Characterization of the Pro-Inflammatory and Pruritogenic Transcriptome in Skin Lesions of the Experimental Canine Atopic Acute IgE-Mediated Late Phase Reactions Model and Correlation to Acute Skin Lesions of Human Atopic Dermatitis.

Intradermal injection of anti-immunoglobulin E (IgE) antibodies in dogs grossly and histologically resemble naturally occurring atopic dermatitis (AD). However, the activated inflammatory and pruritic pathways have not been characterized. The objectives of this study were to characterize the inflammatory transcriptome of experimental acute canine IgE-induced lesions and to determine how these correlate to the transcriptome of naturally occurring human and canine acute atopic dermatitis. Biopsies were collected at 6 and 24 h after intradermal injections of anticanine-IgE antibodies to eight healthy male castrated Beagles; healthy and saline-injected skin served as controls. We extracted total RNA from skin biopsies and analyzed transcriptome using RNA-sequencing. Gene expressions of IgE-induced biopsies were compared to that of controls from the same subject (1.5-fold change, p-adjusted value ≤ 0.05). Acute IgE-mediated lesions had a significant upregulation of pro-inflammatory (e.g., LTB, IL-1B, PTX3, CCL2, IL6, IL8, IL18), T helper-(Th)1/IFNγ signal (e.g., STAT-1, OASL, MX-1, CXCL10, IL-12A) and Th2 (e.g., IL4R, IL5, IL13, IL33 and POSTN) genes, as well as Th2 chemokines (CCL17, CCL24). Pathway analysis revealed strong significant upregulation of JAK-STAT, histamine, IL-4 and IL13 signaling. Spearman correlation coefficient for the shared DEGs between canine anti-canine-IgE and human AD samples revealed a significant moderate positive correlation for anti-canine-IgE 6-h samples (r = 0.53) and 24-h samples (r = 0.47). In conclusion, acute canine IgE-mediated skin lesions exhibit a multipolar immunological axis upregulation (Th1, Th2 and Th17) in healthy dogs, resembling acute spontaneous human AD lesions.

Characterisation of the pruritus responses and pruritic behaviours in an interleukin 31-induced canine model of pruritus.

BACKGROUND: Intravenous administration of interleukin (IL)-31 in healthy dogs has been used as a model to assess antipruritic drugs. However, there is no known in-depth characterisation of pruritic behaviours, and the repeatability of the IL-31-induced pruritus in the individual dogs is currently unknown. OBJECTIVES: To evaluate the immediate/delayed pruritus responses and the pruritic behaviours observed in the IL-31-induced pruritic model in healthy dogs after repeated IL-31 injections. ANIMALS: Fifteen healthy laboratory beagles. METHODS: All dogs were video-recorded for 270 min after two intravenous recombinant IL-31 injections (1.75 µg/kg) and vehicle (phosphate-buffered saline, control) injections, respectively; interventions were randomised and performed with a 2 week wash-out period. Two blinded investigators reviewed the pruritic behaviours of all video recordings. RESULTS: Both canine IL-31 (IL-31_01, IL-31_02) injections significantly increased pruritic seconds and categorical minutes ('YES'/'NO' behaviour per discrete 1 min interval) in healthy dogs compared with both vehicle groups (Vehicle_01, Vehicle_02). The second intravenous canine IL-31 (IL-31_02) administered 14 days after the first IL-31 injection induced a significant increase in pruritic seconds (p = 0.021) and not pruritic categorical minutes (p = 0.231). An increase in pruritic seconds was observed in both IL-31 groups in the first 30 min post-administration, while there was no significant difference between IL-31 and vehicle groups. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, intravenous IL-31 reproducibly induces itch responses in dogs. Future evaluations of the canine IL-31 pruritic model should assess total pruritic behaviours in seconds rather than using a biased 'YES/NO' behaviour per 1 min scoring system.

Establishment of an Intradermal Canine IL-31-Induced Pruritus Model to Evaluate Therapeutic Candidates in Atopic Dermatitis.

