Principles of peripheral blood mononuclear cell apheresis in a preclinical canine model of hematopoietic cell transplantation

BACKGROUND: Preclinical studies of peripheral blood mononuclear cell (PBMC) transplantation conducted in a well-established canine hematopoietic cell transplantation (HCT) model have been successfully translated to human patients over the past 5 decades. OBJECTIVE: We retrospectively investigated the safety and feasibility of PBMC apheresis in the canine model of HCT by analyzing apheresis parameters, cell yields, and the impacts of donor-related and apheresis-related variables on collection yields and donor stability. ANIMALS: One hundred and twenty dogs that underwent PBMC aphereses were evaluated. METHODS: Aphereses were performed with a COBE Spectra blood separator and a central dual-lumen catheter, with or without recombinant canine granulocyte colony-stimulating factor (rcG-CSF) stem cell mobilization. RESULTS: Aphereses from dogs not given rcG-CSF yielded an average volume of 280 +/- 42 mL containing an average of 15,086 +/- 9,834 leukocytes/mL. Aphereses from dogs given rcG-CSF yielded an average volume of 261 +/- 55 mL containing an average of 39,711 +/- 24,488 leukocytes/mL. Higher pre-apheresis white blood cell (WBC) counts correlated with higher apheresis WBC yields (R=0.50, P<.0001). The correlations of collection time, inlet volume, and collection flow rate on WBC yields were statistically significant but only weak to moderate in magnitude (R=0.34, P=.0001; R=0.38, P=.0006; R=0.26, P=.002, respectively) as were the correlations of collection time and inlet volume on collection volumes (R=0.30, P=.002; R=0.42, P<.0001, respectively). All dogs recovered promptly after PBMC aphereses and catheter removal, without complications. CONCLUSIONS AND CLINICAL IMPORTANCE: These data may be useful for translating PBMC apheresis technology to the field of veterinary oncology for the treatment of dogs with hematologic malignancies.     

Transplantation of CD6-depleted peripheral blood stem cells after DLA-haploidentical bone marrow transplantation contributes to engraftment and tolerance in a preclinical model of stem cell transplantation

Human leukocyte antigen (HLA)-haploidentical stem cell transplantation is an opportunity for nearly all patients lacking an HLA matched stem cell donor. However, graft rejection and graft-versus-host disease (GvHD) as well as infectious complications still result in high treatment-related mortality. Here, we used the dog as a preclinical model for the study of tolerance induction with the aim to optimize and to improve a clinical protocol of haploidentical stem cell transplantation. For this purpose CD6-depleted peripheral blood stem cells (PBSCs) were transfused 6d after transplantation of unmodified bone marrow from dog leukocyte antigen (DLA)-haploidentical littermate donors in order to induce immune tolerance. Besides hematopoietic stem cells CD6-depleted PBSC contain, NK cells and a minority of suppressive CD8-positive cells that may suppress activated T lymphocytes. Recipients were conditioned with, cyclophosphamide and antithymocyte globulin (ATG) preceded by a transfusion of donor buffy coat and either 1, 2 or 3 × 3.3 Gy total body irradiation (TBI). Postgrafting immunosuppression was limited to 30 d of cyclosporine and methotrexate. The additional administration of CD6-depleted PBSCs after unmodified marrow could not prevent GvHD, but it may improve engraftment and chimerism after conditioning with 2 × 3.3 Gy TBI. Reasons for incomplete suppression and possible improvements for clinical applications are discussed.     

