Non-fucosylated CB CD34+ cells represent a good target for enforced fucosylation to improve engraftment following cord blood transplantation.

BACKGROUND AIMS: Despite ethnic diversity and ready availability of cryopreserved, human leukocyte antigen-typed cord blood (CB), delayed engraftment remains a significant hurdle to successful CB transplantation. Suboptimal homing of CB hematopoietic stem and progenitor cells (HSPCs) to the hematopoietic microenvironment (HM) is thought to be responsible and due to low levels of HSPC fucosylation. Fucosylation (decoration with sialyl-LewisX) may improve HSPC homing to HM by increasing the strength of HSPC/E-selectin interactions, where E-selectin is constitutively expressed by HM microvasculature. Enforced fucosylation of CB HSPCs using fucosyltransferases, increases the rate and magnitude of engraftment in xenogeneic transplant models. However, it is unclear whether endogenously fucosylated and non-fucosylated CB HSPC are qualitatively identical or whether endogenous fucosylation marks a qualitative difference between CB HSPC. If qualitatively identical, non-fucosylated CB HSPCs represent a good target for enforced fucosylation with improved engraftment conferred on an increased number of otherwise qualitatively identical HSPC. If qualitatively different, then conferring engraftment upon a majority, possibly lower "quality," non-fucosylated HSPCs by enforced fucosylation might inadvertently compromise engraftment. METHODS: Functional (xenogeneic engraftment, colony-forming unit and selectin-binding assays) and phenotypic analyses of fluorescence-activated cell sorting-isolated, endogenously fucosylated and non-fucosylated CB CD34+ cells were performed. RESULTS: Endogenous fucosylation of CB HSPCs exists as a continuum. Endogenously fucosylated HSPCs engrafted more efficiently in a xenogeneic transplantation model than non-fucosylated HSPCs. Outside of the differences in endogenous fucosylation, no other qualitative (functional and/or phenotypic) differences were identified. DISCUSSION: The majority of endogenously non-fucosylated CB HSPCs represent a good target for enforced fucosylation with the goal of improving engraftment following CB transplantation.

Fucosylation with fucosyltransferase VI or fucosyltransferase VII improves cord blood engraftment.

BACKGROUND AIMS: Advantages associated with the use of cord blood (CB) transplantation include the availability of cryopreserved units, ethnic diversity and lower incidence of graft-versus-host disease compared with bone marrow or mobilized peripheral blood. However, poor engraftment remains a major obstacle. We and others have found that ex vivo fucosylation can enhance engraftment in murine models, and now ex vivo treatment of CB with fucosyltransferase (FT) VI before transplantation is under clinical evaluation (NCT01471067). However, FTVII appears to be more relevant to hematopoietic cells and may alter acceptor substrate diversity. The present study compared the ability of FTVI and FTVII to improve the rapidity, magnitude, multi-lineage and multi-tissue engraftment of human CB hematopoietic stem and progenitor cells (HSPCs) in vivo. METHODS: CD34-selected CB HSPCs were treated with recombinant FTVI, FTVII or mock control and then injected into immunodeficient mice and monitored for multi-lineage and multi-tissue engraftment. RESULTS: Both FTVI and FTVII fucosylated CB CD34⁺ cells in vitro, and both led to enhanced rates and magnitudes of engraftment compared with untreated CB CD34⁺ cells in vivo. Engraftment after treatment with either FT was robust at multiple time points and in multiple tissues with similar multi-lineage potential. In contrast, only FTVII was able to fucosylate T and B lymphocytes. CONCLUSIONS: Although FTVI and FTVII were found to be similarly able to fucosylate and enhance the engraftment of CB CD34⁺ cells, differences in their ability to fucosylate lymphocytes may modulate graft-versus-tumor or graft-versus-host effects and may allow further optimization of CB transplantation.

Radiographic evaluation of femoral torsion and correlation with computed tomographic techniques in labrador retrievers with and without cranial cruciate ligament disease.

