Stephen A. Kruth

Derivation and Characterization of Canine Embryonic Stem Cell Lines with In Vitro and In Vivo Differentiation Potential

Embryonic stem cells (ESCs) represent permanent cell lines that can be maintained in an undifferentiated state. In an environment that induces differentiation, they form derivatives of the three embryonic germ layers: mesoderm, ectoderm, and endoderm. These characteristics give ESCs great potential for both basic research and clinical applications in the areas of regenerative medicine and tissue engineering. The establishment of ESCs from large animals that model human diseases is of significant importance. We describe the derivation of permanent canine cell lines from preimplantation‐stage embryos. Similar to human ESCs, canine ESCs expressed OCT3/4, NANOG, SOX2, SSEA‐3, SSEA‐4, TRA‐1–60, TRA‐1–81, and alkaline phosphatase, whereas they expressed very low levels of SSEA‐1. They maintained a normal karyotype and morphology typical of undifferentiated ESCs after multiple in vitro passages and rounds of cryopreservation. Plating cells in the absence of a feeder layer, either in attachment or suspension culture, resulted in the formation of embryoid bodies and their differentiation to multiple cell types. In vivo, canine ESCs gave rise to teratomas comprising cell types of all three embryonic germ layers. These cells represent the first pluripotent canine ESC lines with both in vitro and in vivo differentiation potential and offer the exciting possibility of testing the efficacy and safety of ESC‐based therapies in large animal models of human disease. STEM CELLS 2009;27:329–340

Prevalence and risk factors for Clostridium difficile colonization in dogs and cats hospitalized in an intensive care unit

Clostridium difficile is the most common cause of hospital- and antimicrobial-associated diarrhea in hospitalized humans however the role of C. difficile in diarrhea in dogs has not been defined. A prospective study of C. difficile colonization in dogs and cats was conducted in a veterinary teaching hospital intensive care unit (ICU). Rectal swabs were taken from patients upon admission to the ICU and every third day of hospitalization until discharge or death. C. difficile was isolated from 73/402 (18%) animals; 69% of isolates were toxigenic. Community-associated colonization was identified in 39/366 (11%) of animals that were sampled at the time of admission, while C. difficile was subsequently isolated from 27 of the remaining 327 (8.3%) animals that had a negative admission swab. C. difficile was isolated from seven other dogs during hospitalization, but the origin was unclear because the admission swab was not collected. Administration of antimicrobials prior to admission and administration of immunosuppressive drugs during hospitalization were risk factors for hospital-associated colonization (P=0.006, OR 4.05, 95% CI 1.4-10.8). Acquisition of C. difficile during hospitalization in the ICU was associated with the development of diarrhea (P=0.004). Two ribotypes, one toxigenic and one non-toxigenic, predominated.

In Utero Injection of α-L-Iduronidase-Carrying Retrovirus in Canine Mucopolysaccharidosis Type I: Infection of Multiple Tissues and Neonatal Gene Expression

Canine alpha-L-iduronidase (alpha-ID) deficiency is caused by a single base pair mutation in the alpha-ID gene, resulting in no enzyme activity in homozygous affected pups. The disease clinically resembles human mucopolysaccharidosis type I (MPSI). We used the canine MPSI model system to address the efficacy of a new retroviral vector, MND-MFG, containing the human alpha-ID cDNA (MND-MFG-alpha-ID) for direct in utero gene delivery to MPSI cells. In vitro, the MND-MFG-alpha-ID vector showed high-level, long-term expression of the transgene in both canine and human alpha-ID-deficient fibroblasts. The effectiveness of this vector for in utero gene transfer and expression in multiple tissues was assessed by injecting viral supernatants into MPSI fetuses and evaluating transduction efficiency and enzyme expression at various times after birth. Transduction of a spectrum of cell types and tissues was observed in all seven live-born pups and in one stillborn pup. Although enzyme activity was not detected in adult tissues from the seven surviving pups, significant alpha-ID enzyme activity was detected in both the liver and kidney of the deceased pup. Our combined gene delivery vector and in utero transfer approach, while encouraging in terms of overall gene transfer efficiency to multiple tissues and successful short-term gene expression, was unable to meet the important requirement of sustained in vivo gene expression.

