Canine Health Foundation

AMERICAN KENNEL CLUB

LAY ABSTRACTS

from the

INTERNATIONAL CANINE IMMUNOGENETICS AND

IMMUNOLOGIC DISEASES CONFERENCE

July 31, 1998 - August 2, 1998

University of California, Davis

 

Table of Contents

Click to go to Abstract

 

 

CANINE REACTIVE LANGERHANS CELL HISTIOCYTOSIS: CUTANEOUS AND SYSTEMIC FORM

VK Affolter, AG Cannon, PF Moore

School of Veterinary Medicine,

Department of Pathology, Microbiology, Immunology

UC Davis

Canine histiocytic proliferative diseases manifest themselves either as neoplastic diseases, such as cutaneous histiocytoma and localized and disseminated histiocytic sarcoma (also called malignant histiocytosis), or as reactive diseases, such as cutaneous and systemic histiocytosis. The histiocytic proliferative diseases in the dog are a very frustrating group of diseases, because their etiology and pathogenesis is not known and the clinical presentation and histopathologic features can mimic a variety of other inflammatory processes as well as lymphocytic neoplastic processes. Cutaneous and systemic histiocytosis both present with nodular lesions in the skin and subcutis, but the systemic histiocytosis also affects other organ systems. Cutaneous histiocytosis is seen throughout the entire dog population. Systemic histiocytosis, although observed in a variety of breeds, is more often recognized in certain breeds, including the Bernese Mountain Dog, Rottweilers, Golden Retrievers and Labrador Retrievers. Cutaneous and systemic histiocytosis clinically wax and wane and they respond to immuno-suppressive therapy. The goal of our study was to identify the inflammatory cells involved in cutaneous and systemic histiocytosis based on the expression of molecules on their cell surface. A panel of monoclonal antibodies were used to evaluate the phenotype (characterized by the expression of cell surface molecule) of the lesional inflammatory cells. Characterization of the cells offers new insights in possible etiology and mechanisms involved in the process of reactive histiocytosis in the dog. The lesions were characterized by a mixture of cells. The cellular infiltrate consisted of mainly dendritic antigen presenting cells (dAPC) and lymphocytes which accumulated around vessels and eventually also invaded the vascular walls. The dAPC in canine cutaneous and systemic histiocytosis coexpress some molecules that indicate their activation and their origin from dermal dendritic cells. Accumulations and proliferations of dAPC in humans are categorized as Langerhans cell histiocytosis (LCH). Hence, cutaneous and systemic histiocytosis can be categorized as two different forms of a reactive LCH in the dog. Dendritic APC play a key role in the development of an immune response because they present antigens, such as material from infectious agents, to the lymphocytes. The interaction of the dAPC with the lymphocytes triggers the activation of the lymphocytes and therefore the induction of the immune response. An accumulation and retention of dAPC and lymphocytes suggests a persistent stimulation of the immune system. However, cultures and special stains did not reveal any indication for the presence of an infectious agent. In absence of an infectious agent and based on the response to immuno-suppressive therapy reactive canine LCH most likely represents an immune-dysregulatory process. Current studies axe focusing on evaluating several factors possibly involved in the immune-dysregulatory process of canine reactive LCH. Canine LCH, including the canine cutaneous histiocytoma and the reactive LCH are frequently seen in the dog. Better characterization of the disease and evaluation of a variety of factors possibly involved in the development of LCH in dogs will provide the foundation for a better understanding of these diseases. And it is only a clear understanding of the disease process which will open up new possibilities for more specific and successful treatments.

CHARACTERISATION OF THREE DOG CELL LINES.

Stuart Carter*, Annette Barnes, Sue Bell, Alix Bee, Anne Mee & Chris

Thompson. Connective Tissue Research Group, Faculty of Veterinary Science,

University of Liverpool, UK.

For an adequate understanding of how the dog fights off infections, it is necessary to elucidate the mechanisms of immunity which confer such protection. In man, much of this information has come from studies of what we in the research trade call cell lines. These are continually growing cells, usually taken from patients with various forms of cancer and these are able to continually multiply because they have arrested normal development. This failure to develop means that they cannot age and so become immortal and are self replicating in the laboratory. In each case, the cells which are growing are identical and at a particular stage of development. The availability of these cell lines allows the biologist a unique means of studying how the immune system develops and is a potential source of important reagents for disease studies.

There are very few canine cell lines available and we have studied the characteristics of three which may have considerable use for investigations of dog immunity. These are identified as Kl, K6 and DH 82 and have different malignant origins. We have analysed their cell surface markers, their cytokine production and their secretion of inflammatory mediators. The results show that that they are a rich source of canine materials which will be increasingly useful for studies of how immune dysfunction leads to disease processes.

Acknowledgement. This work is supported by the Wellcome Trust, Petplan Ltd and the Guide Dogs for the Blind Association.

AUTOIMMUNITY, CYTOKINES AND MATRIX METALLOPROTEINASES
IN CANINE RHEUMATOID ARTHRITIS.

Stuart Carter*, Annette Barnes, Sue Bell, Alix Bee, Anne Mee, Chris

Thompson, Andy Coughlan & Peter Clegg. Connective Tissue Research Group,

Faculty of Veterinary Science, University of Liverpool, UK.