Pruritic models in healthy dogs utilizing intravenous administration of interleukin 31 (IL-31) bypass the "natural" itch sensation in AD, which is initiated by pruriceptive primary afferent neurons in the skin. This study aimed to evaluate the immediate/delayed pruritus responses and the pruritic behaviors observed in an intradermal IL-31-induced pruritic model of healthy dogs and the anti-pruritic effect of oclacitinib on said model. In Phase 1, all the dogs were randomized and video-recorded for 300 min after intradermal canine recombinant IL-31 injections (1.75 µg/kg) and vehicle (phosphate-buffered saline) injections. In Phase 2, all the dogs received oral oclacitinib (0.4-0.6 mg/kg, twice daily for 4 consecutive days and once daily on day 5), with the intradermal IL-31 injection performed on day 5. Two blinded investigators reviewed the pruritic behaviors in all the video recordings. Intradermal IL-31 administration to healthy dogs caused a significant increase in the total (p = 0.0052) and local (p = 0.0003) seconds of pruritic behavior compared to the vehicle control. Oral oclacitinib administration significantly reduced the total (p = 0.0011) and local (p = 0.0156) intradermal IL-31-induced pruritic seconds; there was no significant difference in pruritic seconds between the vehicle and oclacitinib within the IL-31 groups. Significant delayed pruritic responses at 150-300 min after IL-31 injections were observed, and intradermal IL-31 failed to induce acute itch (first 30 min). Intradermal injection of IL-31 induces delayed itch responses in dogs that are diminished by the effect of oclacitinib, an oral JAK inhibitor.

Retrospective and prospective assessment of butorphanol, azaperone and medetomidine (BAM™) for immobilisation of feral horses (Equus ferus caballus).

BACKGROUND: Butorphanol-azaperone-medetomidine (BAM™) has not been evaluated in horses. OBJECTIVES: The objective of this study was to evaluate BAM™ for chemical restraint of feral horses. STUDY DESIGN: Retrospective and prospective descriptive studies. METHODS: Data were collected retrospectively from medical records of 28 feral horses immobilised with BAM™ over a 6-year period. Prospectively, 0.0125 mL/kg bwt of BAM™ (butorphanol 27.3 mg/mL, azaperone 9.1 mg/mL and medetomidine 10.9 mg/mL) intramuscularly (im) was administered to eight stallions via dart, and once recumbent, 1.0 mg/kg bwt ketamine was given intravenously (iv). Induction and recovery time and quality via a standardised rubric (1 = very poor; 5 = excellent) and visual analogue scale (VAS), need for additional darts, weight tape measurement and serial physiological parameters were recorded. Serial arterial blood gas analysis was performed during recumbency. Following castration, horses were given 0.1 mg/kg bwt atipamezole (25% iv and 75% im) and allowed to recover unaided. RESULTS: Retrospectively, 28 horses were successfully immobilised with BAM™ without a major complication. Prospectively, eight horses were given a median (range) actual BAMTM dose of 0.0143 (0.0127-0.0510) mL/kg bwt. Three of eight horses needed 1, 2 or 5 additional darts. Median (range) time to recumbency was 11 (2-44) minutes. Median (range) induction (n = 4) and recovery (n = 6) scores via rubric and VAS were 5 (4-5) and 5 (5-5) and 92 (86-93) and 98 (92-99) cm, respectively. Four of seven horses were hypoxaemic at ≥1 time point with otherwise acceptable physiological parameters. Following atipamezole, median (range) time to sternal recumbency and standing was 12 (2-18) and 17 (11-52) minutes, respectively (n = 6). MAIN LIMITATIONS: The sample size was small. Data could not be collected before darting or after recovery. Some data were missing from retrospective analysis. CONCLUSIONS: Intramuscular BAM™ with iv ketamine provided chemical restraint suitable for field castration of feral horses with no mortality. Hypoxaemia occurred in the majority of horses.

Modified-Live Feline Calicivirus Vaccination Elicits Cellular Immunity against a Current Feline Calicivirus Field Strain in an Experimental Feline Challenge Study.

