Lateral ridge augmentation using equine- and bovine-derived cancellous bone blocks: a feasibility study in dogs

OBJECTIVES: The aim of the present study was to histologically evaluate and compare a new prototype collagen type I/III-containing equine- (EB) and a bovine- (BB) derived cancellous bone block in a dog model. MATERIALS AND METHODS: Four standardized box-shaped defects were bilaterally created at the buccal aspect of the alveolar ridge in the lower jaws of five beagle dogs and randomly allocated to either EB or BB. Each experimental site was covered by a native (non-crosslinked) collagen membrane and left to heal in a submerged position for 12 weeks. Dissected blocks were processed for semi-/and quantitative analyses. RESULTS: Both groups had no adverse clinical or histopathological events (i.e. inflammatory/foreign body reactions). BB specimens revealed no signs of biodegradation and were commonly embedded in a fibrous connective tissue. New bone formation and bony graft integration were minimal. In contrast, EB specimens were characterized by a significantly increased cell (i.e. osteoclasts and multinucleated giant cells)-mediated degradation of the graft material (P<0.001). The amount and extent of bone ingrowth was consistently higher in all EB specimens, but failed to reach statistical significance in comparison with the BB group (P>0.05). CONCLUSIONS: It was concluded that the application of EB may not be associated with an improved bone formation than BB.

Lethal toxin of Clostridium sordellii is associated with fatal equine atypical myopathy

The lethal toxin of Clostridium sordellii (TcsL) evokes severe, mostly fatal disease patterns like toxic shock syndrome in humans and animals. Since this large clostridial toxin-induced severe muscle damaging when injected intramuscularly into mice, we hypothesized that TcsL is also associated with equine atypical myopathy (EAM), a fatal myodystrophy of hitherto unknown etiology. Transmission electron microscopy revealed skeletal and heart muscles of EAM-affected horses to undergo degeneration ultrastructurally similar to the damage found in TcsL-treated mice. Performing immunohistochemistry, myofibers of EAM-affected horses specifically reacted with sera derived from horses with EAM as well as an antibody specific for the N-terminal part of TcsL, while both antibodies failed to bind to the myofibers of either healthy horses or those with other myopathies. The presence of TcsL in myofibers of horses with EAM suggests that it plays a role as trigger or even as lethal factor in this disease.

Alpha4beta1 integrin mediates the recruitment of immature dendritic cells across the blood-brain barrier during experimental autoimmune encephalomyelitis

Dendritic cells (DCs) within the CNS are recognized to play an important role in the effector phase and propagation of the immune response in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. However, the mechanisms regulating DC trafficking into the CNS still need to be characterized. In this study, we show by performing intravital fluorescence videomicroscopy of the inflamed spinal cord white-matter microvasculature in SJL mice with EAE that immature, and to a lesser extent, LPS-matured, bone marrow-derived DCs efficiently interact with the CNS endothelium by rolling, capturing, and firm adhesion. Immature but not LPS-matured DCs efficiently migrated across the wall of inflamed parenchymal microvessels into the CNS. Blocking alpha4 integrins interfered with the adhesion but not the rolling or capturing of immature and LPS-matured DCs to the CNS microvascular endothelium, inhibiting their migration across the vascular wall. Functional absence of beta1 integrins but not of beta7 integrins or alpha4beta7 integrin similarly reduced the adhesion of immature DCs to the CNS microvascular endothelium, demonstrating that alpha4beta1 but not alpha4beta7 integrin mediates this step of immature DCs interaction with the inflamed blood-brain barrier during EAE. Our study shows that during EAE, especially immature DCs migrate into the CNS, where they may be crucial for the perpetuation of the CNS-targeted autoimmune response. Thus therapeutic targeting of alpha4 integrins affects DC trafficking into the CNS and may therefore lead to the resolution of the CNS autoimmune inflammation by reducing the number of CNS professional APCs.

