The potential for immunoglobulins and host defense peptides (HDPs) to reduce the use of antibiotics in animal production.

Innate defense mechanisms are aimed at quickly containing and removing infectious microorganisms and involve local stromal and immune cell activation, neutrophil recruitment and activation and the induction of host defense peptides (defensins and cathelicidins), acute phase proteins and complement activation. As an alternative to antibiotics, innate immune mechanisms are highly relevant as they offer rapid general ways to, at least partially, protect against infections and enable the build-up of a sufficient adaptive immune response. This review describes two classes of promising alternatives to antibiotics based on components of the innate host defense. First we describe immunoglobulins applied to mimic the way in which they work in the newborn as locally acting broadly active defense molecules enforcing innate immunity barriers. Secondly, the potential of host defense peptides with different modes of action, used directly, induced in situ or used as vaccine adjuvants is described.

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.

The self-antigen, thyroglobulin, induces antigen-experienced CD4+ T cells from healthy donors to proliferate and promote production of the regulatory cytokine, interleukin-10, by monocytes.

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.

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)-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.

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-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<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-gamma, 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-gamma, 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.

The effect of beta-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.

Evidence against a blood derived origin for transforming growth factor beta induced protein in corneal disorders caused by mutations in the TGFBI gene.

PURPOSE: Several inherited corneal disorders in humans result from mutations in the transforming growth factor beta induced gene (TGFBI), which encodes for the extracellular transforming growth factor beta induced protein (TGFBIp) that is one of the most abundant proteins in the cornea. We previously reported a significant amount of TGFBIp in plasma by immunoblotting using the only TGFBIp antiserum (anti-p68(beta ig-h3)) available at that time (anti-p68(beta ig-h3) was generated against residues Val210-His683 of TGFBIp). This observation raised the possibility that a fraction of corneal TGFBIp may originate from the plasma. However, recent experiments in our laboratory indicated that the anti-p68(beta ig-h3) antiserum cross-reacts with an environmental protein contaminant. Therefore, we investigated the specificity of the originally utilized anti-p68(beta ig-h3) antiserum and re-evaluated the amount of TGFBIp in human plasma by immunoblotting using a new specific antiserum. METHODS: The observed cross-reactivity of the previously utilized anti-p68(beta ig-h3) antiserum was tested by immunoblotting and the antigen identity was determined by mass spectrometry. A part of human TGFBI encoding an NH2-terminal 11.4 kDa fragment of TGFBIp (residues Gly134-Ile236) was amplified by polymerase chain reaction (PCR) and cloned in E. coli. The TGFBIp fragment was expressed in E. coli, purified by Ni2+-affinity chromatography, and used to immunize rabbits to produce a specific antiserum (anti-TGFBIp(134-236)). To enhance the detection of possible TGFBIp in plasma by allowing a higher sample load, albumin and immunoglobulin G (IgG) were specifically depleted from normal human plasma by affinity chromatography. The presence of TGFBIp in plasma was investigated by immunoblotting using the anti-TGFBIp(134-236) antiserum. Purified TGFBIp from porcine corneas was used for estimation of the TGFBIp detection limit. RESULTS: The previously utilized TGFBIp antiserum, anti-p68(beta ig-h3), cross-reacted with human keratin-1, a common environmental protein contaminant. Thus, the anti-p68(beta ig-h3) antiserum recognizes both TGFBIp and keratin-1. In contrast, the anti-TGFBIp(134-236) antiserum reacted with TGFBIp but showed no indication of reactivity with other proteins in plasma. Using this antiserum, TGFBIp was not detected in crude or albumin/IgG-depleted human plasma and the detection limit of TGFBIp using immunoblotting was estimated to be 10 ng. CONCLUSIONS: Our failure to detect TGFBIp in human plasma using a highly specific antiserum suggests that TGFBIp is not present in a physiologically relevant concentration in human plasma. The previous impression that normal human plasma contains a significant amount of TGFBIp by immunoblotting was due to the utilization of a less specific antiserum that recognizes both TGFBIp and human keratin-1. Together with other results, our observation makes it unlikely that TGFBIp is imported into the cornea from the circulation as reported for other abundant extracellular corneal proteins and suggests corneal origin of TGFBIp deposits in individuals with inherited corneal diseases caused by mutations in the TGFBI gene.

Purified natural pig immunoglobulins can substitute dietary zinc in reducing piglet post weaning diarrhoea.

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 14days 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.

Passive immunisation, an old idea revisited: Basic principles and application to modern animal production systems.

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.

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.

Transforming growth factor beta induced protein accumulation in granular corneal dystrophy type III (Reis-Bücklers dystrophy). Identification by mass spectrometry in 15 year old two-dimensional protein gels.

PURPOSE: To investigate the accumulation of TGFBIp in GCDIII and to demonstrate the ability to perform mass spectrometry on old two-dimensional protein gels. METHODS: Proteins were extracted from one cornea with GCDIII from a person with an Arg124Leu mutation in the TGFBI (BIGH3) gene and from one normal human cornea in 1987 and subjected to two-dimensional (2D) gel electrophoresis. After keeping the gels at room temperature for 15 years, protein spots of interest were excised and digested with trypsin. The tryptic-derived peptides were analyzed using mass spectrometry. RESULTS: Four areas of interest were examined and four different proteins (TGFBIp, aldehyde dehydrogenase class 3, actin, and albumin) were identified in the 15-year-old gels. Using image analysis, the amount of TGFBIp was found to be 77 fold higher in the GCDIII affected cornea relative to the normal tissue. In both situations, TGFBIp migrated on the 2D gels as a 63 kDa protein. Mass spectrometry revealed the same nine peptides in TGFBIp from both the normal and the GCDIII affected corneas, including one peptide situated at the amino terminus. Moreover, the cornea with GCDIII contained abundant 40 kDa TGFBIp fragments that were lacking sequences in both the amino and carboxy termini. CONCLUSIONS: Mass spectrometry can be performed on old 2D polyacrylamide gels. In both normal and GCDIII affected corneas, the majority of TGFBIp migrated on 2D gels as a 63 kDa protein with an intact amino terminus. However, the amount of the 63 kDa TGFBIp was 77 fold higher in the GCDIII affected cornea. Furthermore, the GCDIII affected cornea contained abundant 40 kDa fragments that were trunctated in both the amino and carboxy termini.

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.

Purification and structural characterization of transforming growth factor beta induced protein (TGFBIp) from porcine and human corneas.

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.