Canine leukocyte adhesion deficiency colony for investigation of novel hematopoietic therapies

The genetic immunodeficiency disease canine leukocyte adhesion deficiency (CLAD) was originally described in juvenile Irish Setters with severe, recurrent bacterial infections. CLAD was subsequently shown to result from a mutation in the leukocyte integrin CD18 subunit which prevents leukocyte surface expression of the CD11/CD18 complex. We describe the development of a mixed-breed CLAD colony with clinical features that closely parallel those described in Irish Setters. We demonstrate that the early identification of CLAD heterozygotes and CLAD-affected dogs by a combination of flow cytometry and DNA sequencing allows the CLAD-affected animals to receive life-saving antibiotic therapy. The distinct clinical phenotype in CLAD, the ability to detect CD18 on the leukocyte surface by flow cytometry, and the history of the canine model in marrow transplantation, enable CLAD to serve as an attractive large-animal model for the investigation of novel hematopoietic stem cell and gene therapy strategies.     

Clinical effects of bovine lactoferrin on two canine cases with familial neutrophil dysfunction

This study reported detailed clinical effects of bovine lactoferrin on 2 canine littermates (1 female and 1 male) with familial neutrophil dysfunction and an investigation of their genetic background. Clinical signs caused by severe upper respiratory bacterial infections were observed in these dogs. Oral administration of bovine lactoferrin for a long duration improved their clinical signs (severe uveitis in the female dog and coughing from pneumonia in the male dog). Their backcross dogs that have the same father didn't show clinical signs of bacterial infection. Neutrophil function tests revealed that the backcross dogs didn't have any disorders. It is likely that abnormal clinical signs are associated with neutrophil dysfunction in the colony, and the mother dog of these cases might be the genetic carrier of this dysfunction.     

Canine leukocyte adhesion deficiency: presence of the Cys36Ser beta-2 integrin mutation in an affected US Irish Setter cross-breed dog and in US Irish Red and White Setters

Canine leukocyte adhesion deficiency (CLAD) is a primary immunodeficiency disease characterized by recurrent bacterial infections in the presence of marked leukocytosis. The disease was 1st described in the mid-1980s in a cross-breed Irish Setter Dog in the United States. It results from a defective beta-2 subunit of heterodimeric leukocyte adhesion proteins. The causative mutation for CLAD in Irish Setter Dogs from Europe has been identified as a missense mutation at base pair position 107 in the beta-2 integrin subunit gene (ITGB2) that results in an amino acid change from cysteine to serine at amino acid 36 (Cys36Ser) in the beta-2 integrin subunit protein. In the current work, the originally described dog with CLAD has been genetically tested and shown to have the same mutation as the European Irish Setters. This suggests that the mutation has been in the Irish Setter population for many generations spanning more than 2 decades. A related breed, the Irish Red and White Setter, has a history of interbreeding with Irish Setters and shares a common ancestry with the Irish Setter breed. DNA from Irish Red and White Setters residing in the United States was screened either by sequencing or by the newly developed restriction enzyme test for the Irish Setter Cys36Ser CLAD mutation. Seven of 54 dogs tested (13%) were found to be carriers of the Irish Setter CLAD mutation. Five of these were directly related to a sire from the UK, demonstrating the importation of an allele from another continent and establishing the need for genetic testing in this breed in the United States.     

Bovine leukocyte adhesion deficiency (BLAD): a review

Bovine leukocyte adhesion deficiency (BLAD) in Holstein cattle is an autosomal recessive congenital disease characterized by recurrent bacterial infections, delayed wound healing and stunted growth, and is also associated with persistent marked neutrophilia. The molecular basis of BLAD is a single point mutation (adenine to guanine) at position 383 of the CD18 gene, which caused an aspartic acid to glycine substitution at amino acid 128 (D128G) in the adhesion molecule CD18. Neutrophils from BLAD cattle have impaired expression of the beta2 integrin (CD11a,b,c/CD18) of the leukocyte adhesion molecule. Abnormalities in a wide spectrum of adherence dependent functions of leukocytes have been fully characterized. Cattle affected with BLAD have severe ulcers on oral mucous membranes, severe periodontitis, loss of teeth, chronic pneumonia and recurrent or chronic diarrhea. Affected cattle die at an early age due to the infectious complications. Holstein bulls, including carrier sires that had a mutant BLAD gene in heterozygote were controlled from dairy cattle for a decade. The control of BLAD in Holstein cattle by publishing the genotypes and avoiding the mating between BLAD carriers was found to be successful. This paper provides an overview of the genetic disease BLAD with reference to the disease in Holstein cattle.     