Canine rheumatoid arthritis is an autoimmune disease in which the dog's immune system turns against its own tissues, particularly those of the joints, to produce crippling and painful symptoms. Our research into understanding how this process occurs, show that there is an effect on the system of cellular messengers, the cytokines, which in healthy dogs are primarily responsible for repair and normal tissue turnover. In autoimmune diseases such as rheumatoid arthritis, the normal "helpful" role of these molecules changes as control of their activity goes awry.

This imbalance of cytokine activity is reflected in severe inflammatory changes and destruction of cartilage and bone in diseased joints. Samples from arthritic joints show an upregulation of inflammatory cell signals, including certain cytokines and this is seen most markedly in those cases where there is an increase of tissue destroying enzymes (matrix metalloproteinases or MMPs). Furthermore, our work shows that the MMPs are produced early in disease, even in non-autoimmune joint disease and are reduced when successful anti-inflammatory therapy is employed. This data suggests that either the inflammatory cytokines or the MMPs themselves are potential viable targets for pharmacological intervention. Our research group is now working to understand how the cytokine network interacts with the MMPs and is using specific inhibitors to attempt to reduce the inflammatory process and destruction of joint tissues. The outcome of this work will have implications for many diseases where the immune system is responsible for tissue damage.

Acknowledgement. This work is supported by the Wellcome Trust and the Guide Dogs for the Blind Association.

ANTIGEN SPECIFICITY IN CANINE AUTOIMMUNE HAEMOLYTIC

ANAEMIA

M.J. Day

Department of Pathology and Microbiology, University of Bristol,

Langford BS40 SDU, United Kingdom

Autoirnmune haemolytic anaemia (AIHA) is the most commonly diagnosed autoimmune disease in the dog and is a particular problem in breeds such as the cocker spaniel, old English sheepdog and poodle. Clinical episodes of the disease may be precipitated by stress factors such as oestrous or whelping and recent work has suggested that a proportion of cases of AIHA may be triggered by vaccination. There is a spectrum of clinical presentation that ranges from slow onset (days to weeks) of anaemia, to an acute onset (one to two days) of severe haemolytic anaemia. Canine ARIA is generally amenable to supportive and immunosuppressive therapy, however relapse is common. AIHA generally involves destruction of autoantibody-coated red blood cells (RBC) in spleen or liver, or within the circulating blood. The formation of autoantibodies occurs in individuals in which there is some disturbance of the normal control of the immune system.

Canine AIHA has been studied at the University of Bristol over the past decade. Our research has focused upon defining the specific components of RBC to which this abnormal autoimmune response is directed (i.e. the target `autoantigens'). We have also examined the different components of the immune system that are responsible for AIHA; specifically the types of autoantibodies that bind to affected RBC, and the population of regulatory cells (T lymphocytes) that control the production of such autoantibodies. These studies have shown marked similarities between AIHA in humans, dogs and some experimental models of the disease. The target RBC autoantigens are similar, and in each species there is inappropriate activation of the regulatory T lymphocytes that normally lie dormant in healthy individuals. Such knowledge is fundamental to the development of new methods (immunotherapy) for the treatment of autoimmune disorders such as AIHA Clinical trials of different forms of immunotherapy are currently being undertaken in humans, and the fixture development of such approaches for veterinary medicine is predicted.

CHARACTERIZATION OF CYTOKINE GENE TRANSCRIPTION

IN THE SKIN OF DOGS WITH ATOPIC DERMATITIS

Gregg A. Dean, Assistant Professor

Department of Microbiology, Pathology, & Parasitology

College of Veterinary Medicine

North Carolina State University

Atopic dermatitis (AD) is a chronically-relapsing pruritic dermatitis of young adult dogs that is heritable in some breeds. At the North Carolina State University, College of Veterinary Medicine, canine AD is one the top three reasons dogs are presented to the dermatology service. Canine and human AD hold in common i) the presence of an heritable background, ii) the existence of antibodies directed toward environmental allergens including Dermatophagoides house dust mite antigens, iii) the propensity to develop bacterial skin infections, and iv) the occurrence of immune suppression restricted to the skin. Dogs with AD have a reduced quality of life due to persistent skin irritation and secondary skin infections. Current therapy is primarily symptomatic because we lack an understanding of the underlying disease mechanisms. The objectives of our studies are to understand the immune mechanism involved in AD and develop a study system to test therapeutic agents targeting the cause of the disease. In this study we developed a method to evaluate soluble mediators of the immune system called cytokines. It is suspected that cytokines play a key role in AD. We determined the types of immune cells involved in AD lesions and the specific types of cytokines present. With this information we are able to develop a hypothesis on disease mechanism of AD. We have identified some specific points in the progression of disease that may be therapeutic targets and have a preliminary system to test appropriate therapeutic agents. In future studies we will refine our understanding o AD and our model for testing drugs. The goal is to initiate therapeutic trials within one year that may prove more efficacious in curing AD rather than just treating symptoms.