PSGL-1 is dispensible for the development of active experimental autoimmune encephalomyelitis in SJL/J mice

The adhesion molecule P-selectin glycoprotein ligand (PSGL)-1 has been suggested to be involved in the immunopathogenesis of multiple sclerosis (MS). However, in C57BL/6 mice PSGL-1 was found to be dispensible for the development of MOG(aa35-55)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS. To study, if involvement of PSGL-1 to EAE pathogenesis can be observed in another common mouse model, we backcrossed PSGL-1(-/-) mice for at least 12 generations into the SJL/J background and compared PLP(aa139-151) induced EAE in PSGL-1(-/-) SJL/J mice versus wild-type SJL/J mice. Here, we demonstrate that PSGL-1(-/-) SJL/J mice exhibited EAE pathogenesis indistinguishable from wild-type SJL/J mice. Our present study underscores and emphasizes previous observations that PSGL-1 is dispensible for EAE pathogenesis.

Swiss warmblood horse with symptoms of hereditary equine regional dermal asthenia without mutation in the cyclophylin B gene (PPIB)

Hereditary equine dermal asthenia (HERDA) is an autosomal recessive skin disease that affects predominantly Quarter Horses and related breeds. Typical symptoms are easy bruising and hyperextensible skin on the back. The prognosis is guarded, as affected horses cannot be ridden normally and are often euthanised. In the Quarter Horse, HERDA is associated with a mutation in cyclophilin B (PPIB), an enzyme involved in triple helix formation of collagen. Here we describe the case of a Swiss Warmblood filly with symptoms of HERDA without PPIB-mutation and in which we also could exclude Ehlers-Danlos syndrome Type IV, VI, VIIA, VIIB and VIIC (dermatosparaxis type) as etiological diseases.

Estrogen Receptor α Signaling in T Lymphocytes Is Required for Estradiol-Mediated Inhibition of Th1 and Th17 Cell Differentiation and Protection against Experimental Autoimmune Encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

Claudin-1 induced sealing of blood-brain barrier tight junctions ameliorates chronic experimental autoimmune encephalomyelitis

In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), loss of the blood-brain barrier (BBB) tight junction (TJ) protein claudin-3 correlates with immune cell infiltration into the CNS and BBB leakiness. Here we show that sealing BBB TJs by ectopic tetracycline-regulated expression of the TJ protein claudin-1 in Tie-2 tTA//TRE-claudin-1 double transgenic C57BL/6 mice had no influence on immune cell trafficking across the BBB during EAE and furthermore did not influence the onset and severity of the first clinical disease episode. However, expression of claudin-1 did significantly reduce BBB leakiness for both blood borne tracers and endogenous plasma proteins specifically around vessels expressing claudin-1. In addition, mice expressing claudin-1 exhibited a reduced disease burden during the chronic phase of EAE as compared to control littermates. Our study identifies BBB TJs as the critical structure regulating BBB permeability but not immune cell trafficking into CNS during EAE, and indicates BBB dysfunction is a potential key event contributing to disease burden in the chronic phase of EAE. Our observations suggest that stabilizing BBB barrier function by therapeutic targeting of TJs may be beneficial in treating MS, especially when anti-inflammatory treatments have failed.

TET inducible expression of the α4β7-integrin ligand MAdCAM-1 on the blood-brain barrier does not influence the immunopathogenesis of experimental autoimmune encephalomyelitis

Inhibiting the α4 subunit of the integrin heterodimers α4β1 and α4β7 with the mab natalizumab is an effective treatment of multiple sclerosis (MS). Which of the two α4 heterodimers is involved in disease pathogenesis has, however, remained controversial. Whereas the development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, is ameliorated in β7-integrin-deficient C57BL/6 mice, neutralizing antibodies against the β7-integrin subunit or the α4β7-integrin heterodimer fail to interfere with EAE pathogenesis in the SJL mouse. To facilitate α4β7-integrin-mediated immune-cell trafficking across the blood-brain barrier (BBB), we established transgenic C57BL/6 mice with endothelial cell-specific, inducible expression of the α4β7-integrin ligand mucosal addressin cell adhesion molecule (MAdCAM)-1 using the tetracycline (TET)-OFF system. Although TET-regulated MAdCAM-1 induced α4β7-integrin mediated interaction of α4β7(+) /α4β1(-) T cells with the BBB in vitro and in vivo, it failed to influence EAE pathogenesis in C57BL/6 mice. TET-regulated MAdCAM-1 on the BBB neither changed the localization of central nervous system (CNS) perivascular inflammatory cuffs nor did it enhance the percentage of α4β7-integrin(+) inflammatory cells within the CNS during EAE. In conclusion, our study demonstrates that ectopic expression of MAdCAM-1 at the BBB does not increase α4β7-integrin-mediated immune cell trafficking into the CNS during MOG(aa35-55)-induced EAE.

