Impact of platelet lysate on immunoregulatory characteristics of equine mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MSC) play an increasing role in the treatment of immune-mediated diseases and inflammatory processes. They regulate immune cells via cell-cell contacts and by secreting various anti-inflammatory molecules but are in turn influenced by many factors such as cytokines. For MSC culture, platelet lysate (PL), which contains a variety of cytokines, is a promising alternative to fetal bovine serum (FBS). We aimed to analyze if PL with its cytokines improves MSC immunoregulatory characteristics, with the perspective that PL could be useful for priming the MSC prior to therapeutic application. MSC, activated peripheral blood mononuclear cells (PBMC) and indirect co-cultures of both were cultivated in media supplemented with either PL, FBS, FBS+INF-γ or FBS+IL-10. After incubation, cytokine concentrations were measured in supernatants and control media. MSC were analyzed regarding their expression of immunoregulatory genes and PBMC regarding their proliferation and percentage of FoxP3+ cells. Cytokines, particularly IFN-γ and IL-10, remained at high levels in PL control medium without cells but decreased in cytokine-supplemented control FBS media without cells during incubation. PBMC released IFN-γ and IL-10 in various culture conditions. MSC alone only released IFN-γ and overall, cytokine levels in media were lowest when MSC were cultured alone. Stimulation of MSC either by PBMC or by PL resulted in an altered expression of immunoregulatory genes. In co-culture with PBMC, the MSC gene expression of COX2, TNFAIP6, IDO1, CXCR4 and MHC2 was upregulated and VCAM1 was downregulated. In the presence of PL, COX2, TNFAIP6, VCAM1, CXCR4 and HIF1A were upregulated. Functionally, while no consistent changes were found regarding the percentage of FoxP3+ cells, MSC decreased PBMC proliferation in all media, with the strongest effect in FBS media supplemented with IL-10 or IFN-γ. This study provides further evidence that PL supports MSC functionality, including their immunoregulatory mechanisms. The results justify to investigate functional effects of MSC cultured in PL-supplemented medium on different types of immune cells in more detail.

Development of a Web App to Convert Blood Insulin Concentrations among Various Immunoassays Used in Horses.

The measurement of the blood insulin concentration, and comparison to cut-offs, is essential in diagnosing insulin dysregulation, a common equine endocrinopathy. However, different insulin assays provide disparate results. We aimed to ease comparison between assays by compiling original and published data into a web app to convert insulin measurements from one assay to another. Data were available for ADVIA Centaur insulin chemiluminescent immunoassay (CLIA), Beckman Coulter insulin radioimmunoassay (RIA), Immulite 1000 CLIA, Immulite 2000 CLIA, Immulite 2000 XPi CLIA, Mercodia equine insulin enzyme-linked immunosorbent assay (ELISA), and Millipore porcine insulin RIA. Linear models were fitted for 13 assay pairs using non-decreasing splines, and integrated into this app. Assay comparisons including data from several studies showed a lower performance. This indicates technical variation between laboratories, which has not been described before, but is relevant when diagnostic measurements and cut-offs are provided by different laboratories. Nevertheless, the models' overall high performance (median r2 = 0.94; range 0.57-1.00) supports their use to interpret results from diagnostic insulin measurements when the reference assay is unavailable, and to compare values obtained from different assays.

Periodontal structures in horses with pituitary pars intermedia dysfunction: A histological evaluation.

Introduction: Pituitary pars intermedia dysfunction (PPID) and dental disorders are of major concern in horses older than 15 years. Although PPID in geriatric horses and dental disorders in all age groups are well described, a connection between this endocrine disease and pathological changes in equine dental structures has not yet been investigated. In humans, periodontitis is considered to be a complication of systemic diseases like diabetes mellitus type 2, obesity and various conditions leading to an impaired immune response. In PPID, cross links to insulin and immune dysregulations are proven. The aim of this study was to compare histological findings of the gingiva and the sub gingival periodontal ligament of PPID affected horses with control horses. Methods: In a case-control morphometric descriptive study, 145 dental locations of 10 PPID affected horses (27.3 ± 2.06 years) were compared with 147 dental locations of 10 controls (21.4 ± 4.12 years). Histological parameters were leukocyte infiltration, keratinization of gingival epithelium, blood vessel supply of the periodontium and structure of cementum. Results: The distribution and localization of gingival leukocyte infiltrations (LI) in PPID affected horses was more often multifocal to coalescing (p = 0.002) and reached into deeper parts of the periodontium, sometimes down to the sub gingival periodontal ligament (PDL). Aged animals of both groups showed higher prevalence (PPID: OR 1.66; controls: OR 1.15) for severe leukocyte infiltration in the PDL. PPID was not significantly associated with increased LI. The cementum bordering the soft tissue in interdental locations showed four times more irregularities in PPID affected horses than in controls which predisposes for interdental food impaction and periodontal diseases. Discussion: In summary, multifocal to coalescing leukocytes and irregular cementum are seen more often in PPID than in controls - however our findings mainly reflect an association of older age with periodontal disease.