Feline calicivirus (FCV) is a common cat virus associated with oral ulcerations and virulent-systemic disease. Efficacious FCV vaccines protect against severe disease but not against infection. The high genetic diversity of FCV poses a challenge in vaccine design. Protection against FCV has been related to humoral and cellular immunity; the latter has not been studied in detail. This study investigates the cellular and humoral immune response of specified pathogen-free (SPF) cats after modified-live FCV F9 vaccinations and two heterologous FCV challenges by the analysis of lymphocyte subsets, cytokine mRNA transcription levels, interferon (IFN)-γ release assays in peripheral blood mononuclear cells (PBMCs), anti-FCV antibodies, and neutralisation activity. Vaccinated cats developed a Th1 cytokine response after vaccination. Vaccination resulted in antibodies with neutralising activity against the vaccine but not the challenge viruses. Remarkably, IFN-γ-releasing PBMCs were detected in vaccinated cats upon stimulation with the vaccine strain and the first heterologous FCV challenge strain. After the first experimental infection, the mRNA transcription levels of perforin, granzyme B, INF-γ, and antiviral factor MX1 and the number of IFN-γ-releasing PBMCs when stimulated with the first challenge virus were higher in vaccinated cats compared to control cats. The first FCV challenge induced crossneutralising antibodies in all cats against the second challenge virus. Before the second challenge, vaccinated cats had a higher number of IFN-γ-releasing PBMCs when stimulated with the second challenge virus than control cats. After the second FCV challenge, there were less significant differences detected between the groups regarding lymphocyte subsets and cytokine mRNA transcription levels. In conclusion, modified-live FCV vaccination induced cellular but not humoral crossimmunity in SPF cats; innate immune mechanisms, secretory and membranolytic pathways, and IFN-γ-releasing PBMCs seem to be important in the host immune defence against FCV.

The Local and Systemic Humoral Immune Response Against Homologous and Heterologous Strains of the Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

The humoral immune response plays a crucial role in the combat and protection against many pathogens including the economically most important, highly prevalent, and diverse pig pathogen PRRSV - the Porcine Reproductive and Respiratory Syndrome Virus. In addition to viremia and viral shedding analyses, this study followed the local and systemic humoral immune response of pigs for 63 days upon inoculation with one of three types of Type-2 PRRSV (PRRSV-2) strains - one modified live virus (MLV) vaccine strain, and two lineage 1 PRRSV-2 strains, NC134 and NC174. The local response was analyzed by quantifying immunoglobulin (Ig)A in nasal swabs. The systemic response was studied by the quantification of IgG with ELISA and homo- and heterologous neutralizing antibodies (NAs) utilizing a novel method of flow cytometry. In all PRRSV-2 inoculated groups, viral nasal shedding started at 3 dpi, peaked between 3 and 7 days post inoculation, and was cleared at 28-35 dpi with sporadic rebounds thereafter. The local IgA response started 4-7 days after viral shedding occurred and showed a bi-phasic course with peaks at 14 dpi and at 28-35 dpi. Of note, the NC134 and NC174 strains induced a much stronger local IgA response. As reported earlier, main viremia lasted from 7 dpi to 28 dpi (NC174), 42 dpi (NC134) or until the end of the study (MLV). Similar to the local IgA response, the systemic IgG response started 4-7 days after viremia; but in contrast to viremia, serum IgG levels stayed high for all PRRSV-2 inoculated groups until the end of the study. A significant finding was that while the serum NA response in the MLV group was delayed by 28 days, serum NAs in pigs infected with our two NC134 and NC174 strains could be detected as early as 7 dpi (NC134) and 14 dpi (NC174). Compared to homologous NA responses, the NA responses against heterologous strains was strong but slightly delayed between our lineage 1 one strains or non-existent between the MLV and lineage 1 strains. This study improves our understanding of the relationship between local and systemic infections and the humoral immune response induced by PRRSV-2 infection or MLV vaccination. Our data also provide novel insights into the timeline of the development of homologous and heterologous NA levels - by both MLV vaccination or infection with two strains from the currently prevalent PRRSV-2 lineage 1.

Improved neurocognitive performance in FIV infected cats following treatment with the p75 neurotrophin receptor ligand LM11A-31.

