Chris Juul Hedegaard

T helper cell type 1 (Th1), Th2 and Th17 responses to myelin basic protein and disease activity in multiple sclerosis.

Autoreactive T cells are thought to play an essential role in the pathogenesis of multiple sclerosis (MS). We examined the stimulatory effect of human myelin basic protein (MBP) on mononuclear cell (MNC) cultures from 22 patients with MS and 22 sex‐matched and age‐matched healthy individuals, and related the patient responses to disease activity, as indicated by magnetic resonance imaging. The MBP induced a dose‐dependent release of interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and interleukin‐10 (IL‐10) by patient‐derived MNCs. The patients’ cells produced higher amounts of IFN‐γ and TNF‐α, and lower amounts of IL‐10, than cells from healthy controls (P < 0·03 to P < 0·04). Five patients with MS and no controls, displayed MBP‐induced CD4+ T‐cell proliferation. These high‐responders exhibited enhanced production of IL‐17, IFN‐γ, IL‐5 and IL‐4 upon challenge with MBP, as compared with the remaining patients and the healthy controls (P < 0·002 to P < 0·01). A strong correlation was found between the MBP‐induced CD4+ T‐cell proliferation and production of IL‐17, IFN‐γ, IL‐5 and IL‐4 (P < 0·0001 to P < 0·01) within the patient group, and the production of IL‐17 and IL‐5 correlated with the number of active plaques on magnetic resonance images (P = 0·04 and P = 0·007). These data suggest that autoantigen‐driven CD4+ T‐cell proliferation and release of IL‐17 and IL‐5 may be associated with disease activity. Larger studies are needed to confirm this.

Cytokines, autoantibodies and viral antibodies in premorbid and postdiagnostic sera from patients with rheumatoid arthritis: case–control study nested in a cohort of Norwegian blood donors

Objective: To assess the timing of changes in cytokines, cytokine-related markers, autoantibodies and viral antibodies in the pathogenesis of rheumatoid arthritis (RA). Methods: Case–control study nested in a prospective cohort of 31 330 blood donors in Oslo, Norway. Forty-nine donors developed RA up to 23 years after their most recent blood donation. Stored sera from these donors (case sera) and a sex- and age-matched sample of 245 healthy donors (control sera), and postdiagnostic sera from 33 of the 49 RA cases, were analysed for a panel of cytokines and cytokine-related markers, autoantibodies and antibodies against Epstein–Barr virus and parvovirus B19. Results: Cytokines and cytokine-related markers were generally negative in case sera from >5 years before the diagnosis of RA. In the 5-year interval immediately before the diagnosis of RA, more case than control sera were positive (odds ratios >2) for interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist, IL-4, IL-10, tumour necrosis factor-α and soluble tumour necrosis factor receptor I. In postdiagnostic sera, however, 11 of 16 examined cytokines and cytokine-related markers were statistically significantly elevated compared with control sera. Seropositivity for IgG antibodies against cyclic citrullinated peptides and for IgM and IgA rheumatoid factors were seen in case sera from up to 18 years before the diagnosis of RA. IgG antibodies against Epstein–Barr virus and parvovirus B19 did not differ significantly between case and control sera. Conclusions: Cytokines and cytokine-related markers appear to be upregulated rather late in RA pathogenesis. In contrast, IgM rheumatoid factor and IgG anti-cyclic citrullinated peptide autoantibodies may precede the diagnosis of RA by up to two decades.