Adoptive transfer of genetically modified human hematopoietic stem cells into preimmune canine fetuses

To develop a surrogate model system for assaying gene transfer into human hematopoietic stem cells (HSCs) with in vivo repopulating potential, we injected human marrow cells transduced with a reporter retroviral vector in long-term marrow cultures (LTMCs), into the yolk sacs of preimmune canine fetuses. Of eight mid-gestation fetuses injected through the exteriorized uterine wall and under ultrasound guidance, seven were born alive. One puppy died in the neonatal period accidentally. The remaining six puppies are all healthy at 31 months of age. There was no evidence for graft-versus-host disease or any untoward effects of in utero adoptive transfer of transduced human LTMC cells. All puppies were chimeras. Human cells, detected by fluorescence in situ hybridization, were present in blood, declining from 38% to 0.05% between 10 and 44 weeks after birth. Corresponding numbers for marrow were from 20% to 0.05%. Human cells were also detected in assays of hematopoietic cell progenitors and in stimulated blood cultures. All six puppies were positive for the presence of proviral DNA at various time-points after birth. In three dogs, provirus was detected up to 41 weeks after birth in blood or marrow, and in one dog up to 49 weeks in blood. These data support the further development of this large-animal model system for studies of human hematopoiesis.

Independent and combined effects of prednisone and acetylsalicylic acid on thromboelastography variables in healthy dogs

OBJECTIVE To describe the effects of prednisone and acetylsalicylic acid (ASA) on results of thromboelastography in healthy dogs. ANIMALS 16 male mixed-breed dogs. PROCEDURES Dogs were randomly assigned to 3 treatment groups (4 dogs/group) that received prednisone (median dose, 2.07 mg/kg), ASA (median dose, 0.51 mg/kg), or both drugs, PO, every 24 hours from days 0 through 6. Another group received no treatment (control dogs; n = 4). Thromboelastography variables (reaction time, clotting time, α-angle, maximum amplitude [MA], global clot strength, coagulation index, and percentage of clot lysis at 60 minutes [CL(60)]) were evaluated in blood samples collected (prior to drug administration in treated dogs) on days 0 (baseline), 2, 4, and 6. RESULTS Administration of ASA alone did not alter TEG variables. For treatment effect, mean global clot strength was increased in the prednisone and drug combination groups, compared with values for control dogs; MA was also increased in the prednisone and drug combination groups, compared with that of controls. For treatment-by-time effect, median CL(60) was increased in the prednisone group on day 6, compared with baseline value in the same dogs and with median CL(60) of the control group on day 6. Median CL(60) was also increased in the drug combination group on day 6, compared with the baseline value and with that of the control group on day 6. CONCLUSIONS AND CLINICAL RELEVANCE Prednisone administered at approximately 2 mg/kg/d, PO, for 7 days with or without concurrently administered ASA increased clot strength and decreased clot lysis in healthy dogs.

Multiple endocrine diseases in cats: 15 cases (1997–2008)

The objective of this retrospective study was to characterize a population of cats from a tertiary care center diagnosed with multiple endocrine disorders, including the specific disorders and time intervals between diagnosis of each disorder. Medical records of 15 cats diagnosed with more than one endocrine disorder were reviewed. The majority of cats were domestic shorthairs, and the mean age at the time of diagnosis of the first disorder was 10.3 years. The most common combination of disorders was diabetes mellitus and hyperthyroidism. Two cats had concurrent diabetes mellitus and hyperadrenocorticism, one cat had concurrent central diabetes insipidus and diabetes mellitus. A mean of 25.7 months elapsed between diagnoses of the first and second endocrine disorder, but this was variable. This study suggests the occurrence of multiple endocrine disorders is uncommon in cats.

Biological Response Modifiers: Interferons, Interleukins, Recombinant Products, Liposomal Products

The concept of enhancing the normal immune response against infections and neoplasms has been considered for decades. The administration of various natural and synthetic products to simulate systemic infections has largely given over to the idea that specific cytokines can be used effectively when administered systemically. Interferons, interleukins, and hematopoietic growth factors may offer substantial clinical benefit in chronic viral infections, and cancers such as osteosarcoma, melanoma, and lymphosarcoma. Erythropoietin has been shown to have great utility in the management of chronic renal failure. At this point in time, only recombinant products derived from humans are commercially available, and they are expensive and not licensed for use in companion animals. Nevertheless, these products may have significant clinical impact on several highly fatal disorders of dogs and cats. When administered systemically, cytokines perturb complex regulatory pathways, and serious side effects may occur. Innovative delivery methods, such as liposomes, gene therapy, and even oral administration may increase the therapeutic index of these molecules. Biological response modification, cytokine biology, and associated delivery systems are rapidly changing fields, and the small animal veterinarian will need to watch for significant advances in these areas over the next several years.

Comparison of citrated native and kaolin-activated samples for thrombelastographic analysis in healthy dogs.