Evaluation of structural and functional alterations of circulating neutrophils in calves with experimentally induced pneumonic pasteurellosis

OBJECTIVES: To determine the structural and functional alterations in circulating neutrophils that may lead to sequestration in lung microvasculature and endothelial injury in calves with experimentally induced pneumonic pasteurellosis. ANIMALS: 10 healthy, 2- to 4-week-old male Holstein calves. PROCEDURES: Holstein calves were anesthetized and inoculated intrabronchially with Dulbecco phosphate buffered saline (0.9% NaCl) solution (DPBSS; 5 control calves) or 1 x 10(9) Pasteurella haemolytica organisms (5 infected calves). Blood samples were collected before and 1, 2, 4, and 6 hours after inoculation. Total and differential WBC count, dilute whole blood leukocyte deformability, neutrophil size distribution, and neutrophil surface CD11b expression were measured in blood samples. RESULTS: A progressive decrease in leukocyte deformability and increase in neutrophil size was detected 1, 2, 4, and 6 hours after inoculation of P haemolytica. Neutrophil surface CD11b expression was greater than baseline values at 6 hours after inoculation of P haemolytica. Two populations of neutrophils with an increase in size were detected in P haemolytica-infected calves. Both subpopulations had increased CD11b expression, compared with neutrophils that were typical in size. CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophils circulate in an activated and nondeformable state in calves with experimentally induced pneumonic pasteurellosis. A decrease in neutrophil deformability and neutrophil aggregation may contribute to neutrophil trapping in the lung microvasculature during pneumonic pasteurellosis in calves.     

Genetic Variation in Bovine Neutrophil Sensitivity to Glucocorticoid Challenge

Blood neutrophils use CD62L and CD18 adhesion molecules to contact and migrate rapidly through blood vessels in defense against infections in underlying tissues. Previous work showed that glucocorticoid hormones repress expression of CD62L and CD18, causing neutrophilia and increased mastitis susceptibility in dairy cows. The aim of this study was to determine whether bovine neutrophil sensitivity to glucocorticoids exhibits genetic variability. Test animals included 60 pedigreed Holstein bulls treated on 3 consecutive days with a synthetic glucocorticoid (dexamethasone) and five untreated control bulls. Five indicator traits of neutrophil glucocorticoid sensitivity were monitored, including circulating neutrophil counts and two measures on each of CD62L and CD18 expression. Random regression models with treatment-specific serial correlation were used to estimate genetic and non-genetic sources of variation before, during and after glucocorticoid administration. Significant genetic variation was observed for neutrophil CD18 expression, with longitudinal heritability estimates ranging from 0.10 to 0.54 and influenced by dexamethasone. Significant genetic variation was also observed for blood neutrophil counts (heritability estimates ranging from 0.11 to 0.24) but was not influenced by dexamethasone administration. Estimated genetic correlations between circulating neutrophil counts and various indicators of CD62L and CD18 expression were large and negative (-0.44 to - 0.78). These results imply significant genetic variability and pleiotropic effects for neutrophil traits that are important for stress-induced disease susceptibility in dairy cattle and might be exploited by genetic selection.     

Somatic hypermutations and isotype restricted exceptionally long CDR3H contribute to antibody diversification in cattle

Antibody diversification in IgM and IgG antibodies was analyzed in an 18-month old bovine (Bos taurus) suffering from naturally occurring chronic and recurrent infections due to bovine leukocyte adhesion deficiency (BLAD). The BLAD, involving impaired leukocyte β2 integrin expression on leukocytes, develops due to a single point mutation in conserved region of the CD18 gene resulting in substitution of aspartic acid128 with glycine (D128G). Twenty four VDJCμ and 25 VDJCγ recombinations from randomly constructed cDNA libraries, originating from peripheral blood lymphocytes, were examined for the variable-region structural characteristics in IgM and IgG antibody isotypes. These analyses led to conclude that: (a) expression of exceptionally long CDR3H is isotype restricted to cattle IgM antibody; (b) VDJ recombinations encoding IgM with exceptionally long CDR3H undergo clonal selection and affinity maturation via somatic mutations similar to conventional antibodies; (c) somatic mutations contribute significantly to both IgM and IgG antibody diversification but significant differences exist in the patterns of ‘hot spot’ in the FR1, FR3 and CDR1H and, also, position-dependant amino acid diversity; and (d) transition nucleotide substitutions predominate over transversions in both VDJCμ and VDJCγ recombinations consistent with the evolutionary conservation of somatic mutation machinery. Overall, these studies suggest that both somatic mutations and exceptional CDR3H size generation contribute to IgM and IgG antibody diversification in cattle during the development of immune response to naturally occurring chronic and multiple microbial infections.     

