Neutrophil gelatinase-associated lipocalin in dogs with chronic kidney disease, carcinoma, lymphoma and endotoxaemia.

OBJECTIVES: To measure serum and urine neutrophil gelatinase-associated lipocalin (NGAL) concentrations in healthy dogs and dogs with chronic kidney disease, neoplasia and endotoxaemia. METHODS: Serum and urine NGAL concentrations were measured in 42 healthy dogs, 11 dogs with chronic kidney disease, 12 dogs with carcinoma, 20 dogs with lymphoma and 15 dogs with lipopolysaccharide-induced endotoxaemia. In dogs with chronic kidney disease, NGAL was measured 3 and 6 months later. RESULTS: Compared with healthy controls, dogs with chronic kidney disease (PÄ0·0008), carcinoma (PÄ0·0072) and lymphoma (PÄ0·0008) had elevated serum and urine NGAL and urine NGAL-to-creatinine ratio. Serum and urine NGAL was not significantly different between dogs with chronic kidney disease, carcinoma or lymphoma (Pê0·12). In dogs with non-progressive chronic kidney disease, NGAL concentrations did not change significantly over the 6-month study period. CLINICAL SIGNIFICANCE: NGAL can be elevated by chronic kidney disease and neoplasia, compared with healthy controls. Further research is needed to determine if uNGAL or uNGAL-to-creatinine ratio is more specific than serum levels to detect chronic kidney disease.

Biomarkers in the assessment of acute and chronic kidney diseases in the dog and cat.

In both human and veterinary medicine, diagnosing and staging renal disease can be difficult. Measurement of glomerular filtration rate is considered the gold standard for assessing renal function but methods for its assessment can be technically challenging and impractical. The main parameters used to diagnose acute and chronic kidney disease include circulating creatinine and urea concentrations, and urine-specific gravity. However, these parameters can be insensitive. Therefore, there is a need for better methods to diagnose and monitor patients with renal disease. The use of renal biomarkers is increasing in human and veterinary medicine for the diagnosis and monitoring of acute and chronic kidney diseases. An ideal biomarker would identify site and severity of injury, and correlate with renal function, among other qualities. This article will review the advantages and limitations of renal biomarkers that have been used in dogs and cats, as well as some markers used in humans that may be adapted for veterinary use. In the future, measuring a combination of biomarkers will likely be a useful approach in the diagnosis of kidney disorders.

Serum acute phase protein concentrations in dogs with autoimmune hemolytic anemia.

BACKGROUND: Serial monitoring of acute phase protein (APP) concentrations in canine autoimmune hemolytic anemia (AIHA) has not been reported. HYPOTHESES: Acute canine AIHA is accompanied by an acute phase response (APR) characterized by increased C-reactive protein (CRP) and alpha1-acid glycoprotein (AAG) concentrations and decreased albumin concentrations. ANIMALS: Twenty-seven dogs with AIHA and 11 control dogs. METHODS: Prospective, cohort study. CRP, AAG, and albumin concentrations, white blood cell (WBC) count, and packed cell volume (PCV) were determined at admission (day 1), every 48 hours until death or discharge, and on days 30, 90, 180, and 365. RESULTS: Compared with controls, CRP and AAG concentrations were increased and albumin concentration was decreased in dogs with AIHA (days 1-7; P < .002) and normalized with disease stabilization (days 9-365; P > .05). APP concentrations on day 1 were not predictive of survival, duration of hospitalization, or number of blood transfusions (P= .153-.940). PCV correlated with APP concentrations over time (CRP r=-.600, AAG r=-.665, albumin r= .533; P < .0001) as did WBC count (CRP r= .253, AAG r= .486, albumin r=-.246; P < .006). Day 1 CRP concentration was lower for dogs that received corticosteroids before referral (115.3 microg/mL) compared with dogs that did not (191.2 microg/mL; P= .02). CONCLUSIONS: An APR occurs in canine AIHA. Initial APP concentrations are not predictive of acute survival, correlate with hematologic markers of remission, and normalize rapidly with disease stabilization.

The effect of glucocorticoids on canine lymphocyte marker expression and apoptosis.