[Therapy of bullous pemphigoid in a Warmblood gelding]. / Therapie eines bullösen Pemphigoids bei einem Warmblutwallach.

A 15-year-old Warmblood gelding was presented with multiple large, ulcerative, and crusty dermal lesions that had been existing for 4 years. Histopathology of a skin biopsy revealed cleft formation at the dermal-epidermal junction beneath the basal cells and above the basement membrane leading to the diagnosis of bullous pemphigoid. Immunosuppressive therapy with dexamethasone and azathioprine was initiated and after 14 weeks full remission of the ulcers was achieved. Scar tissue formation was evident in the areas of the formerly affected lesions. Following medication tapering over a period of 5 months, long-term therapy was continued with a maintenance dose of 0.5 mg/kg azathioprine daily. The ulcerative lesions recurred after 63 weeks of disease stabilization. Additionally, adverse drug reactions (acute laminitis and increased susceptibility to infections) were evident and the gelding was euthanized due to animal welfare considerations.

The Gingiva of Horses With Pituitary Pars Intermedia Dysfunction: A Macroscopic Anatomical Evaluation.

Pituitary pars intermedia dysfunction (PPID) is a common neurodegenerative disease mainly in horses older than 15 years. The domestic equine population is following the same demographic change as that seen in humans; it is aging and veterinarians are asked to attend to geriatric horses more frequently. Common problems seen regularly in older equines are dental disorders and especially periodontal disease. As a systemic and endocrine disease, associated with delayed wound healing and impaired immune function, PPID should be considered before major dental treatment in aged equines is started. Possible negative effects of PPID on epithelial tissues could also affect the periodontium. Therefore, the aim of the present study was to identify gross changes in the gingiva associated with PPID. Fourteen horses with clinical signs of PPID and adenoma in the pituitary pars intermedia and 13 controls showing neither clinical signs nor PPID-associated histological changes in the pituitary gland were included. PPID-affected horses (26.9 ± 0.73 years) were significantly older than controls (20.0 ± 1.24 years). In the PPID-affected group, significantly more often an irregular and bulky appearance of the gingival texture was observed, as well as an irregular shape of the gingival margin. Furthermore, the sulcus gingivalis of cheek teeth frequently was deeper than 1 mm. These findings indicate a possible association between age, soft tissue alterations, and PPID and suggest a potential predisposition of PPID-affected horses for periodontal diseases.

ECEIM consensus statement on equine metabolic syndrome.

Equine metabolic syndrome (EMS) is a widely recognized collection of risk factors for endocrinopathic laminitis. The most important of these risk factors is insulin dysregulation (ID). Clinicians and horse owners must recognize the presence of these risk factors so that they can be targeted and controlled to reduce the risk of laminitis attacks. Diagnosis of EMS is based partly on the horse's history and clinical examination findings, and partly on laboratory testing. Several choices of test exist which examine different facets of ID and other related metabolic disturbances. EMS is controlled mainly by dietary strategies and exercise programs that aim to improve insulin regulation and decrease obesity where present. In some cases, pharmacologic aids might be useful. Management of an EMS case is a long-term strategy requiring diligence and discipline by the horse's carer and support and guidance from their veterinarians.

A Fluorescent Benzo[g]isoquinoline-Based HIF Prolyl Hydroxylase Inhibitor for Cellular Imaging.

Prolyl hydroxylation domain (PHD) enzymes catalyze the hydroxylation of the transcription factor hypoxia-inducible factor (HIF) and serve as cellular oxygen sensors. HIF and the PHD enzymes regulate numerous potentially tissue-protective target genes which can adapt cells to metabolic and ischemic stress. We describe a fluorescent PHD inhibitor (1-chloro-4-hydroxybenzo[g]isoquinoline-3-carbonyl)glycine which is suited to fluorescence-based detection assays and for monitoring PHD inhibitors in biological systems. In cell-based assays, application of the fluorescent PHD inhibitor allowed co-localization with a cellular PHD enzyme and led to live cell imaging of processes involved in cellular oxygen sensing.