Bovine leukocyte adhesion deficiency in Holstein cattle.

Two Holstein heifers with persistent and recurrent infections including ulcerative gingivitis, periodontitis, pneumonia, loss of teeth and stunted growth associated with marked neutrophilia were evaluated clinically and for neutrophil function, CD18 expression on neutrophils and CD18 genotype analysis by DNA-polymerase chain reaction (PCR) test. Adherence to nylon fibers and phagocytic activity of neutrophils from affected animals were significantly (p < 0.05) impaired as compared with those of controls. Neutrophils from affected heifers had decreased chemiluminescent (CL) responses when stimulated with opsonized zymosan, compared with those of controls. In contrast, neutrophils from affected heifers produced increased CL responses when stimulated with latex beads and phorbol myristate acetate compared with those of controls. The clinical findings, functional leukocyte abnormalities, deficiency in expression of CD18 on neutrophils, and the D128G mutation detected by DNA-PCR testing of affected heifers demonstrated that these heifers have bovine leukocyte adhesion deficiency (BLAD). Although both animals were confirmed to be homozygotes for BLAD by DNA-PCR test, they had differences in clinical, hematological and neutrophil functional characteristics.     

Alpha4-integrin (CD49d) expression on bovine peripheral blood neutrophils is related to inflammation of the respiratory system

Neutrophil emigration from the pulmonary vasculature, is mediated by cellular adhesion molecules (CAM) expressed on the outer membranes of endothelial cells and neutrophils. Although beta(2)-integrin-dependent migration is a major mechanism of neutrophil migration, which was demonstrated by extensive invasion of neutrophils in pulmonary tissue of calves suffering from a genetic deficit in expression of beta(2)-integrins, termed bovine leukocyte adhesion deficiency (LAD), the role of alternative CAM is still unclear. We investigated whether an alternate CAM for beta(2)-integrin function, i.e. the alpha(4)-integrin, was expressed on peripheral blood neutrophils of calves. As we detected basal but significant expression, the effect of naturally acquired pulmonary infection on the expression of either integrin was determined, as an indication for its function in the migration process. In our experiments, basal expression of alpha(4)-integrins on peripheral blood neutrophils from clinically healthy calves was detected. On neutrophils of calves, experiencing field outbreaks of enzootic bronchopneumonia, higher expression of the alpha(4)-integrin was detected, which returned to normal after successful treatment of the disease. In addition, its level of expression was linearly related to plasma acute phase protein (haptoglobin) concentrations, which is a sensitive parameter for severity of respiratory inflammation. Increased expression of the alpha(4)-integrin on peripheral blood neutrophils during pulmonary inflammation indicates a role for this CAM in neutrophil migration in the lung.     

Effect of isoproterenol and dexamethasone on the lipopolysaccharide induced expression of CD11b on bovine neutrophils

The present experiments investigate the changes in expression of CD11b on bovine neutrophils and its modulation by isopropylnoradrenaline (IPN, isoproterenol), dexamethasone (DX), phenylephrine (alpha-agonist) and clenbuterol (beta-agonist). Both IPN and DX caused a dose-dependent inhibition of LPS-induced CD11b expression. A combination of IPN and DX elicited a synergistical decrease of the CD11b expression. Clenbuterol mimicked the effect of IPN, whereas phenylephrine did not. The effect of IPN and DX could at least partly be mediated through a decreased TNF-alpha production by monocytes since tumor necrosis factor-alpha (TNF-alpha) is shown to mediate a dose-dependent CD11b up-regulation. Stimulation of stress hormone receptors partly immuno-suppresses neutrophil functions by inhibition of CD11b expression on the neutrophil surface upon LPS stimulation. This inhibition is probably related to a decrease in TNF-alpha production. A similar mechanism of immuno-suppression could contribute to the higher susceptibility of cattle to Gram-negative bacterial infections of the udder and lung during periods of stress.     