OBJECTIVES: To (1) develop a technique to determine the anteversion angle (AA) of the femur on a single radiograph; (2) determine the correlation between this technique and other published radiographic and computed tomographic (CT) methods; and (3) compare the diagnostic outcome of these methods in determining the level at which femoral torsion occurred in Labrador Retrievers with cranial cruciate ligament (CCL) deficiency. STUDY DESIGN: Cross-sectional clinical study. ANIMALS: Mature pure-bred Labrador Retrievers (n = 30). METHODS: Pelvic limbs (n = 28) of 14 dogs without CCL deficiency were classified as control, whereas limbs of 16 dogs (18 limbs) with CCL deficiency were considered as diseased. Femoral torsion was evaluated using radiography and CT and variables were compared among limb groups by use of a mixed-model ANOVA, with P < .05 considered significant. RESULTS: There was a significant association between biplanar and lateral plane AAs but neither correlated with CT assessment of femoral torsion. On CT, a significant correlation was identified between overall AA and each of the distal, proximal, and femoral head trochanteric angles. Biplanar and lateral plane AAs did not differ between normal and CCL deficient limbs. On CT, overall and distal AAs were increased in CCL deficient limbs compared to control. CONCLUSION: Biplanar determination of femoral torsion can be estimated based on a single lateral radiograph but the results will be inaccurate as only CT identified and localized the site of femoral torsion.

A public health academic-practice partnership to develop capacity for exercise evaluation and improvement planning.

In December 2006, Congress passed the Pandemic and All-Hazards Preparedness Act to improve the nation's public health preparedness and response capabilities. It includes the role of Centers for Public Health Preparedness (CPHPs) to establish a competency-based core curriculum and perform evaluation of impact on newly developed materials. The Heartland Center for Public Health Preparedness (HCPHP) at the Saint Louis University School of Public Health is part of the Centers for Disease Control and Prevention national CPHP network and is engaged with state and regional partners in workforce development, preparedness planning, evaluation, and multi-year exercise and training cycles. This includes development, implementation, and evaluation of the HCPHP Exercise Evaluation Training Program to improve the competence and capacity for exercise evaluation and improvement planning. This program is designed to enhance quality improvement and performance measurement capabilities to identify increase of workforce competence over time (maturity).

Morphometric characteristics of the pelvic limb musculature of Labrador Retrievers with and without cranial cruciate ligament deficiency.

OBJECTIVE: To identify morphometric characteristics of the pelvic limb musculature associated with the development of cranial cruciate ligament (CCL) deficiency in Labrador Retrievers. STUDY DESIGN: Cross-sectional clinical study. ANIMALS: Pure-bred female (n=18) and male (n=12) Labrador Retrievers with (n=16) and without (n=14) CCL deficiency. METHODS: Muscle conformation of pelvic limbs was evaluated by physical examination, radiography (widths of quadriceps, hamstring, and gastrocnemius were expressed relative to tibial length and to each other), and dual-energy X-ray absorptiometry (DEXA, lean contents of quadriceps, hamstring, and gastrocnemius were expressed relative to tibial length and to each other). Pelvic limbs of dogs without CCL deficiency were classified as normal (n=28 limbs), whereas those with CCL deficiency were considered diseased (n=18 limbs) or sound contralateral to CCL deficiency (n=10 limbs). Variables were compared between groups using mixed models analysis of variance, with P<.05 considered significant. RESULTS: The ratios of quadriceps width to tibial length (P=.008), hamstring width (P=.013), and gastrocnemius width (P=.005) on lateral radiographs were lower in diseased limbs than controls. The mass of hamstring muscles in CCL deficient limbs was similar to that of normal limbs. The ratio of the lean content of gastrocnemius to hamstring muscles was greater in diseased (P=.007) and sound contralateral (P=.013) limbs than in normal limbs. CONCLUSIONS: Atrophy associated with CCL deficiency may predominantly affect the quadriceps muscle. Dominance of the gastrocnemius muscle over active restraints to the cranial tibial thrust may be associated with predisposition to CCL deficiency in Labrador Retrievers. CLINICAL RELEVANCE: If confirmed, this dynamic imbalance between muscle groups of the rear limbs could serve as a basis for screening programs and preventive rehabilitation.

Morphometric characteristics of the pelvic limbs of Labrador Retrievers with and without cranial cruciate ligament deficiency.