Autoantibodies to myelin basic protein (MBP) in healthy individuals and in patients with multiple sclerosis: a role in regulating cytokine responses to MBP

Anti‐myelin basic protein (‐MBP) autoantibodies have generally been considered to be absent from sera from healthy individuals, but to be detectable in sera from some patients with multiple sclerosis (MS). However, their pathogenic role is uncertain. We demonstrate the presence of MBP‐reactive autoantibodies in sera from 17 healthy individuals and 17 MS patients. The addition of MBP to the sera caused a dose‐dependent deposition of MBP and co‐deposition of immunoglobulin M (IgM) and fragments of complement component 3 (C3) on allogeneic monocytes. Calcium chelation abrogated the immunoglobulin deposition, indicating that formation of complement‐activating immune complexes played a role in the binding process. Furthermore, MBP elicited tumour necrosis factor (TNF)‐α and interleukin (IL)‐10 production by normal mononuclear cells in the presence of serum from both patients and controls. Mononuclear cells from MS patients responded to MBP with the production of interferon (IFN)‐γ, IL‐4 and IL‐5, in addition to TNF‐α and IL‐10. The production of IFN‐γ and IL‐5 was increased when MS serum was added rather than normal serum. Denaturation of MBP strongly inhibited MBP deposition and the MBP‐induced IgM deposition and cytokine production, indicating that these events were facilitated by autoantibodies recognizing conformational epitopes on MBP. We infer that MBP‐elicited TNF‐α and IL‐10 responses are promoted to equal extents by naturally occurring MBP autoantibodies and autoantibodies contained in MS sera. However, the latter seem to be more efficient in facilitating the production of IFN‐γ and IL‐5.

Targeting the Epidermal Growth Factor Receptor in Solid Tumor Malignancies

The epidermal growth factor receptor (EGFR) is over-expressed, as well as mutated, in many types of cancers. In particular, the EGFR variant type III mutant (EGFRvIII) has attracted much attention as it is frequently and exclusively found on many tumor cells, and hence both EGFR and EGFRvIII have been proposed as valid targets in many cancer therapy settings. Different strategies have been developed in order to either inhibit EGFR/EGFRvIII activity or to ablate EGFR/EGFRvIII-positive tumor cells. Drugs that inhibit these receptors include monoclonal antibodies (mAbs) that bind to the extracellular part of EGFR, blocking the binding sites for the EGFR ligands, and intracellular tyrosine kinase inhibitors (TKIs) that block the ATP binding site of the tyrosine kinase domain. Besides an EGFRvIII-targeted vaccine, conjugated anti-EGFR mAbs have been used in different settings to deliver lethal agents to the EGFR/EGFRvIII-positive cells; among these are radio-labelled mAbs and immunotoxins. This article reviews the current status and efficacy of EGFR/EGFRvIII-targeted therapies.

Effects of spironolactone on human blood mononuclear cells : mineralocorticoid receptor independent effects on gene expression and late apoptosis induction

1 Spironolactone (SPIR) binds to cytoplasmic mineralocorticoid receptors (MR) and functions as an aldosterone antagonist. Recently, the drug was shown to have an early suppressive effect on several immunoactive and proinflammatory cytokines. 2 To elucidate the mechanism behind this, the four MR‐binding steroids SPIR, canrenone, 7α‐thiomethyl‐spironolactone and aldosterone (ALDO) were investigated for effects on lipopolysaccharide‐ and phytohemagglutinin‐A‐activated human blood mononuclear cells. Gene expression was examined after 4 h using microarrays, and SPIR affected 1018 transcripts of the (=) 22,000 probed. In contrast, the SPIR‐related steroids affected 17 or fewer transcripts. Combining SPIR and ALDO resulted in 940 affected transcripts, indicating that SPIR has an early gene‐regulatory effect independent of MR. 3 The affected genes encode a large number of signalling proteins and receptors, including immunoinflammatory response genes and apoptosis and antiapoptosis genes. Apoptosis was evident in CD3‐, CD14‐ and CD19‐positive cells, but only after 18 h of exposure to SPIR. 4 The transcriptional network involving the differentially regulated genes was examined and the results indicate that SPIR affects genes controlled by the transcription factors NF‐κB, CEBPβ and MYC. 5 These observations provide new insight into the non‐MR‐mediated effects of SPIR.

