Search

Aim: The Ostomy Life Study 2019 aimed to obtain a better understanding of the challenges faced by people with stoma. Methods: Online survey with participants from 17 countries. Findings: Of the 54 614 individuals invited to take part, 5187 responded; 62% of the respondents avoided physical and social activities because of their stoma and 37% had never consulted their stoma care nurse to have the fit of their stoma product checked. In a subgroup receiving questions on leakage (n=4209), output under the baseplate and leakage onto clothes were experienced within the previous month by 76% and 26% of respondents, respectively. Higher chance of leakage was associated with an irregular stoma shape and peristomal body profile; a stoma level at or below the skin surface; and the presence of creases, folds and other changes in the peristomal area. Conclusion: Leakage and access to a stoma care nurse to provide the necessary care and guidance remain important concerns for individuals with a stoma.

Leakage of stomal effluents underneath the baseplate or inappropriate removal of stoma appliances can cause peristomal skin complications (PSCs), which are known to have many negative implications for people with a stoma. While numerous studies have examined the causes of PSCs, less is known about what drives the risk of a PSC developing. To address this gap in knowledge, the largest multinational survey to date was conducted, including 4235 people with a stoma on four continents and in 13 countries. The survey revealed that, besides leakage, other factors, such as age, gender, time since surgery and type of stoma, also drive the risk of PSCs. The survey also revealed that having a PSC resulted in greater use of stoma accessories and more frequent contact with health professionals, increasing overall healthcare costs. Collectively, the survey results reveal a need for more awareness on the risk factors for PSCs, which subsequently could have a positive effect on healthcare spending.

Enteric infectious disease in weaner piglets, including postweaning diarrhoea (PWD), are usually treated and/or prevented with antibiotics and/or zinc oxide in the piglet feed. However extensive use of antibiotics and zinc oxide in intensive animal production is unwanted as it may promote microbial antibiotic resistance and pose environmental problems. Recently, in an experimental model of PWD, we observed that oral administration of purified porcine immunoglobulin G (ppIgG) from pooled natural pig plasma could reduce enteric infection. In the present study we were able to reproduce these results as it was observed that oral ppIgG accelerated clearance of faecal haemolytic bacteria in pigs challenged with E. coli in comparison with pigs not receiving ppIgG. This effect was observed upon feeding ppIgG for seven days postweaning suggesting that ppIgG does not have to be used prophylactically for several days preweaning. Furthermore, the effect of oral administration of ppIgG for seven days postweaning was equal to or better than that of dietary zinc oxide in reducing diarrhoea symptoms and in clearing faecal haemolytic bacteria for 14 days postweaning. These observations warrant future trials of dietary ppIgG in intensive swine production units to establish its performance as an alternative to dietary antibiotics and zinc oxide for preventing PWD.

Immunisation by administration of antibodies (immunoglobulins) has been known for more than one hundred years as a very efficient means of obtaining immediate, short-lived protection against infection and/or against the disease-causing effects of toxins from microbial pathogens and from other sources. Thus, due to its rapid action, passive immunisation is often used to treat disease caused by infection and/or toxin exposure. However immunoglobulins may also be administered prior to exposure to infection and/or toxin, although they will not provide long-lasting protection as is seen with active immunisation (vaccination) in which an immunological memory is established by controlled exposure of the host to the pathogen in question. With multi-factorial infectious diseases in production animals, especially those that have proven hard to control by vaccination, the potential of passive immunisation remains big. This review highlights a number of examples on the use of passive immunisation for the control of infectious disease in the modern production of a range of animals, including pigs, cattle, sheep, goat, poultry and fish. Special emphasis is given on the enablement of passive immunisation strategies in these production systems through low cost and ease of use as well as on the sources, composition and purity of immunoglobulin preparations used and their benefits as compared to current measures, including vaccination (also comprising maternal vaccination), antibiotics and feed additives such as spray-dried plasma. It is concluded that provided highly efficient, relatively low-price immunoglobulin products are available, passive immunisation has a clear role in the modern animal production sector as a means of controlling infectious diseases, importantly with a very low risk of causing development of bacterial resistance, thus constituting a real and widely applicable alternative to antibiotics.