BACKGROUND Thrombelastographic (TEG) analysis is a test of global hemostasis in veterinary medicine; however, there have been limited comparisons of analysis of citrated native and kaolin-activated samples. OBJECTIVES The purpose of this study was to determine the variation in TEG variables between citrated native and kaolin-activated whole blood samples and to establish reference intervals for both sample types. METHODS Citrated whole blood samples were obtained from 40 healthy dogs. Thirty minutes after collection, TEG analysis was performed simultaneously on samples with and without kaolin-activation. Reaction time (R), clotting time (K), angle (α), maximum amplitude (MA), global clot strength (G), and clot lysis at 30 minutes (LY30) were recorded, and the concordance correlation coefficient (ρ(c)) was calculated for each sample type. RESULTS Significant differences between results obtained for kaolin-activated and native samples were obtained for R (mean difference -1.3 minute, P = .0009), K (-0.7 minute, P = .0003), α (+5.1º, P = .002), MA (+2.4 mm, P = .002), and G (+568 dyn/cm(2), P = .0009). LY30 was not different between methods. There was substantial agreement between methods for G (ρ(c) = .69) and MA (ρ(c) = .65), moderate agreement for R (ρ(c) = .45) and α (ρ(c) = .44), fair agreement for K (ρ(c) = .29), and slight agreement for LY30 (ρ(c) = .04). CONCLUSIONS The TEG variables were significantly altered by kaolin activation; however, some agreement between sample types suggests a consistent bias. In citrated whole blood activated with kaolin, clot formation time is shortened and the amplitude of the tracing is increased, resulting in a TEG tracing that appears to indicate relative hypercoagulability compared with that obtained using native citrated whole blood samples.

Multiple endocrine diseases in dogs: 35 cases (1996-2009)

OBJECTIVE To characterize a population of dogs from a tertiary care center with 2 or more endocrine disorders, including the specific disorders and time intervals between diagnosis of each disorder. DESIGN Retrospective case series. ANIMALS 35 dogs with 2 or more endocrine disorders. PROCEDURES Medical records were reviewed, and the following was recorded: clinical signs, physical examination findings, and the results of CBC, serum biochemical analysis, urinalysis, aerobic bacterial culture of urine samples, endocrine testing, diagnostic imaging, and necropsy. RESULTS 35 dogs with more than 1 endocrine disorder were identified. Seventy-seven percent (27/35) of the dogs were male, and the mean age at the time of diagnosis of the first endocrinopathy was 7.9 years. Miniature Schnauzer was the most common breed. Twenty-eight of 35 (80%) dogs had 2 disorders; 7 (20%) had 3 disorders. The most common combinations of disorders included diabetes mellitus and hyperadrenocorticism in 57.1 % (20/35) of dogs; hypoadrenocorticism and hypothyroidism in 22.9% (8/35) of dogs; and diabetes mellitus and hypothyroidism in 28.6% (10/35) of dogs. A mean of 14.5 months elapsed between diagnosis of the first and second endocrine disorders, whereas there was a mean of 31.1 months between diagnosis of the first and third endocrine disorders. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that the occurrence of multiple endocrine disorders was uncommon in dogs. The most common combinations of endocrine disorders in this population of dogs were diabetes mellitus and hyperadrenocorticism, followed by hypoadrenocorticism and hypothyroidism.

Laboratory Abnormalities in Patients with Cancer

In this problem-oriented review of abnormalities associated with cancer, we have emphasized distinctive diagnostic points related to pathogenesis for each condition and outlined how the approach to management is determined by pathogenesis. For abnormalities of the complete blood count, it is important to distinguish between abnormalities directly related to marrow malignancy and abnormalities associated with extramarrow malignancy. Hemopoietic tumors consist of developmentally deficient blood cells produced by a clonal population of malignant stem cells. Tumors infiltrating marrow cause overcrowding in the limited marrow microenviroment. Extramarrow malignancies cause blood abnormalities, but the potential for normal marrow function is present. Abnormalities of blood cells secondary to therapy are usually clearly identified by consideration of clinical history. The initial differential diagnosis for hypercalcemia is malignancy. An aggressive diagnostic approach may be needed to identify the neoplasm, and therapy should incorporate measures to prevent renal failure. Hypoproteinemia and hyperproteinemia may be caused by neoplasia. Monoclonal gammopathies should be identified and may be associated with hyperviscosity syndrome. Hypoglycemia in the adult animal is most frequently caused by insulin-secreting tumors, but it has also been associated with hepatic and other tumors. Increased blood urea nitrogen, creatinine, lipase, amylase, and liver enzyme activities may also be caused by malignancy. Inadequate urine concentrating ability may be caused by hypercalcemia or malignancy-associated renal insufficiency. Hematuria in older animals is suggestive of urinary tract neoplasia. Exfoliated tumor cells may be identified in the urine sediment of these patients.