Glatiramer acetate antibodies, gene expression and disease activity in multiple sclerosis

Background: Glatiramer acetate (GA) treatment suppresses disease activity in multiple sclerosis (MS). The immunological response to treatment may differ in patients who are stable on GA therapy and patients with breakthrough disease activity, but the results of previous studies are inconsistent. Objectives: We studied the immunological response to GA and its relationship with disease activity. Methods: Anti-GA antibodies in plasma and the expression of genes encoding cytokines and T-cell-polarizing transcription factors in blood cells were analysed by flow cytometric bead array and polymerase chain reaction (PCR) analysis in 39 untreated and 29 GA-treated relapsing–remitting MS patients. Definition of breakthrough disease was based on the occurrence of relapses, disability progression, or gadolinium (Gd)-enhanced MRI. Results: The expression of T helper type 1 (Th1) and Th17 cytokines and transcription factors was reduced during long-term treatment, but there was no relationship between the expression of cytokines and transcription factors and anti-GA antibodies. High expression of mRNA encoding GATA3 and lymphotoxin-β (LT-β) was associated with low disease activity in Gd-enhanced MRI studies. None of the variables studied were associated with clinical disease activity. GA treatment resulted in the development of IgG and IgG4 anti-GA antibodies during the first months of treatment, persisting during long-term treatment. Conclusions: The observed relationship between the expression of mRNA encoding GATA3 and LT-β expression and MRI disease activity deserves further analysis in future studies. The development of anti-GA antibodies was observed in all patients treated with GA, but this was not related with measures of cellular immunity, clinical or MRI disease activity.

Interferon-beta increases systemic BAFF levels in multiple sclerosis without increasing autoantibody production

Background: Treatment with interferon-beta (IFN-beta) increases B-cell activating factor of the TNF family (BAFF) expression in multiple sclerosis (MS), raising the concern that treatment of MS patients with IFN-beta may activate autoimmune B cells and stimulate the production of MS-associated autoantibodies. Objective: To investigate whether BAFF levels are associated with disease severity/activity in untreated MS patients, and to assess the effect of IFN-beta therapy on circulating BAFF and anti-myelin basic protein (MBP) autoantibody levels. Results: Twenty-three patients with relapsing–remitting MS (RRMS) were followed longitudinally from initiation of IFN-beta therapy. Their blood levels of BAFF correlated positively at baseline with the expanded disability status scale (p < 0.009) and MS severity score (p < 0.05), but not with disease activity as determined by the number of gadolinium-enhanced lesions. The patients were followed for up to 26 months, during which the BAFF levels remained elevated without association to increased disease activity. IFN-beta therapy caused an increase in plasma BAFF levels after both 3 and 6 months of therapy (p < 0.002). However, an 11% decrease in IgM and a 33% decrease in IgG anti-MBP autoantibodies (p < 0.09 and p < 0.009, respectively) was observed after 6 months. Conclusion: Pre-treatment BAFF levels correlate with high disability scores in MS, suggesting that high BAFF expression is a negative prognostic marker. Despite its known beneficial effects, IFN-beta therapy causes a sustained increase in plasma BAFF levels, which does not translate into increased levels of anti-MBP autoantibodies.

Natural Pig Plasma Immunoglobulins Have Anti-Bacterial Effects: Potential for Use as Feed Supplement for Treatment of Intestinal Infections in Pigs.

