Use of a commercial methylcellulose medium with and without recombinant bovine granulocyte colony-stimulating factor for culturing bovine bone marrow cells

A commercial methylcellulose culture medium, with and without the addition of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF), was utilized for culturing bovine bone marrow cells in a colony-forming unit assay. Bone marrow mononuclear cells were isolated and cultured in a commercial methylcellulose-based medium containing several recombinant human cytokines. Cultures were prepared with and without 100 ng/mL of rbG-CSF. The size and mean number of colonies per plate from culture days 3 to 9 were compared. We concluded that bovine bone marrow colony growth was supported by this culture medium. The addition of rbG-CSF yielded larger and more numerous colonies. There were significantly more colonies on day 3 (P < 0.001), day 4 (P < 0.001), and day 5 (P = 0.03) with rbG-CSF. Both culture media had the highest colony counts on day 5.     

Use of autologous bone marrow mononuclear cells and cultured bone marrow stromal cells in dogs with orthopaedic lesions

The aim of the study is to evaluate the clinical application in veterinary orthopedics of bone marrow mononuclear cells (BMMNCs) and cultured bone marrow stromal cells (cBMSCs) for the treatment of some orthopaedic lesions in the dog. The authors carried out a clinical study on 14 dogs of different breed, age and size with the following lesions: 1 bone cyst of the glenoid rime; 2 nonunion of the tibia; 3 nonunion of the femur; 2 lengthening of the radius; 1 large bone defect of the distal radius;1 nonunion with carpus valgus; 4 Legg-Calvé-Perthés disease. In 9 cases the BMMCNs were used in combination with a three dimensional resorbable osteogenic scaffold the chemical composition and size of which facilitates the ingrowth of bone. In these cases the BMMNCs were suspended in an adequate amount of fibrin glue and then distribuited uniformly on a Tricalcium-Phosphate (TCP) scaffold onto which were also added some drops of thrombin. In 1 case of nonunion of the tibia and in 3 cases of Legg-Calvè-Perthés (LCP) disease the cultured BMSCs were used instead because of the small size of the dogs and of the little amount of aspirated bone marrow. X-ray examinations were performed immediately after the surgery. Clinical, ultrasounds and X-ray examinations were performed after 20 days and then every month. Until now the treated dogs have shown very good clinical and X-ray results. One of the objectives of the study was to use the BMMNCs in clinical application in orthopaedic lesions in the dog. The advantages of using the cells immediately after the bone marrow is collected, are that the surgery can be performed the same day, the cells do not need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with the bone and lastly, the technique is easier and the costs are lower.     

Use of autologous bone marrow mononuclear cells and cultured bone marrow stromal cells in dogs with orthopaedic lesions

The aim of the study is to evaluate the clinical application in veterinary orthopedics of bone marrow mononuclear cells (BMMNCs) and cultured bone marrow stromal cells (cBMSCs) for the treatment of some orthopaedic lesions in the dog. The authors carried out a clinical study on 14 dogs of different breed, age and size with the following lesions: 1 bone cyst of the glenoid rime; 2 nonunion of the tibia; 3 nonunion of the femur; 2 lengthening of the radius; 1 large bone defect of the distal radius;1 nonunion with carpus valgus; 4 Legg-Calvé-Perthés disease. In 9 cases the BMMCNs were used in combination with a three dimensional resorbable osteogenic scaffold the chemical composition and size of which facilitates the ingrowth of bone. In these cases the BMMNCs were suspended in an adequate amount of fibrin glue and then distribuited uniformly on a Tricalcium-Phosphate (TCP) scaffold onto which were also added some drops of thrombin. In 1 case of nonunion of the tibia and in 3 cases of Legg-Calvè-Perthés (LCP) disease the cultured BMSCs were used instead because of the small size of the dogs and of the little amount of aspirated bone marrow. X-ray examinations were performed immediately after the surgery. Clinical, ultrasounds and X-ray examinations were performed after 20 days and then every month. Until now the treated dogs have shown very good clinical and X-ray results. One of the objectives of the study was to use the BMMNCs in clinical application in orthopaedic lesions in the dog. The advantages of using the cells immediately after the bone marrow is collected, are that the surgery can be performed the same day, the cells do not need to be expanded in vitro, they preserve their osteogenic potential to form bone and promote the proper integration of the implant with the bone and lastly, the technique is easier and the costs are lower.     