HIV rapidly infects the central nervous system (CNS) and establishes a persistent viral reservoir within microglia, perivascular macrophages and astrocytes. Inefficient control of CNS viral replication by antiretroviral therapy results in chronic inflammation and progressive cognitive decline in up to 50% of infected individuals with no effective treatment options. Neurotrophin based therapies have excellent potential to stabilize and repair the nervous system. A novel non-peptide ligand, LM11A-31, that targets the p75 neurotrophin receptor (p75NTR) has been identified as a small bioavailable molecule capable of strong neuroprotection with minimal side effects. To evaluate the neuroprotective effects of LM11A-31 in a natural infection model, we treated cats chronically infected with feline immunodeficiency virus (FIV) with 13 mg/kg LM11A-31 twice daily over a period of 10 weeks and assessed effects on cognitive functions, open field behaviors, activity, sensory thresholds, plasma FIV, cerebrospinal fluid (CSF) FIV, peripheral blood mononuclear cell provirus, CD4 and CD8 cell counts and general physiology. Between 12 and 18 months post-inoculation, cats began to show signs of neural dysfunction in T maze testing and novel object recognition, which were prevented by LM11A-31 treatment. Anxiety-like behavior was reduced in the open field and no changes were seen in sensory thresholds. Systemic FIV titers were unaffected but treated cats exhibited a log drop in CSF FIV titers. No significant adverse effects were observed under all conditions. The data indicate that LM11A-31 is likely to be a potent adjunctive treatment for the control of neurodegeneration in HIV infected individuals.

Detection of circulating anti-retinal antibodies in dogs with sudden acquired retinal degeneration syndrome using indirect immunofluorescence: A case-control study.

Sudden acquired retinal degeneration syndrome (SARDS) in dogs is proposed to have an immune-mediated etiology. However, there is conflicting evidence regarding the presence of antiretinal antibodies, as assessed by western blotting, in the serum of SARDS patients. Because of the possibility that antibodies recognize only conformational epitopes, we hypothesized that a more sensitive method to investigate circulating retinal autoantibodies in SARDS is immunofluorescence. Sera from 14 dogs with early SARDS, and 14 age- and breed-matched healthy control dogs were screened for circulating antiretinal IgG, IgM, IgE and IgA using indirect immunofluorescence on lightly fixed frozen sections of normal canine retina. Controls without canine serum were also performed. A nuclear counterstain was used to identify cellular retinal layers. Images were obtained using a fluorescence microscope, and 2-3 separate masked observers graded retinal layers for fluorescence staining intensity using a 0-3 scale. Total circulating IgG and IgM was assessed by radial immunodiffusion. Statistical analysis was performed using 2-way ANOVA, paired 2-tailed student's t-test and correlation analysis. Intensity of IgG staining of photoreceptor outer segments was significantly higher using serum from dogs with SARDS compared with healthy controls in 2/3 observers (P < 0.05). Intensity of IgM staining throughout the retina was higher in SARDS dogs compared to matched healthy controls (P < 0.0001), although no specific retinal layer was statistically significant. There were no differences in staining intensity for IgE or IgA. Dogs with SARDS had a comparably lower circulating IgG and higher IgM than healthy controls (P = 0.01 and 0.001 respectively) and IgG and IgM were negatively correlated (r = -0.69, P = 0.007). Despite having decreased serum IgG compared with healthy controls, circulating IgG in dogs with SARDS binds photoreceptor outer segments to a greater extent. Dogs with SARDS have a relatively higher circulating IgM than matched healthy controls. The pathogenic nature of these antibodies is unknown.

Immune dysregulation and osteosarcoma: Staphylococcus aureus downregulates TGF-ß and heightens the inflammatory signature in human and canine macrophages suppressed by osteosarcoma.