CANINE X-LINKED SEVERE COMBINED IMMUNODEFICIENCY

Peter J. Felsburgl, Brian J. Hartnett1, Paula S. Henthornl, Peter F. Moore2,

Steven Krakowka3, and Hans D. Ochs4

1Department of Clinical Studies - Philadelphia, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA.

2Department of Veterinary Medicine, School of Veterinary Medicine, University of California, Davis, CA

3Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH.

4Department of Pediatrics, School of Medicine, University of Washington, Seattle, WA.

A common finding in breeding of dogs are puppies that fail to thrive ("runt of the litter") and often die of infections. In humans, infants with similar conditions often have a primary immunodeficiency. These are defects of the immune system that have a genetic basis. The question is how many of these puppies may also have a primary immunodeficiency.

This study documented the first primary immunodeficiency in the dog with the affected puppies having the typical features of "runts of the litter." The results of the study documented the immunologic defects in the affected puppies thereby determining which clinical immunologic tests can be employed to diagnose other puppies with the disease. We have also determined the genetic defect that causes this disease which has provided a method for diagnosing female earners of the disease. This is an X-linked disease which means that only males get the disease, but females are the earners of the disease. This is impotent since female carriers are clinically and immunologically normal, but half of their female puppies will be carriers and perpetuate the disease, and half their male puppies will be affected and die. The ability to identify female carriers will help eliminate the disease from the breeding population.

Similar clinical immunologic techniques can be used to identify other primary immunodeficiencies in the dog. Once they are identified, the genes responsible for the disease may be determined and genetic tests developed for earner detection.

DETERMINATION OF THE FUNCTIONAL IMPORTANCE OF THE

COMMON GAMMA CHAIN ((c) DURING T CELL DEVELOPMENT

Terry A. Gouthro, Peter J. Felsburg, and Paula S. Henthorn University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA.

X-linked severe combined immunodeficiency (XSCID) is characterized by abnormalities in the development of several lymphoid cell types including T cells. XSCID results from mutations in the gene coding for the common gamma chain ((c). The role of (c as a subunit of multiple cytokine receptors accounts for the severity of the immunodeficiency seen with XSCID. T cell development is coordinately regulated by, among other things, signaling via cytokines and their receptors. It is our hypothesis that T cell development is regulated by stage-specific responses to signaling via (c. To be able to study the mechanisms of this regulation in T cell development, the cell subsets in which (c is necessary for this development must first be identified.

To determine the stage at which (c becomes functionally important in the development of T cells, a naturally occuring canine model of this disease is being utilized. Cells are isolated at various developmental stages from female dogs which are carriers for XSCID. RT-PCR is used to directly detect (c mRNA produced from the normal and/or mutant alleles. The differential expression of the maternally-derived (XSCID) and the paternallyderived (normal) X chromosome is examined in these cells. In females, for genes located on the X chromosome, only one allele is expressed in each cell due to X chromosome inactivation. Consequently, if (c function is necessary at a given developmental stage, those cells in which the active X chromosome carries the mutant copy of the gene will be unable to develop further and will be lost from the subsequent overall populations. Therefore, in carrier females, X expression should be skewed in favor of the paternal X chromosome (with normal (c) at developmental stages in which (c has previously been, or currently is, necessary.

The first population examined was double negative (DN; CD4-CD8-) thymocytes, one of the earliest stages of T cell development in the thymus. In XSCllD dogs, the percentage of DN thymocytes is increased, while the percentage of double positive (DP; CD4+CD8+) thymocytes is decreased, suggesting the presence of a block in development at or about the DN to DP transition. Examination of DN thymocytes from six animals shows a pattern of X-inactivation that is highly skewed (>94% paternal X active). This indicates that the (c chain is functionally necessary earlier in development than at the DN thymocyte stage. Determining when (c first becomes functionally important during T cell development should provide a focus point for subsequent studies, lend insight into the mechanisms by which (c is crucial to the coordinated regulation of thymocyte development, and designate appropriate target populations for intervention.

ALLERGEN-SPECIFIC IgE AND IgGd ANTIBODIES IN ATOPIC

AND NORMAL DOGS

Tan Mei Lian and Richard Halliwell

University of Edinburgh, Edinburgh, Scotland

Allergic skin diseases are the commonest cause for non-routine presentation of dogs to veterinarians for treatment. Flea allergy is the commonest type, followed by allergy to pollens, dusts and molds. The confirmation of the latter type of disease, and the correct identification of the allergens responsible, is a prerequisite for implementation of immunotherapy (desensitization) which is the preferred therapeutic approach. This can be undertaken by skin testing, or by a blood test which detects the specific type of antibody (IgE) that is responsible for allergy.

The use of the blood test was evaluated critically for the first time employing sera from 65 allergic dogs, and 24 normal dogs, all of whom had been skin tested with the same allergens. Surprising findings were that a high proportion of normal dogs gave positive skin tests and positive blood tests, especially to the house dust mite allergens. Levels of antibody, however, were higher in the allergic dogs. Results using mold, whole flea and whole house dust allergens were poor, and these antigens should not be used in the blood test without further purification.

These results emphasize the fact that a careful clinical diagnosis, and elimination of other possible causes of the skin problem, must be undertaken before using either the skin test or the blood test, or misleading results will lead to an incorrect diagnosis and to the wrong therapy.