In vitro effect of recombinant human granulocyte colony-stimulating factor on canine neutrophil apoptosis

Apoptosis is essential in eliminating neutrophils (polymorphonuclear leukocytes: PMNs) in animals. The suppression of PMN apoptosis is believed to be beneficial in eradicating pathogens and is implicated in the pathogenesis of human inflammatory diseases. In the present study, canine PMNs were stimulated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to investigate the in vitro effect on the apoptosis of canine PMNs. Apoptotic cell rates were assessed by flow cytometry in relation to the ability of PMNs to produce reactive oxygen species (ROS). Canine PMN apoptosis was markedly suppressed by rhG-CSF treatment, in association with the retention of the PMN ability to produce ROS. The addition of cycloheximide abolished this suppression by rhG-CSF. Moreover, canine PMNs, which were stimulated by rhG-CSF, expressed high levels of anti-apoptotic mcl-1 gene mRNA, as quantified by real-time polymerase chain reaction method. The results suggest that PMNs, stimulated by G-CSF, could work effectively over a longer period to eliminate pathogens, and that the prolongation of the PMN life-span might occasionally aggravate tissue injuries in dogs. In addition, the suppression of PMN apoptosis seems to be mediated by the induction of anti-apoptotic mcl-1 gene expression.     

Effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on leukocyte count and survival rate of dogs with parvoviral enteritis.

Dogs with clinical signs consistent with parvoviral enteritis and leukopenia (total leukocyte count < 5.0 x 10(9) l(-1)) were included in this randomised double-blind study (treatment group: n = 22; control group: n = 21). The dogs in the treatment group received a subcutaneous daily injection of 10 microg kg(-1) of recombinant human granulocyte colony-stimulating factor (rhG-CSF) for 5 days. Clinical and blood investigations were performed prior to the first injection, daily during the treatment period and on the day after treatment ended, and then once more, 26 days after the first injection. During the study, no significant differences were found between the two groups with respect to survival rate (treatment group: 68 per cent; control group: 71 per cent, P > 0 4, Fisher-Test) and other clinical findings. Similarly the total leukocyte count, neutrophil count and other haematologic and biochemical parameters did not differ significantly between the groups, based on differences from initial values (P > 0 05). Consequently, the use of rhG-CSF in the treatment of dogs with parvoviral enteritis cannot be recommended.     

Effect of recombinant human granulocyte colony stimulating factor on lymphocyte blastogenesis in healthy dogs

Effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the number and blastogenesis of lymphocytes were evaluated in clinically healthy dogs treated subcutaneously with rhG-CSF at a dose of 2.5 microg/kg for 3 days. Significant increases in the number of leukocytes and segmented neutrophils were observed after the administration of rhG-CSF. The number of lymphocytes also increased on days 1 and 2 after the treatment. Activities of phytohemagglutinin, concanavalin A, and pokeweed mitogen-induced lymphocyte blastogenesis (LB) were augmented to twice the pretreatment levels by the administration of rhG-CSF. These results suggested that administration of rhG-CSF activated lymphocyte functions such as LB in healthy dogs.     

Hematologic and bone marrow changes after short- and long-term administration of two recombinant bovine granulocyte colony-stimulating factors.