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.

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.

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 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.

Thyroglobulin (TG), as autoantigen, induces in vitro proliferation of T and B cells from normal individuals, but the cytokine production differs from that in patients with autoimmune thyroid disease. Here, we investigate whether normal T cells responding to TG are naive, or have previously encountered TG in vivo, using their responses to classic primary and secondary antigens, keyhole limpet haemocyanin (KLH) and tetanus toxoid (TT), respectively, for comparison. While TG elicited T-cell proliferation kinetics typical of a secondary response, the cytokine profile was distinct from that for TT. Whereas TT induced pro-inflammatory cytokines [interleukin-2 (IL-2)/interferon-gamma (IFN-gamma)/IL-4/IL-5], TG evoked persistent release of the regulatory IL-10. Some donors, however, also responded with late IFN-gamma production, suggesting that the regulation by IL-10 could be overridden. Although monocytes were prime producers of IL-10 in the early TG response, a few IL-10-secreting CD4(+) T cells, primarily with CD45RO(+) memory phenotype, were also detected. Furthermore, T-cell depletion from the mononuclear cell preparation abrogated monocyte IL-10 production. Our findings indicate active peripheral tolerance towards TG in the normal population, with aberrant balance between pro- and anti-inflammatory cytokine responses for some donors. This observation has implications for autoantigen recognition in general, and provides a basis for investigating the dichotomy between physiological and pathological modes of auto-recognition.

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.

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. British Journal of Pharmacology (2006) 148, 46–53. doi:10.1038/sj.bjp.0706700

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.

Brief description of research area: A common problem in swine production is diarrhoea in newly weaned piglets, and huge quantities of antibiotics go to treat post-weaning diarrhoeas in piglets. The use of antibiotics can lead to the development of multi- and fully resistant bacteria, which consequently pose a great threat to human health. Therefore, sustainable alternatives for treating post-weaning diarrhoea without using antibiotics are in demand. Swine that are old (and big) enough for slaughter have during their upbringing been challenges by many different pathogens and thus have developed immunity towards these pathogens, which include pathogen-specific immunoglobulins (antibodies). We hypothesis that by harvesting natural immunoglobulins from porcine blood plasma, a waste product from swine slaughter, and feeding these immunoglobulins to the piglets this can subsequently (by passive immunisation) prevent and treat post-weaning diarrhoea.Our challenge is to find a suitable method for stabilising the immunoglobulins for oral provision in order for the immunoglobulins to pass as unharmed as possible through the digestive system and still retaining their anti-pathogenic properties.What we know: It is possible to multimerise immunoglobulins, which results in an advantage when binding to their respective antigens in comparison to the non-multimerised immunoglobulins, but too high degree of multimerisation abates immunoglobulin reactivity. Unfortunately, a preliminary study showed that multimerisation destabilises the immunglobulins. On the other hand, proteolytical resistance correlates with increased immunoglobulin concentration.What we need: To investigate the effect of increasing the concentration of multimerised immunoglobulins on proteolytical resistance.To investigate multimerised immunoglobulins’ ability in inhibiting microbial (E. coli) adhesion on relevant matrices, such intestinal villi and/or intestinal cell lines.A toxicological study on (if any) adverse side effects occurs when enteral providing immunoglobulins to piglets.