INDUCTION OF INTERLEUKIN-12 IN CANINE MONOCYTES.

EB Dickerson, ML Padilla, SC Helfand. Department of Medical Sciences, School of

Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive West,

Madison, WI 53706

One of the greatest challenges in modern oncology is learning how to overcome the immune system's tolerance of malignant cells. Inducing effector cells of the immune system to recognize and kill cancer cells has been a dream of oncologists for nearly half a century. In pursuing this goal, it is essential to understand the pathways and controlling factors that turn on antitumor responses of cells of the immune system. This information is fundamental to developing an understanding of why the immune system fails to recognize malignant cells in cancer patients and provides the basis for therapeutic interventions. One approach which has shown promise is the use of monoclonal antibodies (blabs) that can bind to tumor cells promoting their recognition and destruction by killer cells of the immune system. Recently, we discovered that one such monoclonal antibody, R24, not only promotes tumor targeting of canine immune cells, but also directly triggers antitumor responses after specifically binding to some canine immune cells known as monocytes. We have now determined that interleukin=12, a potent immune cell activator, is produced by these monocytes following binding of Mab R24 to them.

This is a novel finding in cancer immunology because it suggests an entirely new role for blab usage in cancer patients. Further, understanding the molecular and biochemical events that are switched on when R24 binds monocytes offers opportunities to clarify pathways potentially important for stimulating antitumor responses by immune cells in general, and to examine abnormalities of these pathways in canine cancer patients. The research has helped to elucidate mechanisms which account for the unique activity of blab R24-activation of immune cells and will provide insight into fundamental responses of immune cells important for controlling cancer.

AN ASSESSMENT OF DIETARY OMEGA-6:OMEGA-3 FATTY ACID RATIOS ON

IMMUNE FUNCTION IN YOUNG AND GERIATRIC DOGS.

Robert J. Kearns, John J. Turek, John R. Burr and Michael G. Hayek. The University of Dayton,

Purdue University and The lams Company.

Companion animals, like humans, are normally able to protect themselves from environmental factors such as infectious disease agents, allergens, and toxins. Protection from these agents is controlled either by natural or acquired resistance mechanisms. Natural or innate resistance involves those non-specific factors provided at birth, and include numerous biochemical, physical and anatomical barriers. In addition, phagocytic cells including macrophages and neutrophils are involved in the engulfinent and destruction of many of these harmful agents. Should innate resistance fall, acquired resistance provides protection for the host. Acquired resistance involves cells of the immune system, and consists of humoral (antibody) and cellular immunity. To survive, the host depends on an elaborate network of communication between cells of the immune system. Ironically, two variables affecting immune function are the aging process and nutritional factors. Aging has been demonstrated to influence both humoral and cellular immune function, as well as cytokine production. Consequently, the aging immune system may contribute to the increased incidence of infectious disease and cancer. Likewise, malnourished animals are immunocompromised and exhibit an increased incidence of acute and chronic illnesses. Dietary lipids have been reported to modulate the immune system either through regulating cytokines, or eicosanoids.

It has recently been reported that dietary lipids, i.e., fatty acids, may not only influence immune function, but these same compounds may reduce the impact of aging on immunological responsiveness. Supplementing the diet with certain fatty acids has resulted in the production of eicosanoids (prostaglandins and leukotrienes) which influence several immune parameters, including T and B cell function, as well as cytokine profiles. The focus of this study was to examine the influence of age and diet on the immune response of young and old Fox Terriers and Labrador Retrievers. Eighteen young and old dogs were utilized for this study. Dogs were fed a basal diet containing an n-6: n-3 fatty acid ratio of 25: i for eight weeks. Half of the dogs were switched to a diet containing an n-6:n-3 ratio of 5:1 and all dogs were fed their respective diets for the following eight weeks. Peripheral blood mononuclear cells were isolated from blood and were examined for proliferative responses to both T and B cell mitogens. Humoral immunity was measured by the animals ability to produce antibody titers to sheep red blood cells. Additionally, macrophages from the peritoneal cavity were isolated and stimulated for the production of cytokines (IL-1, IL-6 and TNF-a).

Results from these studies revealed age x breed differences. Although cytokine production did not differ with age, both breeds demonstrated an age associated decline in sheep red blood cell titers, as well as their ability to respond to different mitogens. Interestingly, this decline yeas greater in Fox Terriers, suggesting a decrease in cellular proliferative capacity in lymphocytes isolated from the larger breed. Diet x breed interactions did not affect IL,-1, IL,-6 or TNF-a production from peritoneal macrophages or peripheral blood mononuclear cells. Feeding a diet containing a ratio of 5:1, however, resulted in an increase in T cell and B cell mitogen responsiveness. In summary, this study indicates that feeding a diet containing a n-6: n-3 fatty acid ratio of 5:1 did not have a negative erect on the immune response of young or geriatric dogs. Additionally, consideration of specific dietary fatty acid profiles may be warranted for different breeds and stages of life.