BACKGROUND: Glucocorticoids are commonly administered to dogs for the treatment of inflammatory disorders, autoimmunity and cancers such as lymphoma. Despite evidence of clinical efficacy, understanding of the effects of glucocorticoids on cells of the canine immune system is limited. HYPOTHESIS: Glucocorticoids affect the expression of phenotypic markers on canine lymphocytes and induce apoptosis. ANIMALS: Fifteen healthy mixed breed dogs. METHODS: Prospective randomized study. Prednisone was administered orally for 3 days, and cells aspirated from the popliteal lymph node before prednisone administration, and on days 1, 3, 10, 17, 24, and 38, were labeled with antibodies against canine CD3, CD4, CD8alpha, CD18, CD21, CD45, CD45RA, and CD90 molecules, and analyzed by flow cytometry. Additional samples were cultured in media with prednisolone for 24 hours and analyzed by cytometry for marker expression, and by gel electrophoresis for DNA fragmentation. RESULTS: Treatment of dogs with glucocorticoids resulted in reduced (p < or = .05) proportions of CD3 (days 1, 3, 17, and 24), CD4 (days 3 and 10), CD21 (day 1, 3, and 38), CD45RA (day 17) and CD90 (days 1, 10, and 17) expressing lymphocytes, and reduced intensity of CD18 (day 17) and CD45 (day 17 and 24) molecules on nodal lymphocytes. Culture oflymphocytes with prednisolone for 24 hours caused a significant reduction in the expression of all markers (p < or = .05) and DNA fragmentation. CONCLUSIONS AND CLINICAL IMPORTANCE: Glucocorticoids significantly alter the expression of phenotypic markers on canine lymphocytes, and in vitro induce apoptosis. These findings identify potential mechanisms for clinical immunosuppression from glucocorticoid treatment.

Telomerase activity in clinically normal dogs and dogs with malignant lymphoma.

OBJECTIVES: To determine whether telomerase activity was present in lymph nodes, buffy coat, and serum samples from dogs with malignant lymphoma (ML) and in liver, lymph node, buffy coat, and serum samples from clinically normal dogs SAMPLE POPULATION: Tissue specimens and blood samples were obtained from 11 clinically normal adult dogs (age range, 1 to 4 years) and 14 client-owned dogs with ML. PROCEDURE: The telomere repeat amplification protocol assay was used to quantify telomerase activity in the tissues from clinically normal dogs and dogs with ML. RESULTS: Of 11 clinically normal dogs, 8 had lymph node samples, 5 had liver samples, and 1 had buffy coat samples with detectable telomerase activity. None of the serum samples from the clinically normal dogs had detectable telomerase activity. Of 14 dogs with ML, 9 had lymph node samples, 3 had buffy coat samples, and 1 had serum samples with measurable telomerase activity. CONCLUSIONS AND CLINICAL RELEVANCE: Telomerase activity was not specific to tumor cells and overlapped with that found in cells from clinically normal dogs. Telomerase activity in neoplastic lymph nodes was not substantially different from that found in lymph nodes from clinically normal dogs. The determination of telomerase activity cannot be used as a sole diagnostic test for cancer. Therapeutic modalities directed toward the telomerase enzyme may not be feasible in dogs, because somatic tissues from clinically normal dogs possess variable amounts of telomerase activity.

The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs.

In this prospective study, feces of dogs with diarrhea were compared with feces of normal dogs for the presence of Clostridium difficile, C difficile toxins A and B, C perfringens, and C perfingens enterotoxin (CPE). C difficile toxins A, B, or both were present in feces of 18 of 87 (21%) dogs with diarrhea and 4 of 55 (7%) normal dogs (P = 0.03), whereas CPE was present in the feces of 24 of 87 (28%) dogs with diarrhea and 3 of 55 (5%) normal dogs (P = 0.01). C difficile was isolated from 2 of 87 (2%) dogs with diarrhea but was not isolated from the feces of 55 normal dogs, possibly because of poor survival of the organism in fecal samples. C perfringens was isolated from the feces of 23 of 24 (96%) CPE-positive dogs with diarrhea, 52 of 63 (83%) CPE-negative dogs with diarrhea, and 39 of 55 (71%) CPE-negative dogs with normal feces. No correlation was found between C perfringens spore number and the presence of CPE.

Large granular lymphocytic leukemia in a mixed breed dog.