Equine CD4(+) CD25(high) T cells exhibit regulatory activity by close contact and cytokine-dependent mechanisms in vitro

Horses are particularly prone to allergic and autoimmune diseases, but little information about equine regulatory T cells (Treg) is currently available. The aim of this study therefore was to investigate the existence of CD4(+) Treg cells in horses, determine their suppressive function as well as their mechanism of action. Freshly isolated peripheral blood mononuclear cells (PBMC) from healthy horses were examined for CD4, CD25 and forkhead box P3 (FoxP3) expression. We show that equine FoxP3 is expressed constitutively by a population of CD4(+) CD25(+) T cells, mainly in the CD4(+) CD25(high) subpopulation. Proliferation of CD4(+) CD25(-) sorted cells stimulated with irradiated allogenic PBMC was significantly suppressed in co-culture with CD4(+) CD25(high) sorted cells in a dose-dependent manner. The mechanism of suppression by the CD4(+) CD25(high) cell population is mediated by close contact as well as interleukin (IL)-10 and transforming growth factor-beta1 (TGF-beta1) and probably other factors. In addition, we studied the in vitro induction of CD4(+) Treg and their characteristics compared to those of freshly isolated CD4(+) Treg cells. Upon stimulation with a combination of concanavalin A, TGF-beta1 and IL-2, CD4(+) CD25(+) T cells which express FoxP3 and have suppressive capability were induced from CD4(+) CD25(-) cells. The induced CD4(+) CD25(high) express higher levels of IL-10 and TGF-beta1 mRNA compared to the freshly isolated ones. Thus, in horses as in man, the circulating CD4(+) CD25(high) subpopulation contains natural Treg cells and functional Treg can be induced in vitro upon appropriate stimulation. Our study provides the first evidence of the regulatory function of CD4(+) CD25(+) cells in horses and offers insights into ex vivo manipulation of Treg cells.

Resistance against strongylid nematodes in two high prevalence Equine Recurrent Airway Obstruction families has a genetic basis

A recent study showed increased resistance against strongylid nematodes in offspring of a stallion affected by recurrent airway obstruction (RAG) compared with unrelated pasture mates. Resistance against strongylid nematodes was associated with RAG affection. Hypothesis: Resistance against strongylid nematodes has a genetic basis. The genetic variants influencing strongylid resistance also influence RAG susceptibility. Faecal samples from the half-sibling offspring of two RAG-affected Warmblood stallions 98 offspring from the first family (family 1) and 79 from the second family (family 2) were analysed using a combined sedimentation-flotation method. The phenotype was defined as a binary trait - either positive or negative for egg shedding. The influence of non-genetic factors on egg shedding was analysed using SAS, the mode of inheritance was investigated using PAP and iBay, and the association between shedding of strongyle eggs and RAG was estimated by odds ratios. Previously established genotypes for 315 microsatellite markers were used for QTL analyses using GRID QTL. The inheritance of "strongylid egg shedding" is influenced by major genes on ECA15 and ECA20. Shedding of strongylid eggs is associated with RAG in family 1 but not in family 2. Conclusions: The status of "shedding of strongyle eggs" has a genetic background. The results were inconclusive as to whether "egg shedding" and RAG share common genetic components. Our results suggest that it may be possible to select for resistance against strongylid nematodes.