[Thrombocytopenia in horses]. / Thrombopenie beim Pferd.

OBJECTIVE: The retrospective study aimed to evaluate the prevalence of true thrombocytopenia and ethylenediaminetetraacetic acid (EDTA)-induced pseudothrombocytopenia in horses and ponies and to assess the diagnostic and prognostic utility of true thrombocytopenia. MATERIAL AND METHODS: In 3592 patients (2008-2015) hematological data (ADVIA® 2120, Siemens) were reviewed. Inclusion criteria were platelet counts < 90 x 109/l (EDTA-blood) and/or < 84 x 109/l (citrate blood). Thrombocytopenia was classified as true, EDTA-induced, and questionable. Patients with true thrombocytopenia were assigned to nine groups according to their history and four groups depending on the main diagnosis (inflammatory disorders, neoplasia, non-inflammatory intestinal disease, others). The frequencies of diagnoses were compared to the overall clinic population. RESULTS: Thrombocytopenia was diagnosed in 123/3592 patients (3.4 %) and classified as true in 60/123 (49 %), EDTA-induced in 6/123 (5 %), and questionable in 57/123 (46 %) of cases. In true thrombocytopenia, the most common reasons for referral were lethargy (23/60, 38 %), fever (19/60, 32 %), and colic (17/60, 28 %). In these patients inflammation, neoplasia, non-inflammatory intestinal disease, and others were diagnosed in 25/60 (42 %), 11/60 (18 %), 10/60 (17 %), and 14/60 (23 %) of cases, respectively. Compared with the overall clinic population, there was an increased frequency of neoplasia (18 % versus 1 %). The mortality rate was significantly higher at 38 % in comparison with the overall population. Remarkably high mortality rates of 32 % and 82 % were observed in patients with inflammatory and neoplastic diseases, respectively. CONCLUSION AND CLINICAL RELEVANCE: True thrombocytopenia is relatively rare in horses with internal medical conditions and should be verified by measurement in samples with citrate as anticoagulant. Thrombocytopenia is rarely the primary reason for referral and is a negative prognostic factor in neoplastic and inflammatory diseases.

Postoperative complications in equine elective, clean orthopaedic surgery with/without antibiotic prophylaxis.

OBJECTIVE: Retrospective analysis of postoperative complications in equines after clean, orthopaedic surgical procedures in order to detect differences between animals treated with antibiotics and horses without receiving these drugs. MATERIAL AND METHODS: Details on 652 patients, surgical procedures and surgery-associated complications were compiled from horses being operated between June 2011 and January 2015. Antibiotic-receiving patients (n = 259) were tested for differences in complication rates and characteristics to controls (n = 393). RESULTS: The total complication rate was 39.1 %. Increased swelling was observed most often (25.6 %), followed by exudation (7.5 %), fever without incisional alterations (2.3 %), suture dehiscence (1.8 %), and seroma (0.8 %). Seven patients (five treated, two controls) developed septic arthritis within a total of 463 arthroscopies (1.5 %). There were no significant differences in the development of postoperative complications, which were seen in 97/259 (37.5 %) antibiotic receiving patients and in 158/393 (40.2 %) controls. The application of perioperative antibiotics was significantly influenced by surgeon (p < 0.0001) and type of surgery (p = 0.0007) and increased with the number of surgical lesions (p = 0.03). In patients undergoing tendovaginoscopy/ bursoscopy, fasciotomy and neurectomy (n = 98), antibiotic prophylaxis was initiated less frequently than in other surgeries, e. g. combinations of surgeries, splint bone extraction, tenotomy, and arthroscopy (n = 554). CONCLUSION: Severe complications in equine clean orthopaedic surgery are rare and complication rates in patients either receiving perioperative antibiotics or not were not significantly different. CLINICAL RELEVANCE: Based on the results the use of antibiotics appears to be non-essential in uncomplicated elective orthopaedic interventions in the horse.

Prevention of equine herpesvirus myeloencephalopathy - Is heparin a novel option? A case report. / Prävention der equinen Herpesvirus-Myeloenzephalopathie ­ Ist Heparin eine vielversprechende Option? Ein Fallbericht.