Colony-stimulating factors are a category of glycoproteins that are instrumental in the regulation of hematopoiesis and inflammation. This investigation documented the clinical bone marrow and peripheral blood responses to short-term and long-term administration of a recombinant bovine granulocyte colony-stimulating factor (rb-GCSF) and an analog, where the cysteine at position 17 was substituted with a serine (rb-GCSF ser17). The colony-stimulating factors produced the expected changes in the hematologic findings of the bovine subjects in the study, and there was a cell-specific response to the compounds. The sustained neutrophilia in the long-term study indicates that the bovine species can tolerate the administration of recombinant forms of bovine GCSF for extended periods of time without detectable adverse side effects. The neutrophils from the short-term study revealed no apparent fluctuation, either as enhanced or reduced capability to reduce nitro blue tetrazolium as compared to pretreatment neutrophils. The administration of both recombinant forms of GCSF produced large increases in the bone marrow myeloid:erythroid (M:E) ratio concomitantly with the neutrophilias. This is the first preliminary report documenting the bone marrow response of cattle to the native and recombinant (rb-GCSF ser17) forms of bovine GCSF.     

Effects of recombinant canine granulocyte colony-stimulating factor on white blood cell production in clinically normal and neutropenic dogs.

Recombinant canine granulocyte colony-stimulating factor (rcG-CSF) was administered to clinically normal dogs, cyclic-hematopoietic dogs, and dogs undergoing autologous bone marrow transplantation, to determine whether rcG-CSF could be used to stimulate WBC production and function in normal and neutropenic dogs. To the normal dogs, rcG-CSF was administered by SC injection at rates of 1 microgram/kg of body weight, q 12 h; 2 micrograms/kg, q 12 h; or 5 micrograms/kg, q 12 h. A significant dose-dependent increase in the WBC count resulted from the stimulation of bone marrow progenitor cells. The increased WBC count was characterized by mature neutrophilia and monocytosis. Neutrophil myeloperoxidase and phagocytic activity were normal in rcG-CSF-treated normal dogs, demonstrating the production of normal functional neutrophils in response to rcG-CSF treatment. Recombinant canine G-CSF prevented neutropenia and associated clinical signs but did not completely eliminate the cycling of neutrophils in cyclic-hematopoietic dogs when it was administered at rates of 1 microgram/kg, q 12 h, and 2.5 micrograms/kg, q 12 h. The time to bone marrow reconstitution was not decreased in dogs treated with rcG-CSF at a rate of 2.5 micrograms/kg, q 12 h, for 13 days following autologous bone marrow transplantation. On the basis of our findings, we suggest that treatment with rcG-CSF is an effective way to stimulate myelopoiesis in dogs, but that the dose of rcG-CSF required to stimulate WBC production will vary depending on the cause of neutropenia. Recombinant canine G-CSF should be useful in stimulating production and maintaining function of WBC for treatment of clinical diseases seen commonly in veterinary practice.     

Recombinant canine granulocyte colony-stimulating factor accelerates recovery from cyclophosphamide-induced neutropenia in dogs

In dogs injected intravenously with 400mg/m(2) cyclophosphamide (CPA), the peripheral neutrophil count decreased to less than 1000 cells/μL in 5-9 days. Treatment with purified recombinant canine granulocyte colony-stimulating factor (rcG-CSF), produced by brevibacillus expression system, at the nadir of the granulocyte count accelerated recovery from the CPA-induced neutropenia by 1-3 days. Therapeutic administration of rcG-CSF at doses of 2.5-10 μg/kg did not show any significant difference on the severity of neutropenia (the period that granulocyte counts were less than 2000 cells/μL). Administration of 2.5 μg/kg rcG-CSF 3 times per day 2-4 days or 3-5 days after CPA treatment not only accelerated recovery but also decreased the severity of neutropenia. No clinical signs of the rcG-CSF were observed. These results showed that the rcG-CSF is effective for treatment of neutropenia in dogs.     

Hematologic effects of subcutaneous administration of recombinant human granulocyte colony-stimulating factor (filgrastim) in healthy alpacas