Canine leishmaniasis transmission: higher infectivity amongst naturally infected dogs to sand flies is associated with lower proportions of T helper cells

The dog is the main reservoir of Leishmania infantum, which is a parasite spread among canine hosts by the bite of sand flies. Phlebotomus perniciosus is the sand fly acting as a major vector in the Mediterranean basin. As a consequence, the dog will suffer from leishmaniasis. In this work the infective capacity of infected dogs, established by direct xenodiagnosis, has been investigated in relation to their immunological status by determining the lymphocyte percentages present in peripheral blood mononuclear cells. We found a significant association between the percentages of T helper cells (CD4/TcRαβ⁺and CD4/CD45RA⁺) and the infection rates detected in the vector, while significant association was not detected in the case of the T cytotoxic cells (CD8/TcRαβ⁺and CD8/CD45RA⁺). The relationship discovered was that the lower the CD4⁺T cell count, the higher the rate of the infection in the vector.     

Factors affecting disease manifestation of toxocarosis in humans: Genetics and environment

Toxocara canis is regarded as the main cause of human toxocarosis but the relative contribution of T. cati is probably underestimated; serological and other diagnostic methods used in most studies of this zoonotic disease do not distinguish between the two parasites. The definitive hosts for T. canis are caniidae. Pups generally have higher infection rates than adult animals and are a major source of eggs in the environment. Humans usually acquire T. canis infection by accidental ingestion of embryonated eggs or encapsulated larvae from the environment or contaminated food, such infections may lead to visceral larva migrans (VLM), ocular larva migrans (OLM) or covert toxocarosis (CT). Although a mixed Th1- and Th2-mediated immunological response, particularly with high levels of IgE and eosinophilia is observed, the underlying mechanisms of molecular and immunopathogenesis for the development of the symptomatic syndromes of VLM, OLM, or of asymptomatic CT are largely unclear. Studies have indicated that immunological defences against various infectious diseases may be highly influenced by complex interactions of environmental and host genetic factors e.g. MHC class I and II, also known as human leucocyte antigen (HLA). Toxocara spp. infections are associated with a polarized CD4⁺ Th2 response with high IgE levels and eosinophilia, mediated mainly by HLA class II molecules. Associations have been made between HLA class II and pathological severity and host genetic effects on exposure to infection. Recent research suggests Foxp3⁺ CD4⁺CD25⁺-expressing T regulatory (Treg) cells play a role in regulation of the immunopathology of granulomas in experimental toxocaral granulomatous hepatitis and in enhanced expression of TGF-β1, which is an important factor for the local survival and function of Treg observed during T. canis invasion in the mouse small intestine, liver, muscle, and brain. Since the potential susceptibility loci HLA class II molecules, are considered involved in the regulation of a Th2-dominant immunity which is highly controlled by Foxp3⁺ CD4⁺CD25⁺ Treg cells by stimulation through TGF-β1, which thus provides a beneficial environment to T. canis larvae but severe injuries to local organs. However, TGF-β1 variant Leu10Pro known to be involved in disease severity warrants further elucidation as this too may have a role in the severity of human toxocarosis. Exploration of TGF-β1 polymorphism, Foxp3⁺ CD4⁺CD25⁺ Treg cells, and MHC polymorphisms may allow insight into the contribution made by environmental and genetic factors in influencing disease syndrome type and severity in humans with toxocarosis.     

The comparative profile of lymphoid cells and the T and B cell spectratype of germ-free piglets infected with viruses SIV,PRRSV or PCV2

Lymphocyte subsets isolated from germ-free piglets experimentally infected with swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) or porcine circovirus type 2 (PCV2) were studied and the profile of these subsets among these three infections was monitored. Germ-free piglets were used since their response could be directly correlated to the viral infection. Because SIV infections are resolved even by colostrum-deprived neonates whereas PRRSV and PCV2 infections are not, SIV was used as a benchmark for an effectively resolved viral infection. PRRSV caused a large increase in the proportion of lymphocytes at the site of infection and rapid differentiation of B cells leading to a high level of Ig-producing cells but a severe reduction in CD2^—CD21⁺ primed B cells. Unlike SIV and PCV2, PRRSV also caused an increase in terminally differentiated subset of CD2⁺CD8α⁺ γδ cells and polyclonal expansion of major Vβ families suggesting that non-specific helper T cells drive swift B cell activation. Distinct from infections with SIV and PRRSV, PCV2 infection led to the: (a) prevalence of MHC-II⁺ T cytotoxic cells, (b) restriction of the T helper compartment in the respiratory tract, (c) generation of a high proportion of FoxP3⁺ T cells in the blood and (d) selective expansion of IgA and IgE suggesting this virus elicits a mucosal immune response. Our findings suggest that PRRSV and PCV2 may negatively modulate the host immune system by different mechanisms which may explain their persistence.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0091-x) contains supplementary material, which is available to authorized users.     

