paired t‐test, P = 0.005, 0.0003, 0.002, 0.03 and 0.03, respectively). BMD was not significantly decreased in patients. The difference of total body BMD between patients and controls (i.e. BMD in patients minus BMD in controls) was negatively correlated with age. There was no correlation between androgen levels and either BMD or bone turnover. The total dose of steroid taken in the previous 2 years was not correlated with BMD, bone turnover or androgen levels. The was no correlation between BMD adjusted by age and bone turnover or initial age at diagnosis.

CONCLUSIONS We conclude that (1) bone turnover is decreased in congenital adrenal hyperplasia, (2) bone mineral density is not decreased in congenital adrenal hyperplasia and (3) patients initially have higher bone mineral density but later have lower bone mineral density than controls, especially in women.