CANINE GENETIC MARKERS

ASSOCIATED WITH AUTOIMMUNE DISEASE

Loma Kennedy

The overall aim is to look for genetic markers associated with autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus in the dog. It is known that in humans, many autoimmune diseases are associated with genes in the major histocompatibility complex (MMC). We want to find out if similar associations exist in the dog.

However, as yet, no suitable methods for identifying these genes in the dog exist. We have developed DNA based systems which are simple, rapid, and cost effective. These can identify the major variants of two genes in the canine MHC.

We are establishing the frequencies of these variants in the normal dog population, to provide a base line control group with which to compare our disease groups. If we can identify variants associated with particular autoimmune diseases, it may be possible to use this information to identify animals at risk of developing the disease. These animals can then be excluded from breeding programs, and therefore improve the overall health of the breed.

IDENTIFICATION OF POSSIBLE ALLERGENS

IN FLEA SALIVARY GLAND EXTRACTS

Susan Lee, Faculty of Science, Griffith University, Brisbane Australia

Flea bite hypersensitivity (FBH) is a major cause of dermatitis in dogs but relatively little has been reported on the allergens responsible. Without such information it is difficult to design reliable tests to identify flea allergy or to attempt hyposensitisation or immunisation of dogs against the flea. Lee et al. (1997) found that antibodies generated in mice exposed to feeding fleas identified four protein bands at apparent MW 56, 54, 42 and 40 K in salivary gland extracts. This work has been extended by using salivary gland extracts to study the immune response of dogs suffering from FBH.

Dogs were classified as either sensitive to feeding fleas (FF+) or insensitive (FF-) by use of a provocative test (Lee et al. 1995). Their sera were tested in an indirect ELISA against flea salivary gland extracts. This failed to produce any pattern of reactivity for either IgE or IgG antibodies that could distinguish FF+ dogs from FF- dogs. The bands noted as important when tested previously with sera from mice exposed to feeding fleas were not particularly prominent.

The reactivity of the skin of the two groups of dogs was then assessed by intradermal testing with fractionated extract of salivary glands, prepared as follows. More than 2000 flea salivary glands were extracted into phosphate buffered saline (PBS) and fractionated using gel permeation chromatography. The chromatogram revealed 11 prominent peaks containing protein, with MW ranging from 56K down to less than 6K. The fractions (1 mL) were injected intradermally (50 mL portions) and the response assessed by measurement of the wheal/bleb at the injection site, compared to the response to injection of PBS alone. None of the dogs reacted to the protein peak at -56K, or to the proteins with MW < 6K. Of the nine FF+ dogs tested, six reacted strongly to a protein peak at -40K, and seven reacted to a protein peak at -12K. This reaction was manifested by a red wheat >4 mm larger than the control blebs. The FF- dogs (7) showed either no reactivity to any of the fractions or slight transient reactions to the fractions from the 40K and 12K peak.

The protein bands at 40 and 42 K separated using SDS-PAGE, were subjected to amino acid sequencing. Twelve amino acids were identified and the bands were found to be identical in the portion that was sequenced. The sequence matched that obtained by Frank et al. (1997) for a protein obtained from flea salivary deposits eluted off a nitrocellulose membrane. The sequence obtained has no particular homology with sequences on protein data bases and may be a protein unique to the flea. The proteins in salivary gland extracts have been further characterised for carbohydrate content and protease activity.

The results of tests on canine serum and skin indicate that serum antibodies are not useful to detect FBH, in contrast to skin tests with a specific extract. The skin tests highlight the possible relevance of a proteins) with MW -40K and -12K, and indicate that flea allergic dogs may develop hypersensitivity to more than one component of flea saliva. This work and that of Frank et al. (1997) have identified target proteins for development of more reliable tests for FBH and for possible hyposensitisation/immunisation procedures.

Frank GR, Hunter SW & Wallenfels L (1997). Ectoparasite saliva proteins and apparatus to collect such proteins (to Heska Corporation) United States Patent 5,646,115, issued 8 July 1997

Lee SE, Jackson LA & Opdebeeck JP (1997). Salivary antigens of the cat flea, Ctenocephalides felis felis. Parasite Immunology 19:13-19

Lee SE, Johnstone IP & Opdebeeck JP (1995). A simple method of rearing fleas for a provocative hypersensitivity test in dogs and cats.Australian Veterinary Journal 72(10): 390-392

 

GENETIC SUSCEPTIBILITY TO GLUTEN-SENSITIVE ENTEROPATHY
IN IRISH SETTER DOGS

OA Garden1, A Polvi2, RS Houiston3, M Maki4, J Partanen2,

RM Batt1,

1Department of Small Animal Medicine and Surgery, Royal Veterinary College, University of London, Hatfield, Hertfordshire, UK

2Tissue Typing Laboratory, Finnish Red Cross Blood Transfusion Service, Helsinki, Finland

3Section of Epidemiology, Institute of Cancer Research, Sutton, UK

4IMT, University of Tampere, and Department of Pediatrics, Tampere University Hospital, Tampere, Finland

As many as one in 125 Irish setters in UK (0.8%) is estimated to have gluten-sensitive enteropathy (GSE). In GSE, ingested wheat gluten, present in some dog foods, causes the symptoms, namely in puppies weight loss or poor weight gain and chronic intermittent diarrhoea and in adults poor appetite, thinness and diarrhoea. In affected dogs intestinal permeability has increased and partial villous atrophy is seen. Diagnosis is mainly based on permeability measurements and biopsy. Permanent withdrawal of the gluten from the died leads to an improvement of the condition.