OBJECTIVE-To evaluate skeletal characteristics of pelvic limbs with and without cranial cruciate ligament (CCL) deficiency in Labrador Retrievers. ANIMALS-30 adult purebred Labrador Retrievers. PROCEDURES-Pelvic limbs (n = 28) of 14 dogs without CCL deficiency were classified as control limbs, whereas the limbs of 16 dogs with CCL deficiency were considered affected by (18 limbs) or predisposed to (10 contralateral limbs of dogs with 1 affected limb) CCL deficiency. Skeletal characteristics were evaluated via physical examination, radiography, and computed tomography. Radiographic and computed tomographic variables were compared among limb groups by use of a mixed-model ANOVA. RESULTS-The tibial plateau slope was steeper in CCL-deficient limbs but not in predisposed limbs, compared with the slope in control limbs. The angle between diaphyseal and proximal tibial axes was increased in both CCL-deficient and predisposed limbs. The relative width of the proximal portion of the tibia and the inclination of the patellar ligament did not differ among limb groups. The overall and distal femoral anteversion angles were greater in CCL-deficient and predisposed limbs, whereas the femoral condyle trochanteric angle was decreased in those limb groups, compared with findings in control limbs. CONCLUSIONS AND CLINICAL RELEVANCE-Cranial angulation of the proximal portion of the tibia, excessive steepness of the tibial plateau, and distal femoral torsion appeared more likely to be associated with CCL deficiency than femoral angulation, tibial torsion, intercondylar notch stenosis, and increased inclination of the patellar ligament.

Proximodistal alignment of the canine patella: radiographic evaluation and association with medial and lateral patellar luxation.

OBJECTIVES: To evaluate the contribution of proximodistal alignment of the patella to patellar luxation, and to evaluate the structures contributing to proximodistal alignment of the patella relative to the femoral trochlea. STUDY DESIGN: Retrospective study using a convenience sample. ANIMALS: Medium to giant breed dogs (n=106). METHODS: Medical records and stifle radiographs of 106 dogs were reviewed. Radiographic measurements evaluated the proximodistal alignment of the patella with respect to the femoral trochlea, distal aspect of the femur, and proximal aspect of the tibia. Measurements were compared between dogs with clinically normal stifles (controls; n=51 dogs, 66 stifles), and dogs with a clinical diagnosis of medial patellar luxation (MPL, n=46 dogs, 65 stifles) or lateral patellar luxation (LPL, n=9 dogs, 11 stifles) using ANOVA. RESULTS: In dogs with MPL, the ratio of patellar ligament length (PLL) to patellar length (PL) was increased, as was the ratio of the distance from the proximal aspect of the patella to the femoral condyle (A) to PL (P<.0001). Dogs with LPL had a decreased A:PL (P=.003) and an increased ratio of the proximal tibial length (PTL) to distal tibial width (DTW; P=.009). CONCLUSIONS: MPL is associated with a relatively long patellar ligament and patella alta in medium to giant breed dogs. LPL is associated with a relatively long proximal tibia and patella baja. Values for PLL:PL>2.06 and A:PL>2.03 are suggestive of the presence of patella alta, whereas a value for A:PL<1.92 is suggestive of patella baja. CLINICAL RELEVANCE: Measurements of both PLL:PL and A:PL are recommended in dogs with patellar luxation, and surgical correction should be considered in those with abnormal values.

Clinical applications of demineralized bone matrix: a retrospective and case-matched study of seventy-five dogs.

OBJECTIVES: To evaluate the outcome in dogs treated with demineralized bone matrix (DBM) as an adjunct to orthopedic procedures. STUDY DESIGN: Retrospective and case-match study. ANIMALS: Dogs (n=75). METHODS: Medical records (1999-2006) and radiographs of dogs that had orthopedic procedures (comminuted fractures, tibial plateau leveling osteotomy [TPLO] where correction for tibial rotation created an osteotomy gap, arthrodeses, open corrective osteotomies) where DBM was used were reviewed for signalment, quantity of DBM implanted, duration of exercise restriction, radiographic healing, and complications. Dogs that had TPLO and correction of tibial torsion (n=15), or arthrodesis (n=16) were compared with case-matched controls. Data were analyzed using Kruskal-Wallis test, ANOVA, Tukey's HSD test, and logistic regression analysis. RESULTS: Mean (+/-SD) healing time for orthopedic surgeries with DBM augmentation were 15+/-6.97 (weeks) and complication rate was 19% (14 dogs). Dogs with a TPLO gap filled with DBM were allowed to return to normal exercise 2 weeks earlier than dogs with a well-apposed TPLO site. Radiographic healing, duration of exercise restriction, and timing of destabilization were similar in dogs undergoing carpal and tarsal arthrodesis whether they received DBM, autogenous graft, or both. CONCLUSIONS: DBM can be used to treat uncomplicated bone defects associated with comminuted fracture repairs, open osteotomies, and arthrodeses in dogs. Under these circumstances, clinicians might expect similar clinical outcomes without the possibility of side effects associated with the harvest of autogenous cancellous bone. CLINICAL RELEVANCE: DBM is safe for use in dogs.