Non-Hodgkin lymphoma (NHL) has been associated with elevated levels of inflammatory and immune-regulating cytokines, and polymorphisms in the genes encoding interleukin (IL)-10 and tumor necrosis factor (TNF)-α have been associated with increased incidence of certain subtypes of NHL. The aim of the present study was to screen for a broader spectrum of growth factors and inflammatory mediators and to compare the profiles in different subtypes of NHL in pediatric patients. Serum samples were collected at diagnosis from 31 pediatric patients diagnosed with NHL admitted at Rigshospitalet, Copenhagen, between 1995 and 2008. Cytokines and growth factors were measured in serum using the Luminex platform by application of a 30-plex kit. Levels of IL-6, IL-2R, IL-10, TNF-RI, and macrophage inflammatory protein-1α were significantly higher in patients with anaplastic large-cell lymphoma compared with patients diagnosed with B-cell lymphomas and lymphoblastic lymphomas. High levels of IL-4, IL-13, TNF-RI, and epidermal growth factor were associated with a poorer general condition at diagnosis. The present study suggests that NHL subgrouping and the general condition of pediatric patients at diagnosis are associated with plasma levels of growth factors and inflammatory mediators at presentation.

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.

Variations in the gene for the nucleotide-binding oligomerisation domain (NOD) 2 have been associated with Crohn's disease but not multiple sclerosis (MS). Here we investigate the effect of three polymorphisms in the NOD2 gene (rs5743277, rs2066842 and rs5743291) on cytokine production and CD4+ T cell proliferation elicited by human myelin basic protein (MBP) in blood mononuclear cell (MNC) cultures from 29 patients with MS. No polymorphism was observed at rs5743277. No associations with the rs2066842 polymorphism were found. Concerning rs5743291, none were homozygous for the minor allele. Seven of 29 (24%) patients were heterozygous, and five of these (71%) exhibited increased MBP-induced CD4+ T cell proliferation versus four of 22 (18%), who were homozygous for the major allele (p

Udgivelsesdato: 2010-Nov-2 Background: The results of several cross-sectional studies suggest a relationship between periodontitis and higher serum levels of C- reactive protein (CRP). Most of these studies have been restricted to adult study groups with severe periodontal inflammation and the potential effect of confounding factors has been frequently overlooked. Methods: A case-referent study comprising 87 adolescent cases presenting with clinical attachment loss ≥ 3 mm in at least 2 of 16 teeth recorded and 73 non-cases that did not fulfill these criteria was nested in a fully enumerated adolescent population screened for signs of early periodontitis. Venous blood samples were obtained and CRP levels quantified using a high-sensitivity human CRP antibody bead kit and Luminex technology. The Mann-Whitney test was used to assess overall differences between groups. Results: The median serum CRP values and interquartile range for the cases and non-cases were 64 ng/ml (27-234) and 55 ng/ml (31-183), respectively (p=0.8). Conclusions: Serum levels of CRP were not significantly higher among subjects with early periodontitis than among non-cases. However, a statistically significant positive association between the percentage of sites with BOP and log transformed CRP values was observed.

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)-alpha 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)-gamma, IL-4 and IL-5, in addition to TNF-alpha and IL-10. The production of IFN-gamma 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-alpha 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-gamma and IL-5.

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-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) by patient-derived MNCs. The patients' cells produced higher amounts of IFN-gamma and TNF-alpha, and lower amounts of IL-10, than cells from healthy controls (P