The aim of this study was to find out the genetic basis of canine GSE. If genes affecting GSE are found, genetic testing will help to avoid breeding dogs that carry GSE susceptibility genes. As canine GSE is similar to human celiac disease (CD) and certain alleles of the MHC-DQA1 and -DQB 1 genes predispose the individuals carrying them to CD, we studied if some alleles of similar genes in dogs predispose to GSE.

We found out that unlike in human CD in canine GSE no alleles of MHC-DQA1 and -DQB1 genes seem to predispose to GSE. Our results show that Irish setter breeders do not need to pay attention to MHC-DQAl and -DQBl genes when trying to avoid the born of GSE affected puppies. The search for actual genes affecting the GSE continues.

IMMUNE RESPONSE IN DOGS: VARIATION OF DLA WITH BREED

Wendy Thomson

We are interested in looking at the immune response in dogs. The level of immune response to all bacteria and viruses varies considerably between individuals within the same species. This variation is largely explained by variation in the genetic factors which regulate the immune response. A collection of genes called the Major Histocompatibility complex (MHC) has been identified in all higher species and these are important in regulating immunity. MHC genes are found in many different forms and for any particular pathogen some will be associated with good immune responses and others poor. It is important to determine how these different forms vary between breeds. This may help us to decide which breeds are likely to be more susceptible to infection/autoimmune disease and the ability to mount an immune response to vaccination.

We have developed molecular based methods to identify the different forms for two of the MHC genes. We have typed a random cohort of dogs covering over fifty breeds and compared the phenotypes of the different breeds. We have established that some forms of these genes are found in virtually all breeds, whereas others are breed specific. In addition, some breeds possess many forms of the genes whereas others have a smaller, more distinctive range. These studies need to be extended to include larger numbers of breeds and larger numbers within each breed.

This work may help in predicting which animals are at increased risk of infection/autoimmune disease and which breeds will respond well to a particular vaccine. Any studies of this nature will have to ensure very careful matching of cases and controls. Long term we may be able to improve particular breeds in terms of their overall immune response via suitable breeding programmes.

AN EMMUNOPHENOTYPIC STUDY OF CANINE LEUKEMIAS

W. Vernau and P.F. Moore

Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine

UC Davis

Lymphoma (cancer of lymphocytes in tissue) and lymphocytic leukemia (cancer of lymphocytes in blood) are some of the most common and serious (but often treatable) malignant diseases of dogs. Early and accurate diagnosis is a fundamental prerequisite for successful therapy. Until now, diagnosis of these diseases in dogs has relied largely on routine blood work and microscopic assessment. However, an accurate diagnosis is often not possible using these tools alone. There are many new technologies available that could greatly assist veterinarians in the diagnosis and management of canine leukemia and lymphoma. One of these tools is immunophenotyping. The utility of immunophenotypic assessment of leukemias (and lymphomas) has been firmly established in people. Immunophenotyping can assist in the provision of an accurate diagnosis and provide useful prognostic and therapeutic information. Additionally, immunophenotyping can provide insight into the cause of some leukemias. The veterinary literature contains little information about the immunophenotypes present in canine leukemias. Similar to the situation in people, this information could potentially be useful diagnostically, prognostically and therapeutically. A comprehensive panel of dog specific antibodies has been developed over the last 12 years in our laboratory. Utilizing a panel of thirty of these antibodies, we immunophenotyped a large series (>70) of canine leukemias.

In people, chronic lymphocytic leukemia (CLL) is a relatively common, often indolent leukemia affecting older adults, particularly males. Canine CLL occurs in older dogs (mean age 9.5 years; range 1.5- 15 years). Blood lymphocyte counts ranged from 15, 000 1 uL to 1,200,000 / uL ( normal range for peripheral lymphocytes in dogs= 800 - 3,600 / uL). Surprisingly, 70% of CLL cases involved proliferation of T lymphocytes and 57% of CLL cases had large granular lymphocyte (LGL) morphology. B cell CLL accounted for only 30% of canine CLL cases. These results are in marked contrast to people where more than 95% of CLL's are of B cell origin. These differences likely reflect fundamental differences in the canine immune system (versus human). Similar to the situation in people, canine CLL was also an indolent, slowly progressive disease. With appropriate therapy, many, of the dogs with CLL have survived (and done well clinically) for more than three years. Numerous acute leukemias were also phenotyped. Contrary to previous information in the veterinary literature, the majority of these leukemias had a phenotype most consistent with a myeloid origin (which confers a poorer prognosis). We identified some markers that appear to be quite useful diagnostically, such as CD 34.