Since William Coley utilized bacterial immunotherapy to treat sarcomas in the late 19th century, an association between infection and improved survival has been reported for human and canine osteosarcoma patients. One of the reasons for this improved survival is likely a reactivation of the host immune system towards an inflammatory anti-tumour response, and one of the key players is the macrophage. Yet, despite their importance, the response of macrophages to infectious agents in the context of osteosarcoma has not been thoroughly evaluated. The aim of this study was to evaluate how in vitro exposure to a bacterial agent (Staphylococcus aureus) influenced canine and human macrophage differentiation in the presence of osteosarcoma. Our hypothesis was that S. aureus would, in the presence of osteosarcoma, induce a macrophage phenotype with significantly increased inflammatory signatures. Consistent with our hypothesis, human macrophages co-cultured with osteosarcoma and S. aureus exhibited increased IFN-γ, TNF-α and IL-12p70 cytokine secretion, decreased TGF-ß cytokine secretion and increased mRNA expression of TNF-α when compared with macrophages co-cultured with osteosarcoma and to macrophages cultured alone. Canine macrophages similarly exhibited increased IFN-γ and TNF-α cytokine secretion, decreased TGF-ß cytokine secretion, increased mRNA expression of TNF-α and increased surface receptor expression of CD80 when co-cultured with osteosarcoma and S. aureus. Collectively, the findings of this study suggest that infection upregulates the inflammatory immune response to counteract osteosarcoma-induced immune suppression. This work informs a potential therapeutic strategy to optimize inflammatory stimuli for triggering an anti-osteosarcoma macrophage response.

The T-Cell Response to Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause severe reproductive and respiratory pathologies resulting in immense monetary and welfare costs for the swine industry. The vaccines against PRRSV are available; but they struggle with providing protection against the plethora of heterologous PRRSV strains. To improve PRRSV vaccine development, the aim of this study was to provide an in-depth analysis of the crucial heterologous T-cell response to type-2 PRRSV. Following PRRSV modified live virus (MLV) vaccination or infection using one high- or one low-pathogenic PRRSV-strain, this nine-week study evaluated the T-cell response to different PRRSV strains. Our results demonstrate an important role for T cells in this homo- and heterologous response. Specifically, the T-helper cells were the main responders during viremia. Their peak response at 28 dpi correlated with a reduction in viremia, and their homing receptor expression indicated the additional importance for the anti-PRRSV response in the lymphatic and lung tissue. The cytocoxic T lymphocyte (CTL) response was the strongest at the site of infection-the lung and bronchoalveolar lavage. The TCR-γδ T cells were the main responders post viremia and PRRSV induced their expression of the lymph node homing the chemokine receptor, CCR7: This indicates a crucial role for TCR-γδ T cells in the anti-PRRSV response in the lymphatic system.

Antibiotic Therapy Does Not Alter the Humoral Response to Vaccination for Porcine Circovirus 2 in Weaned Pigs.

Recent reports suggest that antibiotic therapy may either reduce or enhance the immune response to various porcine vaccines. Based upon these findings, we asked if antibiotic therapy alters immune cell populations, as measured by flow cytometry and/or vaccine-specific humoral immunity, as measured by sample to positive (S/P) antibody ratios. Here, we investigated the immuno-modulatory effects of enrofloxacin, ceftiofur, and tulathromycin on the immune response to a Mycoplasma hyopneumoniae (M. hyopneumoniae) and porcine circovirus type 2 (PCV-2) combination vaccine in weaned pigs. Maternal antibody likely interfered with the induction of immunity to M. hyopneumoniae. Antibiotic administration did not affect immune cell populations, as assessed by flow cytometry and did not affect the induction of humoral immunity to PCV-2.

ACVIM consensus statement on the diagnosis of immune-mediated hemolytic anemia in dogs and cats.

Immune-mediated hemolytic anemia (IMHA) is an important cause of morbidity and mortality in dogs. IMHA also occurs in cats, although less commonly. IMHA is considered secondary when it can be attributed to an underlying disease, and as primary (idiopathic) if no cause is found. Eliminating diseases that cause IMHA may attenuate or stop immune-mediated erythrocyte destruction, and adverse consequences of long-term immunosuppressive treatment can be avoided. Infections, cancer, drugs, vaccines, and inflammatory processes may be underlying causes of IMHA. Evidence for these comorbidities has not been systematically evaluated, rendering evidence-based decisions difficult. We identified and extracted data from studies published in the veterinary literature and developed a novel tool for evaluation of evidence quality, using it to assess study design, diagnostic criteria for IMHA, comorbidities, and causality. Succinct evidence summary statements were written, along with screening recommendations. Statements were refined by conducting 3 iterations of Delphi review with panel and task force members. Commentary was solicited from several professional bodies to maximize clinical applicability before the recommendations were submitted. The resulting document is intended to provide clinical guidelines for diagnosis of, and underlying disease screening for, IMHA in dogs and cats. These should be implemented with consideration of animal, owner, and geographical factors.