Aim: The aim of this study is firstly to examine if the D2C7-immunotoxin (D2C7-IT) specifically binds to the epidermal growth factor receptor (EGFRwt) and the EGFR-mutant EGFRvIII subsequently inducing cell death. Secondly, to study the internalization of D2C7-IT after binding to EGFRwt/EGFRvIII. Background: Glioblastoma multiforme (GBM) is a malignant primary brain tumor and from diagnose median survival remains around 15 months. Tumor burden can be decreased by operation but eventually the tumor recurs. Thus, novel treatment-strategies for GBM patients are in great demand that would result prolonged median survival and increased quality of life. While EGFRwt and EGFRvIII are not found in normal brain tissue, expression of these are observed in malignant tissue of GBM, and consequently the D2C7-IT has been designed to target both EGFRwt and EGFRvIII. D2C7-IT comprises single chain disulphide stabilized fragment variables (scdsFv) of the bivalent anti-EGFR/EGFRvIII antibody D2C7, a 38 kDa fragment of the pseudomonas exotoxin A (PE38), and finally a C-terminal ‘endoplasmatic reticulum (ER) retention motif’: KDEL. In theory, the D2C7-IT is internalized after binding to EGFR/EGFRvIII and the PE38 moiety is proteolytically cleaved in the endosomal compartment, which releases the C-terminal toxic part of the PE38 into the cytosol. In the cytosol, binding of the KDEL-receptor assures transport to the ER where the toxic moiety inhibits protein synthesis and causes cell death. Methods: Western blotting was applied for examining D2C7-IT-binding to EGFRwt and EGFRvIII in different human malignant cell lines expressing varying levels of EGFRwt or EGFRvIII. MTT assays were used for assessing the impact of D2C7-IT on cell viability in these cell lines. Results: We observed that D2C7-IT binds to cells expressing EGFRwt or EGFRvIII. In addition, the binding on these cells could be inhibited by co-incubating with either EGF or the D2C7 antibody in surplus. Furthermore, D2C7-IT induced a moderate Tyr1173 phosphorylation (marker for internalization) on EGFRwt expressing cells. Concordantly, EGF induced a high level of EGFR-Tyr1173 phosphorylation on EGFRwt expressing cells, which was reduced to a moderate level when co-incubating with D2C7-IT in surplus. D2C7-IT reduced viability in cell cultures expressing EGFR or EGFRvIII while D2C7-IT had no effect on EGFRwt/EGFRvIII negative cells. We are currently trying to visualize the subcellular localizations of D2C7-IT after internalization by fluorescent microscopy. Conclusion: These preliminary results show that D2C7-IT specifically binds to EGFR and EGFRvIII, and reduces viability of EGFRwt/EGFRvIII expressing cells in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2732. doi:1538-7445.AM2012-2732

Interferon‐α/β (IFN‐α/β) is increasingly used as antiviral and immunomodulatory therapies. Unfortunately, bioavailability varies with IFN species and mode of administration, and all IFN species are potentially immunogenic. Assays for antiviral activity (IFN) and antiviral neutralization (antibodies, NAb) have been used for some time to monitor patients on IFN biologicals. These assays require laborious titrations making them unsuitable for large‐scale clinical use. Myxovirus A (MxA) is a resistance GTP‐binding protein that is specifically induced by treatment with type 1 IFNs. For example, IFN‐β‐induced MxA in blood leucocytes has been used as a biomarker in IFN‐β‐treated patients with multiple sclerosis. However, the degree of specificity of MxA in this regard is unclear, and measurements of MxA protein and/or mRNA are not yet suitable for routine clinical use. In an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for IFN‐α and ‐β), microarray screenings with U133A GeneChips (Affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line A549. We studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant IFN‐α and IFN‐β and other antiviral and immunomodulatory cytokines. The results will be presented at the conference.

Interferon-α/β (IFN-α/β) is increasingly used as antiviral and immunomodulatory therapies. Unfortunately, bioavailability varies with IFN species and mode of administration, and all IFN species are potentially immunogenic. Assays for antiviral activity (IFN) and antiviral neutralization (antibodies, NAb) have been used for some time to monitor patients on IFN biologicals. These assays require laborious titrations making them unsuitable for large-scale clinical use. Our laboratories have therefore modified the antiviral assays for IFN bioactivity and Nab, so that they are suitable for large-scale screening in specialized laboratories. The read-out is survival of a subcloned A549 cell line in the presence of an otherwise lethal amount of virus. Thus, survival increases in the presence of type 1 IFN and decreases in the presence of NAb against the IFN added to the cells. MxA is induced by type 1 IFN and can be used for measuring the Nab activity. In another assay, the MxA level in the A549 cell line is measured. In an attempt to find a new and better reporter gene for type 1 IFN than MxA and genes specific for either IFN-α or -β, a micro array screen was carried using the U133A chip from Affymetrix. The expression of 22,000 genes can be studied simultaneous with this technology. The results will be presented at the conference.