Equine herpesvirus myeloencephalopathy (EHM) is a severe manifestation of equine herpesvirus 1 (EHV-1) infection. Prevention and treatment of EHM during EHV-1 outbreaks is critical, but no reliable and tested specific medication is available. Due to the thromboischemic nature of EHM and due to the fact that EHV-1 entry in cells is blocked by heparin, it was hypothesized that this compound may be useful in reduction of EHM incidence and severity. Therefore, during an acute EHV-1 outbreak with the neuropathogenic G2254/D752 Pol variant, metaphylactic treatment with heparin to prevent EHM was initiated. Clinical signs were present in 61 horses (fever n = 55; EHM n = 8; abortion n = 6). Heparin (25000 IU subcutaneously twice daily for 3 days) was given to 31 febrile horses from day 10 of the outbreak, while the first 30 horses exhibiting fever remained untreated. Treatment outcome was analyzed retrospectively. Heparin-treated horses showed a lower EHM incidence (1/31; 3.2%) than untreated horses (7/30; 23.3%; p = 0.03). Results indicate that heparin may be useful for prevention of EHM during an EHV-1 outbreak. These promising data highlight the need for randomized and possibly blinded studies for the use of heparin in EHV-1 outbreaks.

Clinical observations and management of a severe equine herpesvirus type 1 outbreak with abortion and encephalomyelitis.

Latent equine herpesvirus type 1 (EHV-1) infection is common in horse populations worldwide and estimated to reach a prevalence nearing 90% in some areas. The virus causes acute outbreaks of disease that are characterized by abortion and sporadic cases of myeloencephalopathy (EHM), both severe threats to equine facilities. Different strains vary in their abortigenic and neuropathogenic potential and the simultaneous occurrence of EHM and abortion is rare. In this report, we present clinical observations collected during an EHV-1 outbreak caused by a so-called "neuropathogenic" EHV-1 G(2254)/D(752) polymerase (Pol) variant, which has become more prevalent in recent years and is less frequently associated with abortions. In this outbreak with 61 clinically affected horses, 6/7 pregnant mares aborted and 8 horses developed EHM. Three abortions occurred after development of EHM symptoms. Virus detection was performed by nested PCR targeting gB from nasal swabs (11 positive), blood serum (6 positive) and peripheral blood mononuclear cells (9 positive) of a total of 42 horses sampled. All 6 fetuses tested positive for EHV-1 by PCR and 4 by virus isolation. Paired serum neutralization test (SNT) on day 12 and 28 after the index case showed a significant (≥ 4-fold) increase in twelve horses (n = 42; 28.6%). This outbreak with abortions and EHM cases on a single equine facility provided a unique opportunity for the documentation of clinical disease progression as well as diagnostic procedures.

Chlamydophila spp. infection in horses with recurrent airway obstruction: similarities to human chronic obstructive disease.

BACKGROUND: Recurrent airway obstruction (RAO) in horses is a naturally occurring dust-induced disease mainly characterized by bronchiolitis which shows histological and pathophysiological similarities to human chronic obstructive pulmonary disease (COPD). In human COPD previous investigations indicated an association with Chlamydophila psittaci infection. The present study was designed (1) to clarify a possible role of this infectious agent in RAO and (2) to investigate the suitability of this equine disorder as a model for human COPD. METHODS: Clinico-pathological parameters of a total of 45 horses (25 horses with clinical signs of RAO and 20 clinically healthy controls) were compared to histological findings in lung tissue samples and infection by Chlamydiaceae using light microscopy, immunohistochemistry, and PCR. RESULTS: Horses with clinical signs of RAO vs. controls revealed more inflammatory changes in histology (p = 0.01), and a higher detection rate of Chlamydia psittaci antigens in all cells (p < 0.001) and bronchiolar epithelial cells alone (p < 0.001) by immunohistochemistry. The abundance of chlamydial inclusions increased with the severity of disease. PCR was positive in 60% of horses with RAO vs. 45% of the controls (p = 0.316). OmpA sequencing identified Chlamydophila psittaci (n = 9) and Chlamydophila abortus (n = 13) in both groups with no significant differences. Within the group of clinically healthy horses subgroups with no changes (n = 15) and slight inflammation of the small airways (n = 5) were identified. Also in the group of animals with RAO subgroups with slight (n = 16) and severe (n = 9) bronchiolitis could be formed. These four subgroups can be separated in parts by the number of cells positive for Chlamydia psittaci antigens. CONCLUSION: Chlamydophila psittaci or abortus were present in the lung of both clinically healthy horses and those with RAO. Immunohistochemistry revealed acute chlamydial infections with inflammation in RAO horses, whereas in clinically healthy animals mostly persistent chlamydial infection and no inflammatory reactions were seen. Stable dust as the known fundamental abiotic factor in RAO is comparable to smoking in human disease. These results show that RAO can be used as a model for human COPD.