A mixed breed dog was diagnosed with large granular lymphocytic leukemia. Immunophenotypic analysis indicated the lymphocytes were CD3+, CD8+ T cells expressing the alpha beta T cell receptor and a leukointegrin, alpha d. Chemotherapy and splenectomy resulted in an initial reduction in the lymphocyte count.

The effects of prednisone and azathioprine on circulating immunoglobulin levels and lymphocyte subpopulations in normal dogs.

This study investigates serum immunoglobulin (SIg) levels and lymphocyte subpopulations in normal dogs in response to putative immunosuppressive doses of prednisone and/or azathioprine. The objectives were to quantify SIg levels and lymphocyte subpopulations, including Thy-1+, CD4+, CD8+ and B cells, in normal dogs both before and after the administration of prednisone and/or azathioprine at 2 mg/kg, PO, each. Eighteen beagles were divided into 3 groups of 6 dogs each. Blood samples for radial immunodiffusion assay of IgG, IgM and IgA, complete blood count (CBC)and flow cytometry were collected prior to the administration of any drugs and again after 14 d of azathioprine, prednisone or azathioprine and prednisone. Peripheral blood mononuclear cells were isolated using density centrifugation and were incubated with monoclonal antibodies reacting with CD4+, CD8+, Thy-1+ and membrane immunoglobulin. Lymphocyte subsets were quantified using flow cytometry. Azathioprine-treated dogs had no significant changes in SIg levels or lymphocyte subpopulations. Prednisone-treated dogs had significant (P < 0.05) decreases in all SIg levels, all lymphocyte subpopulations and erythrocyte numbers, and had an increase in neutrophil counts. Prednisone and azathioprine-treated dogs had significant (P < 0.05) decreases in serum IgG levels and Thy-1+ and CD8+ lymphocyte subpopulations, with an increase in the CD4:CD8. These dogs also had a significant decrease in erythrocyte number and a significant increase in the monocyte count. These findings suggest that azathioprine and prednisone in combination or prednisone alone may be useful for the treatment of T cell-mediated diseases since decreased circulating T cell levels were demonstrated following treatment. The combination of drugs or azathioprine alone may not be appropriate for treatment of acute or autoantibody-mediated immune disease, because SIg levels were minimally affected by treatment.

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.

Amputation and carboplatin for treatment of dogs with osteosarcoma: 48 cases (1991 to 1993).

Forty-eight dogs with histologically confirmed appendicular osteosarcoma (OSA) entered a prospective clinical trial evaluating treatment with amputation and up to 4 doses of carboplatin given every 21 days. The median disease-free interval (DFI) was 257 days, with 31.2% of the dogs disease-free at 1 year. The median survival time was 321 days, with 35.4% of the dogs alive at 1 year. Dogs with proximal humeral OSA had shorter DFI (P = .016) and survival (P = .037) times than dogs with OSA at other locations. Dogs with lower body weights ( < 40 kg) had longer DFI (P = .0056) and survival (P = .007) times than larger dogs. Survival times for dogs that received carboplatin were statistically longer than those previously reported for amputation alone (P < .001). DFI and survival times are similar to those previously reported for 2 to 4 doses of cisplatin. Carboplatin appears to be a well-tolerated chemotherapeutic drug that can be given safely every 21 days at a dose of 300 mg/m2. Neutropenia was the dose-limiting toxicity in this study.

0-7-21 radiation therapy for the palliation of advanced cancer in dogs.

The 0-7-21 radiation therapy protocol was investigated as a palliative treatment in dogs with advanced malignancies. Twenty-four dogs with a variety of tumor types were irradiated using 800 cGy fractions given on days 0, 7, and 21. Twenty-three dogs were evaluated. Palliative response was assessed using a quality of life instrument developed for veterinary use. This pain score was based on owner response to questions regarding analgesic requirement, activity level, appetite, and degree of lameness in the affected dogs. Seventeen (74%) of the 23 dogs experienced complete pain relief, and 3 (13%) obtained partial relief. Of the 17 dogs that achieved a complete response, pain recurred in 8 at a median time of 70 days. Six dogs were alive and free of pain up to 557 days after irradiation. The 0-7-21 protocol was well tolerated; pain relief occurred quickly, and acute radiation reactions were negligible.

0-7-21 radiation therapy for the treatment of canine oral melanoma.