Even with sophisticated tests such as immunophenotyping, it is not always easy to decide if a lymphocyte proliferation is benign and reactive (to an agent) or malignant, although the outcome in each case is profoundly different. We therefore also proposed to develop a genetic test that would greatly facilitate accurate diagnosis, prognosis and treatment of canine lymphocyte malignancies. This is very relevant to all forms of lymphoma and leukemia but particularly to chronic lymphocytosis / leukemia, in which it can be especially difficult to establish a definitive diagnosis because of the indolent or slowly progressive nature of the disease. The test is a polymerise chain reaction (PCR) based test that requires only very small samples and can be performed on a variety of samples including blood, aspirates, punch biopsies and even paraffin embedded tissue used previously for histopathology. Preliminary data indicated that our test is effective and is capable of differentiating a neoplastic (malignant) from a reactive (benign) lymphoproliferative process. This test will serve many purposes. It should significantly improve our ability to provide an accurate diagnosis and prognosis for canine lymphoproliferative disease. It will also expedite extremely sensitive detection of minimal residual disease and early relapse, thereby improving our ability to treat these diseases. Additionally, and very importantly, it has similar application to a wider variety of other clinically important and common diseases, including inflammatory bowel disease and granulomatous meningoencephalitis.

CANINE MYASTHENIA GRAVIS - LESSONS FROM THE PAST TEN YEARS

G. Diane Shelton DVM, PhD

Associate Adjunct Professor

Department of Pathology

University of California, San Diego

La Jolla, CA X2093-0612

Our knowledge of acquired myasthenia gravis (MG) in dogs has advanced significantly over the past ten years and it is now well established that MG is not an uncommon clinical problem in this species. Acquired MG is an autoimmune disease in which the body's immune system mistakenly attacks and destroys special proteins (acetylcholine receptors) located on the muscle surface where the nerve attaches to the muscle. These proteins respond to the chemical acetylcholine, which is released by the stimulated nerve, and this response starts the process which causes the muscles to contract. It is not known what initiates the autoimmune reaction, but if acetylcholine receptors are lost because of it, the response of the muscle to nerve impulses is poor and muscle weakness may occur. The muscles affected are called voluntary or striated muscles and include limb muscles involved with ambulation, pharyneal and esophageal muscles involved in swallowing and delivery of food into the stomach, laryngeal muscles involved in respiration and vocalizing, and extraocular muscles involved in eye movements. Involuntary heart muscle and smooth muscles of the gut, blood vessels, and uterus are not involved in MG. Different muscle groups may be affected in different dogs and clinical signs are not the same in all dogs. This makes the clinical diagnosis of MG tricky for the inexperienced. A specific and sensitive blood test is available for the diagnosis of canine MG at the Comparative Neuromuscular Laboratory, University of California, San Diego.

In humans, autoimmune diseases such as MG, thyroid disease, lupus, and rheumatoid arthritis seem to run in families, and these families statistically (but not invariably) share certain tissue markers. New and promising experimental treatments for autoimmune diseases are based on the presence of such markers. In collaboration with the Center for Companion Animal Health at the University of California, Davis, the Akita dog is the first breed in which genetic markers are being evaluated in canine MG. The prognosis in canine MG remains guarded since many dogs succumb to aspiration pneumonia resulting from megaesophagus. With early recognition of a megaesophagus and appropriate treatment, the prognosis is improving. A unique feature of canine MG is the presence of spontaneous remission of the disease. Studies involving regulation of the immune response in canine MG may provide a direction for future treatment modalities aimed at a natural process of disease remission.

VACCINE-INDUCED AUTOANTIBODIES IN BEAGLE DOGS.

Harm HogenEsch, Department of Veterinary Pathobiology, School of Veterinary

Medicine, Purdue University, West Lafayette, IN 47907.

Vaccination is a very efficient and cost-effective way to control infectious diseases. Diseases caused by distemper and parvovirus would continue to kill many dogs if effective vaccines were not available. However, although the short-term safety records of vaccines are excellent, there is increasing concern that current vaccination protocols have long term adverse effects on the health of dogs. In particular, vaccines have been implicated in the apparent increase of autoimmune and immune-mediated diseases in dogs. However, these allegations are based on anecdotal and circumstantial evidence, and there is presently no direct evidence for a causal relationship between vaccination and immune-mediated disease. Until such information becomes available, it will remain unclear if vaccination protocols need to be changed.

In an attempt to address this issue, female Beagle dogs (5 per group) were vaccinated with a multivalent vaccine and a rabies vaccine (group A), with a multivalent vaccine only (group B) or with a rabies vaccine only (group C). Group D remained unvaccinated. Blood samples were collected at various time points following vaccinations for immunologic, serologic, hematologic and endocrine assays.

All dogs (now 20 months of age) remained clinically healthy, and there were no differences between groups with regard to hematologic and routine immunologic values. Autoantibodies developed after the first or second vaccination, and persisted during the course of the study. Vaccinated dogs (groups A-C) had significantly increased concentrations of serum autoantibodies against various antigens compared to the unvaccinated control dogs.

In summary, vaccination of dogs with a multivalent and/or rabies vaccine results in the production of autoantibodies. We will continue to study these dogs to determine if the vaccine-induced autoantibodies will cause clinical disease.