OBJECTIVE: To determine the effects of SC administration of filgrastim on cell counts in venous blood and bone marrow of healthy adult alpacas. ANIMALS: 10 healthy alpacas. PROCEDURES: Alpacas were randomly assigned to receive treatment with filgrastim (5 microg/kg, SC; n=5) or an equivalent volume of physiologic saline (0.9% NaCl) solution (5) once a day for 3 days. Blood samples were obtained via jugular venipuncture 1 day prior to treatment and once a day for 5 days commencing 24 hours after the first dose was administered. Complete blood counts were performed for each blood sample. Bone marrow aspirates were obtained from the sternum of each alpaca 48 hours before the first treatment was administered and 72 hours after the third treatment was administered. Myeloid-to-erythroid cell (M:E) ratio was determined via cytologic evaluation of bone marrow aspirates. RESULTS: In filgrastim-treated alpacas, substantial increases in counts of WBCs and neutrophils were detected within 24 hours after the first dose was administered. Band cell count and percentage significantly increased 24 hours after the second dose. Counts of WBCs, neutrophils, and band cells remained high 48 hours after the third dose. Red blood cell counts and PCV were unaffected. The M:E ratio also increased significantly after treatment with filgrastim. CONCLUSIONS AND CLINICAL RELEVANCE: Filgrastim induced rapid and substantial increases in numbers of circulating neutrophils and M:E ratios of bone marrow in healthy alpacas. Therefore, filgrastim may be useful in the treatment of camelids with impaired bone marrow function.     

Prevalence of leukemic blood and bone marrow in dogs with multicentric lymphoma

Multicentric lymphoma was diagnosed in 53 dogs. A study was performed to evaluate the prevalence of leukemic involvement in blood samples, bone marrow aspirates, and bone marrow core biopsy specimens at the time of initial diagnosis. Data indicated that 57% (30/53) of the dogs were leukemic when all materials were considered relative to the presence of cellular atypia or immaturity and abnormal tissue distribution. In the 30 leukemic dogs, detection was made in the specimens with the following frequency: 15 in blood (50%), 18 in bone marrow aspirates (60%), and 29 in bone marrow core biopsy specimens (97%). Five cases (17%) were only detected by core biopsy examination, even when dogs with bone marrow lymphocytosis of greater than 15% of nucleated cells were considered leukemic. Nondiffuse histologic colonization patterns accounted for the lack of correlation between the type of bone marrow specimens. Clinical staging for treatment response and prognosis was best determined by evaluation of concurrently obtained blood samples, bone marrow aspirates, and bone marrow core biopsy specimens.     

Cardiovascular changes after bone marrow transplantation in dogs with mucopolysaccharidosis I

Five dogs with mucopolysaccharidosis I, 3 of which had been treated with bone marrow transplantation (BMT), were evaluated for 20 months with electrocardiography, thoracic radiography, and M-mode and 2-dimensional echocardiography. Treated and untreated (control) dogs had widened P waves on ECG. Thoracic radiographs remained normal for all dogs throughout the study. Thickening of the mitral valve was observed on echocardiograms of dogs in both groups, but the untreated dogs appeared to have thicker valves. Concentrations of glycosaminoglycans in the mitral valves and myocardium were higher in control dogs than in treated dogs. Markedly large aortic root diameters were observed on echocardiograms in both untreated dogs, but aortic root diameters remained normal in treated dogs. Echocardiography, but not electrocardiography, was useful in monitoring heart enlargement in each dog. Dogs treated with BMT generally had less severe cardiac changes and slower disease progression than control dogs.     

Hematologic changes after total body irradiation and autologous transplantation of hematopoietic peripheral blood progenitor cells in dogs with lymphoma

Dogs with and without lymphoma have undergone hematopoietic cell transplantation in a research setting for decades. North Carolina State University is currently treating dogs with B- and T-cell lymphoma in a clinical setting with autologous peripheral blood progenitor cell transplants, using peripheral blood CD34+ progenitor cells harvested using an apheresis machine. Complete blood counts were performed daily for 15 to 19 days posttransplantation to monitor peripheral blood cell nadirs and subsequent CD34+ cell engraftment. This study documents the hematologic toxicities of total body irradiation in 10 dogs and the subsequent recovery of the affected cell lines after peripheral blood progenitor cell transplant, indicating successful CD34+ engraftment. All peripheral blood cell lines, excluding red blood cells, experienced grade 4 toxicities. All dogs had ≥ 500 neutrophils/μl by day 12, while thrombocytopenia persisted for many weeks. All dogs were clinically normal at discharge.     