Associations of natural variation in the CD163 and other candidate genes on host response of nursery pigs to porcine reproductive and respiratory syndrome virus infection

Pigs with complete resistance to porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) have been produced by genetically knocking out the CD163 gene that encodes a receptor of the PRRSV for entry into macrophages. The objectives of this study were to evaluate associations of naturally occurring single nucleotide polymorphisms (SNPs) in the CD163 gene and in three other candidate genes (CD169, RGS16, and TRAF1) with host response to PRRSV-only infection and to PRRS vaccination and PRRSV/porcine circovirus 2b (PCV2b) coinfection. SNPs in the CD163 gene were not included on SNP genotyping panels that were used for previous genome-wide association analyses of these data. An additional objective was to identify the potential genetic interaction of variants at these four candidate genes with a mutation in the GBP5 gene that was previously identified to be associated with host response to PRRSV infection. Finally, the association of SNPs with expression level of the nearby gene was tested. Several SNPs in the CD163, CD169, and RGS16 genes were significantly associated with host response under PRRSV-only and/or PRRSV/PCV2b coinfection. The effects of all SNPs that were significant in the PRRSV-only infection trials depend on genetic background. The effects of some SNPs in the CD163, CD169, and RGS16 genes depend on genotype at the putative causative mutation in the GBP5 gene, which indicates a potential biological interaction of these genes with GBP5. In addition, genome-wide association results for the PRRSV-only infection trials revealed that SNPs located in the CDK5RAP2 or MEGF9 genes, near the TRAF1 gene, had suggestive effects on PRRS viral load, which indicates that these SNPs might contribute to PRRSV neuropathogenesis. In conclusion, natural genetic variants in the CD163, CD169, and RGS16 genes are associated with resistance to PRRSV and/or PCV2b infection and appear to interact with the resistance quantitative trait locus in the GBP5 gene. The identified SNPs can be used to select for increased natural resistance to PRRSV and/or PRRSV-PCV2b coinfection.     

The Effect of Experimental Infectious Mastitis on Leukocyte Subpopulations and Cytokine Production in Non-lactating Ewes

The interactions between leukocytes and cytokines during the acute response to intramammary infections in the dry mammary gland of sheep were studied. Dry ewes were experimentally infected in one udder half with either Staphylococcus aureus or Escherichta coli, or infused with saline as control. Udder secretion samples, blood samples and udder tissue samples were collected before and 4, 8 and 24 h after infections/infusions. Total and differential leukocyte counts were calculated in both blood and mammary secretions, and flow cytometry was used to detect the presence of CD4+, CD8+, WC1+, IL-2R+, CD18+ or L-selectin+ lymphocytes, CD18+ or L-selectin + neutrophils, and CD14+ leukocytes. Moreover, the concentrations of interleukin-1β+ (IL-1β), interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) in udder secretions were measured using ELISA, and RT-PCR was used to detect the presence of corresponding cytokine mRNA in udder tissue biopsies. The results suggest an association between the concentrations of IL-1β, IL-8 and the intensity of neutrophil infiltration of the infected gland. Immunologically relevant changes in proportions of lymphocyte subpopulations might also occur in the acute phase of the inflammatory reaction of the udder. Greater cellular and cytokine responses to E. coli infection may have contributed to the milder clinical picture and more rapid resolution of infection than that seen for S. aureus. Enhancing the production of pro-inflammatory cytokines may improve defence against bacterial mastitis.     

Association among filamentous actin content, CD11b expression, and membrane deformability in stimulated and unstimulated bovine neutrophils

OBJECTIVE: To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule-independent sequestration of neutrophils in inflamed lungs of cattle. ANIMALS: Healthy 2- to 4-week-old male Holstein calves. PROCEDURES: Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-alpha (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The Factin content and CD11 b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood. RESULTS: The subpopulation of the smallest-sized neutrophils (>90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11 b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils. CONCLUSIONS AND CLINICAL RELEVANCE: Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells.     