Assessment of a novel nanoparticle hyperthermia therapy in a murine model of osteosarcoma.

OBJECTIVE: To evaluate the effects of nanoparticle hyperthermia therapy on monocyte function and tumor-derived factors associated with macrophage polarization in a murine osteosarcoma model. STUDY DESIGN: Experimental study. ANIMALS: Female C3H mice. METHODS: Peripheral blood monocyte cell surface phenotype, monocyte chemotaxis, tumor messenger RNA expression, and survival were compared among osteosarcoma (OS)-bearing mice treated with nanoparticle hyperthermia therapy, OS-bearing mice with osteomyelitis, OS-bearing mice, vehicle control mice, and normal control mice. RESULTS: OS-bearing mice with osteomyelitis had a higher proportion of "nonclassical" monocytes (Ly6Clo ) compared with all other experimental groups. There were alterations in monocyte expression of multiple chemokine receptors among experimental groups including CXCR2, CCR2, and CXCR4. Monocytes from OS-bearing mice treated with hyperthermia therapy exhibited greater chemotaxis compared with monocytes from OS-bearing mice with osteomyelitis. CONCLUSION: OS likely induced alterations in monocyte phenotype and function. Nanoparticle hyperthermia therapy increased in vitro monocyte chemotaxis. CLINICAL IMPACT: Enhancing monocyte/macrophage function in dogs with OS may enhance antitumor immunity.

T Regulatory Cell Induced Foxp3 Binds the IL2, IFNγ, and TNFα Promoters in Virus-Specific CD8+ T Cells from Feline Immunodeficiency Virus Infected Cats.

Polyfunctional CD8+ T cells play a critical role in controlling viremia during AIDS lentiviral infections. However, for most HIV-infected individuals, virus-specific CD8+ T cells exhibit loss of polyfunctionality, including loss of IL2, TNFα, and IFNγ. Using the feline immunodeficiency virus (FIV) model for AIDS lentiviral persistence, our laboratory has demonstrated that FIV-activated Treg cells target CD8+ T cells, leading to a reduction in IL2 and IFNγ production. Furthermore, we have demonstrated that Treg cells induce expression of the repressive transcription factor, Foxp3, in CD8+ T cells. Based upon these findings, we asked if Treg-induced Foxp3 could bind to the IL2, TNFα, and IFNγ promoter regions in virus-specific CD8+ T cells. Following coculture with autologous Treg cells, we demonstrated decreased mRNA levels of IL2 and IFNγ at weeks 4 and 8 postinfection and decreased TNFα at week 4 postinfection in virus-specific CD8+ T cells. We also clearly demonstrated Treg cell-induced Foxp3 expression in virus-specific CD8+ T cells at weeks 1, 4, and 8 postinfection. Finally, we documented Foxp3 binding to the IL2, TNFα, and IFNγ promoters at 8 weeks and 6 months postinfection in virus-specific CD8+ T cells following Treg cell coculture. In summary, the results here clearly demonstrate that Foxp3 inhibits IL2, TNFα, and IFNγ transcription by binding to their promoter regions in lentivirus-specific CD8+ T cells. We believe this is the first description of this process during the course of AIDS lentiviral infection.

Micro-RNA 10a Is Increased in Feline T Regulatory Cells and Increases Foxp3 Protein Expression Following In Vitro Transfection.