Evolution of multidrug-resistant Staphylococcus aureus infections in horses and colonized personnel in an equine clinic between 2005 and 2010

A total of 70 Staphylococcus aureus isolates from postoperative infections in hospitalized horses were isolated between January 2005 and January 2011. Among them, 12 isolates were methicillin-susceptible S. aureus (MSSA), 18 were borderline-oxacillin-resistant S. aureus (BORSA), and 40 were methicillin-resistant S. aureus (MRSA). During the same period, the equine clinic personnel were screened for nasal carriage of BORSA and MRSA. Genotyping revealed that BORSA ST1(MLST)-t2863(spa) isolates were responsible for most equine infections and were the main isolates found in colonized members of the personnel between 2005 and 2007, and that in 2007, MRSA ST398-t011-IVa(SCCmec) emerged in infection sites and personnel, replacing BORSA. Besides decreased susceptibility to oxacillin, all MRSA and BORSA of these two major clonal lineages displayed resistance to gentamicin and kanamycin conferred by the aac(6')-Ie-aph(2')-Ia gene and to trimethoprim conferred by dfr(K) in MRSA and dfr(A) in BORSA. All MRSA had additional resistance to tetracycline conferred by tet(M), whereas BORSA generally also display resistance to streptomycin conferred by str. The number of hospital-acquired MRSA infections in horses could be limited after the introduction of basic hygiene measures and personnel decolonization. Two MRSA carriers could not be decolonized using mupirocin, and a year after decolonization, additional members were recolonized with MRSA. Hygiene measures should, therefore, be maintained to limit the transmission of S. aureus between personnel and horses.

Selective cloning, characterization, and production of the Culicoides nubeculosus salivary gland allergen repertoire associated with equine insect bite hypersensitivity

Salivary gland proteins of Culicoides spp. have been suggested to be among the main allergens inducing IgE-mediated insect bite hypersensitivity (IBH), an allergic dermatitis of the horse. The aim of our study was to identify, produce and characterize IgE-binding salivary gland proteins of Culicoides nubeculosus relevant for IBH by phage surface display technology. A cDNA library constructed with mRNA derived from C. nubeculosus salivary glands was displayed on the surface of filamentous phage M13 and enriched for clones binding serum IgE of IBH-affected horses. Ten cDNA inserts encoding putative salivary gland allergens were isolated and termed Cul n 2 to Cul n 11. However, nine cDNA sequences coded for truncated proteins as determined by database searches. The cDNA sequences were amplified by PCR, subcloned into high level expression vectors and expressed as hexahistidine-tagged fusion proteins in Escherichia coli. Preliminary ELISA results obtained with these fusions confirmed the specific binding to serum IgE of affected horses. Therefore, the putative complete open reading frames derived from BLAST analyses were isolated by RACE-PCR and subcloned into expression vectors. The full length proteins expressed in Escherichia coli showed molecular masses in the range of 15.5-68.7 kDa in SDS-PAGE in good agreement with the masses calculated from the predicted protein sequences. Western blot analyses of all recombinant allergens with a serum pool of IBH-affected horses showed their ability to specifically bind serum IgE of sensitized horses, and ELISA determinations yielded individual horse recognition patterns with a frequency of sensitization ranging from 13 to 57%, depending on the allergen tested. The in vivo relevance of eight of the recombinant allergens was demonstrated in intradermal skin testing. For the two characterized allergens Cul n 6 and Cul n 11, sensitized horses were not available for intradermal tests. Control horses without clinical signs of IBH did not develop any relevant immediate hypersensitivity reactions to the recombinant allergens. The major contribution of this study was to provide a repertoire of recombinant salivary gland allergens repertoire from C. nubeculosus potentially involved in the pathogenesis of IBH as a starting basis for the development of a component-resolved serologic diagnosis of IBH and, perhaps, for the development of single horse tailored specific immunotherapy depending on their component-resolved sensitization patterns.

Bacterial infections in horses: a retrospective study at the University Equine Clinic of Bern

Bacterial infections present a major challenge in equine medicine. Therapy should be based on bacteriological diagnosis to successfully minimize the increasing number of infections caused by multidrug-resistant bacteria. The present study is a retrospective analysis of bacteriological results from purulent infections in horses admitted at the University Equine Clinic of Bern from 2004 to 2008. From 378 samples analyzed, 557 isolates were identified, of which Staphylococcus aureus, Streptococcus equi subsp. zooepidemicus and coliforms were the most common. Special attention was paid to infections with methicillin-resistant S. aureus (MRSA) ST398 and a non-MRSA, multidrug-resistant S. aureus clone ST1 (BERN100). Screening of newly-admitted horses showed that 2.2 % were carriers of MRSA. Consequent hygiene measures taken at the Clinic helped to overcome a MRSA outbreak and decrease the number of MRSA infections.

Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.