Laminar leukocyte accumulation in horses with carbohydrate overload-induced laminitis

Background: While there is evidence of laminar leukocyte infiltration in black walnut extract (BWE)‐induced laminitis, there is no such evidence for carbohydrate overload (CHO) laminitis. Objective: To assess presence of leukocytes and signs of epidermal stress/injury in the laminar tissue from horses with CHO‐induced laminitis. Animals: Twenty‐four adult horses. Methods: Immunohistochemistry for myeloid cell markers calprotectin (CP) and monocyte‐specific marker (CD163) was performed on laminar sections obtained from 2 groups of horses in the CHO model: the developmental time point (DTP) group (n = 6) and the onset of lameness (LAM) group (n = 6), and a control (CON) group (n = 8). Results: DTP was characterized by an increase in CP⁺ leukocytes (7.8‐fold increase versus CON, P < .001), and LAM time point was characterized by a more marked increase in laminar CP⁺ (108.5‐fold, P < .001) and mild increase in CD163⁺ (1.9‐fold, P= .007) cell counts. Increased CP epidermal signal (indicating epidermal stress or injury) occurred consistently at the LAM time point, although histological evidence of basement membrane (BM) detachment was minor, only being present in 3/6 horses. Conclusions and Clinical Relevance: Maximal laminar leukocyte infiltration and epithelial stress occurred at the onset of lameness in the CHO model showing a different temporal pattern from the BWE model, where maximal leukocyte infiltration clearly precedes epithelial stress. Leukocyte infiltration before major histological changes in the CHO model indicates that leukocyte infiltration can be a cause of and not a reaction to BM degradation and structural failure.     

D-lactic acid interferes with the effects of platelet activating factor on bovine neutrophils

D-lactic acidosis occurs in ruminants, such as cattle, with acute ruminal acidosis caused by ingestion of excessive amounts of highly fermentable carbohydrates. Affected animals show clinical signs similar to those of septic shock, as well as acute laminitis and liver abscesses. It has been proposed that the inflammatory response and susceptibility to infection could both be caused by the inhibition of phagocytic mechanisms. To determine the effects of d-lactic acid on bovine neutrophil functions, we pretreated cells with different concentrations of D-lactic acid and measured intracellular pH using 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and calcium flux using FLUO-3 AM-loaded neutrophils. Reactive oxygen species (ROS) production was measured using a luminol chemiluminescence assay, and MMP-9/gelatinase-B granule release was measured by zymography. CD11b and CD62L/l-selectin expression, changes in cell shape, superoxide anion production, phagocytosis of Escherichia coli-Texas red bioparticles, and apoptosis were all measured using flow cytometry. Our results demonstrated that D-lactic acid reduced ROS production, CD11b upregulation and MMP-9 release in bovine neutrophils treated with 100 nM platelet-activating factor (PAF). D-lactic acid induced MMP-9 release and, at higher concentrations, upregulated CD11b expression, decrease L-selectin expression, and induces late apoptosis. We concluded that D-lactic acid can interfere with neutrophil functions induced by PAF, leading to reduced innate immune responses during bacterial infections. Moreover, the increase of MMP-9 release and CD11b expression induced by 10mM D-lactic acid could promote an nonspecific neutrophil-dependent inflammatory reaction in cattle with acute ruminal acidosis.     

Horses with equine recurrent uveitis have an activated CD4+ T‐cell phenotype that can be modulated by mesenchymal stem cells in vitro

Equine recurrent uveitis (ERU) is an immune‐mediated disease causing repeated or persistent inflammatory episodes which can lead to blindness. Currently, there is no cure for horses with this disease. Mesenchymal stem cells (MSCs) are effective at reducing immune cell activation in vitro in many species, making them a potential therapeutic option for ERU. The objectives of this study were to define the lymphocyte phenotype of horses with ERU and to determine how MSCs alter T‐cell phenotype in vitro. Whole blood was taken from 7 horses with ERU and 10 healthy horses and peripheral blood mononuclear cells were isolated. The markers CD21, CD3, CD4, and CD8 were used to identify lymphocyte subsets while CD25, CD62L, Foxp3, IFNγ, and IL10 were used to identify T‐cell phenotype. Adipose‐derived MSCs were expanded, irradiated (to control proliferation), and incubated with CD4⁺ T‐cells from healthy horses, after which lymphocytes were collected and analyzed via flow cytometry. The percentages of T‐cells and B‐cells in horses with ERU were similar to normal horses. However, CD4⁺ T‐cells from horses with ERU expressed higher amounts of IFNγ indicating a pro‐inflammatory Th1 phenotype. When co‐incubated with MSCs, activated CD4⁺ T‐cells reduced expression of CD25, CD62L, Foxp3, and IFNγ. MSCs had a lesser ability to decrease activation when cell‐cell contact or prostaglandin signaling was blocked. MSCs continue to show promise as a treatment for ERU as they decreased the CD4⁺ T‐cell activation phenotype through a combination of cell‐cell contact and prostaglandin signaling.