The effects of femoral stem and neck length on cement strains in a canine total hip replacement model

OBJECTIVE: To determine the effects of femoral prosthesis stem length and head size on cement strains in a canine hip replacement system. STUDY DESIGN: An in vitro experimental model. SAMPLE POPULATION: (1) Three standard and 3 1-cm shortened femoral implants with +3 femoral heads. (2) Two standard implants with +0, +3, and +6 femoral heads. METHODS: Femoral stems were embedded in polymethylmethacrylate cement. A uniaxial proximodistal-oriented strain gauge was applied to the cement on the medial and lateral aspects of the construct 1.5 cm, 6.0 cm and 7.0 cm distal to the collar. Each construct with a +3 femoral head was mounted in a materials testing system. An axial compressive load (0-200 N) was applied to the femoral head and cement strains were recorded. Additionally, 2 standard length constructs were also tested with +0 and +6 femoral heads. The effects of stem length and neck length on cement strains were assessed with analysis of variance. RESULTS: Strains increased at all locations with increasing loads for all constructs. Shorter implants had higher strains by 152% and 171%, lateral (P =.003) and medial (P =.0025) to the stem tip. No significant strain differences were noted, at any strain gauge location, between different neck lengths (P values ranged from.20 to.67). CONCLUSIONS: Although a shorter implant stem has a potential to improve implant fit, it led to significantly higher cement strains that may increase the risk for aseptic loosening. Changes in femoral neck length did not significantly affect cement strains under the conditions tested. CLINICAL RELEVANCE: Shortening of the femoral stem currently cannot be recommended in canine total hip replacement. The existing use of variable neck lengths likely does not increase the risk of failure of the femoral stem.     

In vivo cell tracking of canine allogenic mesenchymal stem cells administration via renal arterial catheterization and physiopathological effects on the kidney in two healthy dogs

Stem cell therapy is being special premise for various renal diseases. However, there is limited literature on localization and pathologic and functional effects of allogenic mesenchymal stem cells (MSCs) in healthy dogs. Two healthy dogs were included in this study. Canine MSCs (cMSCs) were cultured from canine bone marrow and incubated with superparamagnetic iron oxide (SPIO) for in vivo cell tracking via MR imaging. The dogs were given the MSC (3 × 10(6) cells) into a renal artery via femoral artery catheterization. Follow-up serial renal assessments included ultrasonography and MRI, serum chemistry, urine analysis, and renal clearance tests. The dogs were euthanized at days 8 and 35 respectively for histopathologic evaluation of kidney. Strong hypointensity in MRI was detected in the treated renal cortex the day after cMSCs infusion. However they disappeared from MR image by the 8th day. Of the serum chemistry tests, serum hepatic enzymes (ALT, AST) were significantly elevated for one week after cMSCs treatment. Histopathological findings also revealed infiltration of SPIO-containing cells into the parenchyma of kidney. On 35th day, histopathology, glomerular atrophy, tubular necrosis, and mineralization were found in the subcapsular cortex, with fibrosis of the interstitial tissues. In vivo MRI studies of stem cells were useful in determining the sequential location of stem cells in the renal parenchyma of healthy dogs. Allogenic stem cells administered via renal artery caused inflammation, tubular necrosis, mineralization, and fibrosis without functional complications.     

Early exposure to probiotics in a canine model of atopic dermatitis has long-term clinical and immunological effects

Probiotics modulate the immune response and may have protective effects against atopic dermatitis (AD). Clinical trials using dogs with spontaneous disease are limited by confounding factors such as different diets, environments and sensitizations while a more controlled evaluation is possible using experimental models. A validated model of canine AD showed that early exposure to Lactobacillus rhamnosus GG (LGG) significantly decreases allergen-specific IgE and partially prevents AD in the first 6 months of life. This study is a follow-up three years after discontinuation of LGG. Clinical signs were evaluated after allergen challenge with ragweed, timothy, Dermatophagoides farinae. Allergen-specific IgE, IL-10 and TGF-β were measured on the 1st day of challenge, before allergen exposure. Normal dogs were included as controls. Analyses included seven dogs in the non-probiotic and nine in the probiotic litter. For clinical scores, a 2-Group × 9-Time Analysis of Variance showed significant effects of group (p=0.0003, probiotic相似文献   

Evaluation of the usefulness of sensitization to aeroallergens as a model for canine atopic dermatitis in genetically predisposed Beagles