ANTINUCLEAR ANTIBODIES - PRESENCE AND SPECIFICITY
IN AUTOIMMUNE CONNECTIVE TISSUE DISEASE

Helene Hansson, Department of Small Animal Clinical Sciences

Swedish University of Agricultural Sciences, Uppsala, Sweden

My work deals with certain types of rheumatic diseases called autoimmune connective tissue diseases. Patients suffering from these disorders, dogs as well as humans, usually show musculoskeletal symptoms. Other clinical signs may however also develop, including for example dermatological disorders and fever.

These diseases are usually accompanied by a "disturbance" in the immune system with an abnormal production of a special kind of antibodies (so called autoantibodies). Our studies are mainly concerned with the diagnostic features and we thus investigate the presence and the nature of the autoantibodies. We have shown that the commonly used diagnostic techniques for human patients may be used for canine patients as well. However, dogs usually present "log-specific" autoantibodies that somehow differ from the human ones. This finding indicates that dogs may present "dog-specific" autoimmune connective tissue diseases.

LEFLUNOMIDE EFFECTIVELY TREATS NATURALLY-OCCURRING
IMMUNE MEDIATED AND INFLAMMATORY DISEASES OF DOGS THAT
ARE UNRESPONSIVE TO CONVENTIONAL THERAPIES

Clare Gregory, U.C. Davis

Our laboratory has been studying the effects of an new drug, Leflunomide, for the management of immune-mediated and inflammatory diseases of dogs. In particular, we were interested in the treatment of diseases that were not controlled by conventional medicines or, the medicines used had harmful side effects. As background, we had found that leflunomide, combined with another immunosuppressive drug, cyclosporine, had the ability to control organ transplant rejection in dogs.

Dogs who were treated with leflunomide had a wide range of immune-mediated and inflammatory diseases. Most had not had a good response to traditional treatments. Diseases that destroy red blood cell and platelets in the blood, inflammation of the tissues of the brain, and diseases of the skin were treated. In most cases, leflunomide effectively and safely controlled these diseases. It was the first medication to be effective in the treatment of canine histiocytosis, and reduced or eliminated the need for steroid therapy in the treatment of inflammatory brain diseases.

In conclusion, we found that leflunomide was very effective and safe for the treatment of a wide variety of immune-mediated and inflammatory diseases of dogs. Soon, we hope to begin a clinical renal transplantation program using leflunomide in combination with cyclosporine.

 

COMPARISON OF IMP INTRONIC SINE IN DOGS
WITH HOMOLOGUES IN OTHER SPECIES

John Gerlach

Presentation of antigens to the cells of the immune system is the key to recognition of self and non-self and the basis of an immune response. The processing of proteins from within a cell may allow the presentation of peptides, or small protein fragments, from self or non-self (parasite or virus) proteins. Part of the cellular machinery that processes these proteins for presentation are the 20S proteosomes and a sub-piece, the low molecular mass proteosome (UP).

Searching for the canine homologue of the LMT gene has lead to proof of its presence. Further characterization of this region on the genomic level has also shown the presence of a genomic repeat in an intron (non-coding region) of the canine LMP-2 gene. This repeat is species specific and review of available data indicates that other species also have species specific repeats in the same location.

These similarities across various species maintained throughout evolution further enforces the concept that what is learned in one species on a molecular level can be applied to other species.

CLONING OF THE CANINE INTERLEUKIN-2 RECEPTOR a SUBUNIT.

EB Dickerson, ML Padilla, SC Helfand. Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive West, Madison, WI 53706

New mechanistic information about the immune system has resulted in many of opportunities for basic and clinical research leading to improved diagnosis and therapy of disease. Adapting discoveries from marine and human research, and advancing the field of immunology in veterinary medicine have been severely limited by the lack of reagents specific for the immune systems of the animals we care for. The human IL-2Ra subunit, a membrane receptor for IL-2 expressed by activated T lymphocytes, has been studied with specific monoclonal antibodies (blabs) in a wide variety of human diseases. The cellassociated form of IL-2Ra has proven to be an important molecule for antibody-targeting on cancer cells and highly activated T lymphocytes in some autoimmune diseases. In its shed form in serum, IL-2Ra is a highly prognostic marker for some cancers, and serves as a surrogate for detection of early cancer relapse, and rejection of transplanted organs. We believe that analysis of IL-2Ra in canine disease is a promising endeavor for veterinary medicine. Our previous report of inappropriate expression of IL-2Ra in canine lymphoma is just one example of IL-2Ra dysfunction in the dog. After examining cross-reactivity of a variety of marine anti-human IL-2Ra blabs against canine immune cells, we concluded that available blabs against human IL-2Ra do not recognize canine IL-2Ra. We have decided to develop marine blabs that are specific for canine IL-2Ra in order to screen canine lymphoid cancer for IL-2Ra expression as a potential target for cancer immunotherapy. We also have an interest in examining shed IL,-2Ra in the blood of dogs with lymphoma and other cancers, and expect measurement of IL-2Ra in the blood of dogs with autoimmune disease could also be developed into a clinical monitoring tool of immune activation status. In the current study, we determined the genetic code for the canine IL-2Ra protein in preparation for development of blabs against it and have begun the analysis of IL-2Ra using molecular techniques with this gene sequence. This approach is highly sensitive and specific for detection of canine II,-2Ra.