CD4⁺CD25⁺Foxp3⁺ T regulatory (Treg) cells are activated during the course of lentiviral infection and exhibit heightened suppressor function when compared to Treg cells from uninfected controls. Foxp3 is essential to Treg cell function and multiple studies have documented that lentivirus-activated Treg cells exhibit heightened Foxp3 expression when compared to Treg cells from uninfected controls. Our hypothesis was that lentivirus-induced micro-RNAs (miRNAs) contribute to heightened Treg cell suppressor function by stabilizing Foxp3 expression. We demonstrated that CD4⁺CD25⁺ T cells from both feline immunodeficiency virus infected (FIV⁺) cats and uninfected control cats exhibit increased miRNA 10a and 21 levels compared to autologous CD4⁺CD25- T cells but there was no difference in the levels of these miRNAs when Treg cells from FIV⁺ cats were compared to Treg cells from uninfected controls. Further, there was no increase in Foxp3 mRNA following transfection of miRNA 10a or 21 into a feline cell line. However, transfection with miRNA 10a resulted in increased Foxp3 protein expression.

Extreme lymphocytosis with myelomonocytic morphology in a horse with diffuse large B-cell lymphoma.

An 11-year-old, 443-kg Haflinger mare was presented to the North Carolina State University Veterinary Teaching Hospital with a 2-week history of lethargy and a 3-day duration of anorexia, pyrexia, tachycardia, and ventral edema. Severe pitting edema, peripheral lymphadenopathy, and a caudal abdominal mass were noted on physical examination. An extreme leukocytosis (154.3 × 103 /µL) and microscopic hematologic findings suggestive of myelomonocytic leukemia were observed. Serum protein electrophoresis revealed a monoclonal gammopathy and urine protein electrophoresis revealed a monoclonal light chain proteinuria. Necropsy and histopathology confirmed widespread neoplastic infiltration in many organs with a heterogenous population of cells; there was no apparent evidence of bone marrow involvement. Immunohistochemistry confirmed presence of a majority of B cells with a limited antigen expression, admixed with a lower number of T cells. Molecular clonality analysis of IgH2, IgH3, and kappa-deleting element (KDE, B cell) on whole blood and KDE on infiltrated tissues revealed clonal rearrangements, and the KDE intron clones that amplified in blood and in infiltrated tissue were identical. In contrast, the clonality analysis of T-cell receptor γ revealed no clonality on blood cells and infiltrated tissues. In conjunction with the histopathologic changes, the lesion was interpreted to be composed of neoplastic B cells with a reactive T-cell population. Polymerase chain reaction testing for equine herpes virus 5 was negative. The final diagnosis was diffuse large B-cell lymphoma with a marked hematogenous component.

Efficacy of various Marek's disease vaccines protocols for prevention of Marek's disease virus-induced immunosuppression.

Marek's disease virus (MDV) induces tumors and severe immunosuppression in chickens. MDV-induced immunosuppression (MDV-IS) is very complex and difficult to study. In particular, the late MDV-IS (late-MDV-IS) is of great concern since it can occur in the absence of lymphoid organ atrophy or gross tumors. We have recently developed a model to reproduce late-MDV-IS under laboratory conditions. This model measures MDV-IS indirectly by assessing the effect of MDV infection on the efficacy of infectious laryngotracheitis (ILT) vaccination; hence the name late-MDV-IS ILT model. In this study, we have used the late-MDV-IS ILT model to evaluate if MD vaccination can protect against late-MDV-IS. One experiment was conducted to determine whether serotype 1 MD vaccines (CVI988 and Md5ΔMEQ) could induce late-MDV-IS by themselves. Three additional experiments were conducted to evaluate efficacy of different MD vaccines (HVT, HVT+SB-1, CVI988, and Md5ΔMEQ) and different vaccine protocols (day-old vaccination, in ovo vaccination, and double vaccination) against late-MDV-IS. Our results show that none of the currently used vaccine protocols (HVT, HVT+SB-1, or CVI988 administered at day of age, in ovo, or in double vaccination protocols) protected against late-MDV-IS induced by vv+MDV strains 648A and 686. Experimental vaccine Md5ΔMEQ administered subcutaneously at one day of age was the only vaccine protocol that significantly reduced late-MDV-IS induced by vv+MDV strain 686. This study demonstrates that currently used vaccine protocols confer high levels of protection against MDV-induced tumors (protection index=100), but do not protect against late-MDV-IS; thus, commercial poultry flocks could suffer late-MDV-IS even in complete absence of tumors. Our results suggest that MDV-IS might not be related to the development of tumors and novel control methods are needed. Further evaluation of the experimental vaccine Md5ΔMEQ might shed light on protective mechanisms against late-MDV-IS.