There is an increasing demand for non-antibiotics solutions to control infectious disease in intensive pig production. Here, one such alternative, namely pig antibodies purified from slaughterhouse blood was investigated in order to elucidate its potential usability to control post-weaning diarrhoea (PWD), which is one of the top indications for antibiotics usage in the pig production. A very cost-efficient and rapid one-step expanded bed adsorption (EBA) chromatography procedure was used to purify pig immunoglobulin G from slaughterhouse pig plasma (more than 100 litres), resulting in >85% pure pig IgG (ppIgG). The ppIgG thus comprised natural pig immunoglobulins and was subsequently shown to contain activity towards four pig-relevant bacterial strains (three different types of Escherichia coli and one type of Salmonella enterica) but not towards a fish pathogen (Yersinia ruckeri), and was demonstrated to inhibit the binding of the four pig relevant bacteria to a pig intestinal cell line (IPEC-J2). Finally it was demonstrated in an in vivo weaning piglet model for intestinal colonization with an E. coli F4+ challenge strain that ppIgG given in the feed significantly reduced shedding of the challenge strain, reduced the proportion of the bacterial family Enterobacteriaceae, increased the proportion of families Enterococcoceae and Streptococcaceae and generally increased ileal microbiota diversity. Conclusively, our data support the idea that natural IgG directly purified from pig plasma and given as a feed supplement can be used in modern swine production as an efficient and cost-effective means for reducing both occurrence of PWD and antibiotics usage and with a potential for the prevention and treatment of other intestinal infectious diseases even if the causative agent might not be known.

The effect of β-interferon therapy on myelin basic protein-elicited CD4+ T cell proliferation and cytokine production in multiple sclerosis

Interferon (IFN)-beta therapy has well-established clinical benefits in multiple sclerosis (MS), but the underlying modulation of cytokine responses to myelin self-antigens remains poorly understood. We analysed the CD4+ T cell proliferation and cytokine responses elicited by myelin basic protein (MBP) and a foreign recall antigen, tetanus toxoid (TT), in mononuclear cell cultures from fourteen MS patients undergoing IFN-beta therapy. The MBP-elicited IFN-gamma-, TNF-alpha- and IL-10 production decreased during therapy (p<0.007-0.03), while the IL-6 production increased (p<0.03). No significant change was observed in the MBP-induced CD4+ T cell proliferation, or in the production of IL-4, IL-5 and brain-derived neurotrophic factor. In comparison, IFN-beta therapy reduced IFN-gamma and IL-4 responses to TT (p<0.003 and p<0.04). Thus, IFN-beta inhibits IFN-gamma production in general, presumably alleviating the detrimental influence of IFN-gamma in MS. However, the increase in proinflammatory IL-6 and the decrease in anti-inflammatory IL-10 responses suggest that IFN-beta has more diverse effects than previously assumed.

Endogenous Interferon-β-Inducible Gene Expression and Interferon-β-Treatment Are Associated with Reduced T Cell Responses to Myelin Basic Protein in Multiple Sclerosis

Autoreactive CD4+ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used for treatment of multiple sclerosis, and some untreated multiple sclerosis patients have increased expression levels of type I interferon-inducible genes in immune cells. The role of endogenous type I interferons in multiple sclerosis is controversial: some studies found an association of high expression levels of interferon-β-inducible genes with an increased expression of interleukin-10 and a milder disease course in untreated multiple sclerosis patients, whereas other studies reported an association with a poor response to treatment with interferon-β. In the present study, we found that untreated multiple sclerosis patients with an increased expression of interferon-β-inducible genes in peripheral blood mononuclear cells and interferon-β-treated multiple sclerosis patients had decreased CD4+ T-cell reactivity to the autoantigen myelin basic protein ex vivo. Interferon-β-treated multiple sclerosis patients had increased IL10 and IL27 gene expression levels in monocytes in vivo. In vitro, neutralization of interleukin-10 and monocyte depletion increased CD4+ T-cell reactivity to myelin basic protein while interleukin-10, in the presence or absence of monocytes, inhibited CD4+ T-cell reactivity to myelin basic protein. Our findings suggest that spontaneous expression of interferon-β-inducible genes in peripheral blood mononuclear cells from untreated multiple sclerosis patients and treatment with interferon-β are associated with reduced myelin basic protein-induced T-cell responses. Reduced myelin basic protein-induced CD4+ T-cell autoreactivity in interferon-β-treated multiple sclerosis patients may be mediated by monocyte-derived interleukin-10.