Eighteen dogs with malignant melanoma of the oral cavity were treated with high-dose per fraction (0-7-21) radiation therapy. Eight hundred cGy was administered on days 0, 7, and 21 for a total dose of 2,400 cGy in 3 weeks. Of 17 dogs evaluated, 9 (53%) had a complete remission and 5 (30%) achieved a partial remission with an overall response rate of 83%. Local failure occurred in 2 of the 9 dogs where a complete response was initially observed. One dog died of intercurrent disease, and one died of metastatic disease without evidence of local recurrence. Five dogs are alive and free of disease 9 to nineteen months from the initiation of therapy. The 0-7-21 protocol was well-tolerated, and acute radiation reactions were low-grade and limited to the skin. The results of this study demonstrate that oral melanomas in dogs are responsive to radiation. 0-7-21 radiation therapy offers a viable alternative to radical excision, especially when tumor volume or location would require cosmetically or functionally debilitating surgery.

Gene transfer into hematopoietic stem cells: long-term maintenance of in vitro activated progenitors without marrow ablation.

Adoptive transfer of genetically modified somatic cells will play an increasingly important role in the management of a wide spectrum of human diseases. Among the most appealing somatic cells as potential gene transfer vehicles are hematopoietic cells, because of their wide distribution and their well-characterized capacities for proliferation, differentiation, and self-renewal. Genes can be readily transferred into short-lived and lineage-restricted hematopoietic cells, but there remains a need to develop reliable methods for gene transfer into hematopoietic stem cells in large animals. In this work, we used a gene transfer approach in which hematopoietic cells in long-term marrow cultures were exposed to the replication-defective retrovirus N2, bearing the reporter gene neo, on multiple occasions during 21 days of culture. Genetically marked cultured autologous cells were infused into 18 canine recipients in the absence of marrow-ablative conditioning. neo was detected by Southern blotting and/or the polymerase chain reaction in the marrow, blood, marrow-derived granulocyte/macrophage and erythroid progenitors, and cultured T cells in dogs after infusion. In most dogs, the proportion of long-term marrow culture cells contributing to hematopoiesis rose during the first 3 months after infusion and peaked within the first 6. The maximal levels attained were between 10% and 30% G418-resistant (neo-positive) granulocyte/macrophage progenitors. At 12 months, five dogs maintained greater than 10% G418-resistant progenitors, and for two of them this level exceeded 20%. Two dogs had greater than 5% G418-resistant hematopoietic progenitors at 24 months after infusion. Our data suggest that very primitive hematopoietic progenitors are maintained in long-term marrow cultures, where they can be triggered into entering the cell cycle. In vivo, these activated cells likely continue normal programs of proliferation, differentiation, and self-renewal. Their progeny can be maintained at clinically relevant levels for up to 2 years without the requirement that endogenous hematopoiesis be suppressed through chemo- or radiotherapy prior to adoptive transfer. Long-term marrow culture cells may thus be ideal targets for gene therapy involving adoptive transfer of transduced hematopoietic cells.

Preparation and transfusion of canine platelet concentrates.

A protocol was developed for preparation of platelet concentrates (PC) to support thrombocytopenic dogs. Four clinically normal dogs with platelet counts that ranged from 200 to 330 x 10(9) platelets/L were used as donors. One unit (450 ml) of blood was collected by venipuncture into a double blood bag. Whole blood (WB) was centrifuged for 4 minutes at 1,000 x g (braking time = 2 minutes, 30 seconds) to prepare platelet-rich plasma (PRP). The PRP was expressed into the satellite bag and was centrifuged for 10 minutes at 2,000 x g (braking time = 2 minutes, 36 seconds). The platelet-poor plasma was expressed, leaving 40 to 70 ml of plasma and the pelleted platelets in the satellite bag. The resulting PC was left undisturbed for 60 minutes to promote disaggregation, and the platelets were then resuspended by gentle manual agitation. Forty-eight PC were prepared. Mean (+/- SD) platelet yield from WB to PRP was 78 (+/- 13)% (range, 35 to 97%); yield from PRP to PC was 94 (+/- 6)% (range, 75 to 100%); and overall yield (PC from WB) was 74 (+/- 13)% (range, 36 to 91%). Mean PC platelet count was 8.0 (+/- 3.0) x 10(10) platelets/PC (range, 2.3 to 13.4 x 10(10) platelets/PC). The WBC content was 0.1 to 2.3 x 10(9) platelets/PC, representing 3 to 74% of WBC in the WB. Hematocrit was 0.1 to 26.2%. Results of bacterial and fungal culturing were negative. The PC were irradiated (18 Gy) and transfused to 5 cross-matched dogs undergoing bone marrow transplantation that developed profound thrombocytopenia of up to 8 weeks' duration.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical and pathological findings in dogs following supralethal total body irradiation with and without infusion of autologous long-term marrow culture cells.