OBJECTIVE: To evaluate a model for atopic dermatitis (AD) and to measure the effect of sensitization in Beagles genetically predisposed to produce high serum concentrations of allergen specific IgE. ANIMALS: 22 laboratory Beagles. PROCEDURE: Seventeen dogs were sensitized from birth to 3 allergens (recombinant birch pollen, Dermatophagoides pteronyssinus, and D farinae). Five nonsensitized dogs from the same litters served as controls. Clinical scoring, regular intradermal testing, measurement of serum concentrations of allergen-specific IgE, and collection of biopsy specimens of skin at 23, 32, and 43 weeks of age were performed. Serial tissue sections were stained for identification of IgE+ cells, mast cells and their subtypes, T-cells, Langerhans cells, and major histocompatibility complex class-II+ cells. At the age of 15 months, dogs were continuously exposed to 2 microg of mite allergen/g of dust. RESULTS: Sensitized dogs had positive intradermal test reactions and significantly higher serum concentrations of allergen specific IgE, compared with nonsensitized dogs. In sensitized and nonsensitized dogs, a significantly higher number of mast cells was found at predilection sites, compared with the control biopsy site. The number of mast cells at predilection sites increased with age. Sensitization significantly increased the number of epidermal Langerhans cells by 23 weeks of age. The number of epidermal Langerhans cells significantly increased in nonsensitized dogs by 32 weeks of age. Clinical scoring only revealed mild transient erythema in some dogs. CONCLUSIONS: increases in concentrations of serum allergen-specific IgE and exposure to allergens is not sufficient to induce clinical signs of AD in genetically predisposed dogs.     

Designing clinical trials in canine spinal cord injury as a model to translate successful laboratory interventions into clinical practice

Many interventions have been shown to improve outcome after experimental spinal cord injury in laboratory animals. The challenge now is to determine whether any of these can be translated to become an efficacious therapy for clinical lesions - a process that is often difficult and frequently fails. Here, we discuss the steps that are required to make this transition and the need for rigorous clinical trials. A key component is an outcome measure that is amenable to statistical analysis; we describe methods that we have developed to accurately measure function after spinal cord injury in dogs. The general methodology may have parallels in the development of veterinary models to test putative therapies for other diseases of humans and animals.     

Cellular localization of Visudyne® as a function of time after local injection in an in vivo model of squamous cell carcinoma: an investigation into tumor cell death

Objective To evaluate the effects of time on cellular localization of Visudyne^® after local injection. Animals Twenty athymic nude mice. Procedures A squamous cell carcinoma (SCC) cell line (A‐431) was injected into right and left dorsolumbar subcutaneous tissue of each mouse, representing treatment (T) and control (C) tumors. In experiment 1 (Exp 1; n = 10) and 2 (Exp 2; n = 10), the T tumors received a local injection of Visudyne^® (0.1 mg/cm³), and C tumors received an equal dose of 5% dextrose in water (D5W). Mice were randomly subdivided into two groups (A and B; n = 5 per group). Mice in Exp 1A and B were sacrificed 1 and 30 min after local injection, respectively. Experiment 1A and B tumors were evaluated by fluorescence microscopy to determine drug localization. Experiment 2A and B tumors were exposed to LED illumination 1 and 30 min after injection, respectively, and evaluated by transmission electron microscopy (TEM) to determine ultrastructural tumor cell damage. Results Fluorescence was detected within the cytoplasm of T tumors in both Exp 1A and B. Significance was detected in fluorescence intensity between T1 min vs. T30 min (P = 0.03) and between T1 min and C1 min tumors (P = 0.01), respectively. Tumors in Exp 2A and B demonstrated evidence of apoptotic cell death. Conclusions Fluorescence microscopy demonstrated higher Visudyne^® concentration within SCC cytoplasm of 1 min compared with 30‐min tumors. Transmission electron microscopy results revealed that tumors treated by photodynamic therapy (PDT) within 30 min of local injection undergo cellular apoptosis.     

Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background

In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro.

Methods

Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 μM for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples ≥ 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors.

Results

Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction ≥ 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed.

Conclusion

Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.