Multiple sclerosis (MS) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. A candidate autoantigen, myelin basic protein (MBP), has especially attracted attention. The presence of anti-MBP antibodies is a predictor of definite MS, but their role in the pathogenesis remains obscure. T cells have long been known to play a pivotal role in the pathogenesis of MS. Recently, an important role for B cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. The uptake of MBP by B cells and the presentation of MBP-derive peptides to T helper (Th) cells by B cells may be promoted by the formation of complement (C) activating immune complexes (ICs) between MBP and natural autoantibodies in healthy individuals and disease-associated anti-MBP antibodies in MS patients, respectively. We have investigated the formation of MBP-containing IC, the binding of MBP to B cells, the MBP-elicited induction of Th-cell and B-cell proliferation and the cytokine production in peripheral blood mononuclear cells (PBMCs) from healthy donors grown in the presence of intact or C-inactivated serum from healthy donors or patients with MS. While MBP did not induce measurable proliferation of B cells nor CD4+ T cells, we observed the production of TNF-α, IFN-γ and IL-10 by PBMC in response to incubation with MBP in the presence of sera from healthy controls as well as sera from MS patients. By contrast, no production of IL-2, IL-4 and IL-5 was detected. We are currently investigating the capability of MS sera to promote the formation of MBP-containing IC and thereby enhance the cytokine responses, by virtue of elevated anti-MBP contents.

Post-weaning diarrhoea (PWD) is a common condition in intensive swine production, resulting in reduced welfare of weaners and economic losses for the farmer as a result of illness, death, and treatment costs. It is also one of the main causes of antibiotics- and zinc use in the pig production industry. We aim at developing a sustainable product for protection against PWD based on natural antibodies (immunoglobulins) derived directly from inexpensive raw materials. The availability of such an inexpensive and highly active immunogloulin product would allow swine producers to reduce expenses and minimize the on antibiotics and zinc usage. Swine immunoglobulins were isolated directly from slaughterhouse swine plasma-waste by expanded bed chromatography. It was shown that the isolated Immunoglobulin fraction bound enterotoxigenic Escherichia coli (ETEC) and Salmonella ssp. and inhibited their adhesion to porcine epithelial cells in vitro. As the immunoglobulin fraction is intended for oral use as a feed supplement, we also tested the safety of feeding 4 grams of natural immunoglobulins to 4-5 week old weaner piglets for 14 days and observed no adverse effects. In an experimental model of E. coli F4+ induced PWD, we observed that piglets given IgG as a feed supplement cleared the E coli infection significantly faster than control weaner piglets not receiving an immunoglobulin feed supplement. Furthermore, deep sequencing of the ileal microbiota showed a significantly lowered colonization of the family Enterobactericea in immunoglobulin fed piglets as compared to the control group. Thus pig slaughterhouse plasma is indicated as a potential source resource of antibodies for the control of PWD.

Mutations in the TGFBI (BIGH3) gene that encodes for transforming growth factor beta induced protein (TGFBIp) are the cause of several phenotypically different corneal dystrophies. While the genetics of these protein misfolding diseases are well documented, relatively little is known about this extracellular matrix protein itself. In this study, we have purified TGFBIp from normal human and porcine corneas using nondenaturing conditions and standard chromatography techniques. The two homologues were shown to be monomers, and we did not find evidence for posttranslational additions. The C-terminal of both human and porcine TGFBIp is truncated predominantly after the integrin binding sequence Arg(642)-Gly(643)-Asp(644) (RGD). However, using an antibody against the C-terminal fragment (residues 648-683), we also detected a small amount of full-length TGFBIp in corneal extracts. Approximately 60% of TGFBIp was covalently associated with insoluble components of the extracellular matrix in both human and porcine corneas through a disulfide bridge.