Clinical application of recombinant human bone morphogenetic protein-2 in 4 dogs

OBJECTIVE: To describe outcome in dogs with insufficient bone healing treated with recombinant human bone morphogenetic protein-2 (rhBMP-2). STUDY DESIGN: Retrospective study. ANIMALS: Four dogs clinically affected with delayed union or nonunion bone healing. METHODS: Medical records were reviewed for signalment, clinical problem, treatment, and outcome. RESULTS: Four dogs that had delayed- or nonunion of bone fracture, osteotomy, or arthrodesis were treated with either minimally invasive, fluoroscopically guided, percutaneous administration or direct surgical application of rhBMP-2. Doses used ranged from 0.2 to 1.6 mg of rhBMP-2. In 3 dogs, a calcium phosphate matrix (CPM) carrier was used whereas in 1 dog commercially prepared rhBMP-2 impregnated in an absorbable collagen sponge (INFUSE Bone Graft) was used. This latter dog had osteomyelitis associated with implant infection before rhBMP-2 administration. Rapid radiographic union was noted in all dogs with excellent long-term outcome. Adverse effects were minimal and included transient worsening of lameness after percutaneous administration of rhBMP-2 in 2 dogs. CONCLUSIONS: rhBMP-2 stimulated rapid bone formation at delayed- or nonunion sites resulting in radiographic bone union with minimal adverse effects and excellent long-term outcome in 4 dogs. CLINICAL RELEVANCE: Direct intraoperative administration or fluoroscopically guided, minimally invasive delivery of rhBMP-2 may be an effective treatment modality for bone delayed- or nonunions and could potentially be used to stimulate new bone production in a variety of orthopedic surgical conditions in dogs.     

Pathological changes in the bone marrow of dogs with leishmaniosis

Bone marrow aspiration smears from 15 dogs naturally infected with leishmania were evaluated. Three of the dogs showed no clinical signs, six had up to three clinical signs and six had more than three. The most common pathological features of the bone marrow were megakaryocytic dysplasia in 10 of the dogs, erythrophagocytosis in eight, erythroid dysplasia in two and emperipolesis in two. The megakaryocytic and erythroid dysplasia were probably related to an increased number of marrow macrophages producing high levels of tumour necrosis factor alpha and interferon gamma. Six of the dogs with clinical signs showed bone marrow dysplastic features and erythrophagocytosis, suggesting that leishmaniosis could be the unique cause of both conditions.     

A combination chemotherapy protocol with dose intensification and autologous bone marrow transplant (VELCAP-HDC) for canine lymphoma

Twenty-eight dogs with lymphoma were treated with a 12-week, 5-drug chemotherapy protocol concluding with high-dose cyclophosphamide supported by autologous bone marrow transplants. A dose escalation design was used to determine the maximum tolerated cyclophosphamide dose (MTD) in this setting. Three cyclophosphamide dose levels were given: 300 mg/ m2 IV (groupl, 3 dogs), 400 mg/m2 IV (group 2, 12 dogs), and 500 mg/m2 IV (group 3, 13 dogs); and the MTD was 500 mg/m2 IV. Toxicity was common but mild, and the dose-limiting toxicity was myelosuppression, specifically neutropenia. No dog died as a result of treatment-related toxicity. One dog in group 3 developed fever, neutropenia, and presumed sepsis and responded promptly to routine management. No other dog required hospitalization. Lower stage and higher cyclophosphamide dose (both increasing dose [study groups 1-3], and the highest dose [group 3]) compared with the lower doses combined (groups 1 and 2) were significantly associated with longer remission duration (all P < .0001). Median remission duration for dogs in group 3 was 54 weeks, compared with 21 weeks for dogs in groups 1 and 2 combined. Factors associated with longer survival time were lower stage (P = .042) and higher cyclophosphamide dose (both increasing dose [study groups 1-3], and the highest dose [group 3] compared with the lower doses combined [groups 1 and 2]) (P = .027). Median survival time for dogs in group 3 was 139 weeks, compared with 43 weeks and 68 weeks for dogs in groups 1 and 2, respectively.