We developed a canine model for autologous bone marrow transplantation (AuBMT) with long-term marrow culture (LTMC) cells. Marrow was harvested from nine normal dogs. Harvests from dogs 2-7 were placed into 21 day LTMC. Cells in LTMC from dogs 4-7 were labelled with the neomycin phosphotransferase gene neo. Dogs were given 60Co total body irradiation (TBI) and then infused with LTMC cells: dog 1 received 500 cGy TBI and 2.08 x 10(8)/kg uncultured marrow cells. Dogs 2-7 received 600-800 cGy TBI and 0.07-0.45 x 10(8)/kg LTMC cells. Dogs 8 and 9 received 600 and 800 cGy TBI, respectively, but no infusion of marrow or LTMC cells. For all dogs, profound myelosuppression developed during week 1 and pyrexia developed during week 2. Enrofloxacin was given from one day before TBI until a peripheral neutrophil count > 1.0 x 10(9)/L was achieved, which eliminated Escherichia coli from feces. Dogs 1, 2 and 5-9 also received gentamicin and/or combination beta-lactam antibiotics. Numerous platelet transfusions were needed to control hemorrhages in all dogs except dog 1. Dog 1 achieved neutrophils > 1.0 x 10(9)/L on day 15, while dogs 2 and 5-9 achieved this count on days 33-48. Dogs 3 and 4 died on days 17 and 18, respectively, of beta-hemolytic streptococcal sepsis and hemorrhage, with no evidence of hematopoiesis at necropsy. The marker gene, neo, was documented in lymphoid and myeloid cells of dogs 5-7 up to 21 months post-AuBMT. Our studies indicate that dogs can recover following supralethal TBI and can survive the delayed engraftment associated with AuBMT using LTMC cells, if they receive intensive platelet and antimicrobial therapy. Used prophylactically for such therapy, enrofloxacin achieved selective intestinal decontamination, but did not prevent sepsis when used as the sole antimicrobial agent during myelosuppression. Furthermore, our studies indicate that infused LTMC cells, at the above doses, can contribute to hematopoietic recovery, but are not essential for recovery following TBI, and do not shorten the period of prolonged profound myelosuppression induced by TBI.

The use of an implantable central venous (Hickman) catheter for long-term venous access in dogs undergoing bone marrow transplantation.

Methods were developed for the insertion and maintenance of long-term central venous catheters in dogs in order to provide reliable venous access during bone marrow transplantation. Single-lumen, 9.6 Fr Hickman catheters with a VitaCuff were used. The catheter was inserted into the jugular vein via a surgical cut-down, and tunnelled subcutaneously to exit over the thoracic spine. Fluoroscopic guidance was necessary to ensure proper positioning of the catheter tip in the right atrium. The catheter was secured at the venous entrance site with a grommet and at the cutaneous exit site with a finger-cuff suture. The exit site was bandaged; dressings were changed daily. Five dogs were studied. Catheter insertion and maintenance techniques were developed using two dogs. For the other three dogs, which developed 7 wk of profound myelosuppression induced by total body irradiation, the catheters were used for blood sampling and infusions of antibiotics, fluids, and blood products. For these three dogs there were 261 total catheter-days. Complete catheter obstruction did not occur. Partial obstruction (inability to withdraw blood) occurred for 13 days with one catheter. The tip of this catheter was in the cranial vena cava. One irradiated dog had a staphylococcal exit site infection for several days after catheter insertion, which resolved with antibiotic therapy. Infections of the subcutaneous tunnel, and catheter associated bacteremia, were not identified. Infectious and hemorrhagic complications of myelosuppression were less severe than in six other dogs where intermittent venipuncture was used for vascular access during radiation induced myelosuppression. In conclusion, long-term central venous catheterization is feasible in dogs during profound myelosuppression and markedly facilitates patient management.

Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors.

Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony-forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst-forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood-derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.