Modulating DNA methylation in activated CD8+ T cells inhibits regulatory T cell-induced binding of Foxp3 to the CD8+ T Cell IL-2 promoter.

We have previously demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) activated during the course of feline immunodeficiency virus (FIV) infection suppress CD8(+) CTL function in a TGF-ß-dependent fashion, inhibiting IFN-γ and IL-2 production and inducing G1 cell-cycle arrest. In this article, we describe the molecular events occurring at the IL-2 promoter leading to suppression of IL-2 production. These experiments demonstrate that Foxp3 induced by lentivirus-activated Tregs in the CD8(+) target cells binds to the IL-2 promoter, actively repressing IL-2 transcription. We further demonstrate that the chronic activation of CD8(+) T cells during FIV infection results in chromatin remodeling at the IL-2 promoter, specifically, demethylation of CpG residues. These DNA modifications occur during active transcription and translation of IL-2; however, these changes render the IL-2 promoter permissive to Foxp3-induced transcriptional repression. These data help explain, in part, the seemingly paradoxical observations that CD8(+) T cells displaying an activation phenotype exhibit altered antiviral function. Further, we demonstrate that blocking demethylation of CpG residues at the IL-2 promoter inhibits Foxp3 binding, suggesting a potential mechanism for rescue and/or reactivation of CD8(+) T cells. Using the FIV model for lentiviral persistence, these studies provide a framework for understanding how immune activation combined with Treg-mediated suppression may affect CD8(+) T cell IL-2 transcription, maturation, and antiviral function.

CD4+CD25+ T regulatory cells activated during feline immunodeficiency virus infection convert T helper cells into functional suppressors through a membrane-bound TGFß / GARP-mediated mechanism.

BACKGROUND: We and others have previously reported that cell membrane-bound TGFß (mTGFß) on activated T regulatory (Treg) cells mediates suppressor function. Current findings suggest that a novel protein known as Glycoprotein A Repetitions Predominant (GARP) anchors mTGFß to the Treg cell surface and facilitates suppressor activity. Recently, we have described that GARP+TGFß+ Treg cells expand during the course of FIV infection. Because Treg cells are anergic and generally exhibit poor proliferative ability, we asked how Treg homeostasis is maintained during the course of feline immunodeficiency virus (FIV) infection. RESULTS: Here, we report that Treg cells from FIV+ cats express GARP and mTGFß and convert T helper (Th) cells into phenotypic and functional Treg cells. Th to Treg conversion was abrogated by anti-TGFß or anti-GARP treatment of Treg cells or by anti-TGFßRII treatment of Th cells, suggesting that Treg cell recruitment from the Th pool is mediated by TGFß/TGFßRII signaling and that cell-surface GARP plays a major role in this process. CONCLUSIONS: These findings suggest Th to Treg conversion may initiate a cascade of events that contributes to the maintenance of virus reservoirs, progressive Th cell immunosuppression, and the development of immunodeficiency, all of which are central to the pathogenesis of AIDS lentivirus infections.

Infection with feline immunodeficiency virus directly activates CD4+ CD25+ T regulatory cells.

Lentivirus infection activates CD4(+) CD25(+) T regulatory (Treg) cells. Activation of Treg cells may be due to direct virus infection or chronic antigenic stimulation. Herein we demonstrate that in vitro feline immunodeficiency virus (FIV) infection, but not UV-inactivated virus, activates Treg cells as measured by immunosuppressive function and upregulation of GARP, FoxP3, and membrane-bound transforming growth factor ß (TGF-ß). These data demonstrate for the first time that AIDS lentiviruses infect and activate Treg cells, potentially contributing to immune dysfunction.