Comparison of cold GP/sealer and resin bonded obturation techniques in canine teeth in dogs.

An in vitro study compared two obturation materials in the canine teeth in dogs. The teeth were instrumented with rotary instruments and obturated with either gutta percha and a sealer or resin-based materials, utilizing the gutta percha apical plug/master cone technique. Radiographs were used for evaluation of the overall appearance of the finalfill. A modified apical dye leakage method was used to evaluate the ability of each material to provide an adequate barrier to apical leakage. When comparing the two obturation materials, the differences in the radiographic appearance scores and the apical dye leakage test results were not statistically significant. The rate of apical leakage is comparable to other obturation methods that have been reported. The use of a rotary system in conjunction with the gutta percha apical plug/master cone technique is valid for the endodontic treatment of indicated teeth.

Utility of postoperative CEA for surveillance of recurrence after resection of primary colorectal cancer.

INTRODUCTION: To evaluate the usefulness of postoperative CEA levels in the surveillance of colorectal cancer patients. METHODS: Over a 56 month period a total of 569 patients with measured CEA levels underwent curative resection for colorectal cancer. The median follow up was 40 months, during which period recurrence occurred in 149. Serum CEA levels were measured at 6 monthly intervals starting from 3 months post resection. ROC was used to calculate the optimum cut-off of CEA (5 ng/ml). RESULTS: Postoperative elevation of CEA levels were more frequent in patients with an aggressive primary colorectal cancer (grade, T stage and nodal disease; p < 0.05). In patients found to have colorectal recurrence, a significantly higher proportion of patients were resectable in the group with a non-elevated CEA (diagnosed by CT with PET imaging p < 0.05). The median interval between CEA elevation and diagnosis of recurrence (diagnostic interval) was 4 weeks. CEA elevation led to a change in the routine surveillance program by bringing imaging forward by 2 months. CEA levels were a significant predictor of survival following resection of colorectal primary (CEA ≤5-38 months, CEA >5-27 months; p < 0.05). CEA (p < 0.05) retained its significance on multivariate analysis along with the T stage (p < 0.05). CONCLUSION: CEA is a predictor of recurrence, resectability and survival following resection of colorectal cancer. Furthermore, an elevated CEA has a short diagnostic interval (4 weeks) for detecting recurrent disease and therefore should mandate adjustment of the routine surveillance program with the next planned imaging being brought forward (2 months).

Expression of a surface antigen (MA6) by peripheral blood CD34+ cells is correlated with improved platelet engraftment and may explain delayed platelet engraftment following cord blood transplantation.

CD34(+) cell dose provides a measure of hematopoietic tissue that predicts the rate of engraftment upon transplant. It is positively correlated with multiple measures of hematopoietic recovery, including platelet engraftment. Here we identify a subpopulation of CD34(+) cells that coexpress a surface antigen--MA6, which is more positively correlated with platelet engraftment in a clinical setting than CD34(+) alone. The specific identity and function of MA6 remain to be determined, however, it is expressed by primitive megakaryocyte (MK) progenitors, but is lost with differentiation and is not expressed by platelets. Commitment of CD34(+)MA6(+) cells to the MK lineage was confirmed by in vitro assays and their significance in hematopoietic transplantation explored by flow cytometric analysis of cryopreserved samples of granulocyte colony stimulating factor-mobilized peripheral blood progenitor cell (PBPC) products along with a retrospective analysis of platelet engraftment data. Platelet engraftment by day 21 was predicted by receipt of ≥ 6 × 10(6) CD34(+) cells/kg or ≥ 0.3 × 10(6) CD34(+)MA6(+) cells/kg. Subsequent analysis of cord blood (CB) CD34(+) cells revealed <0.2% coexpressed MA6(+), compared to 8% of PBPC CD34(+) cells. This low proportion of CD34(+)MA6(+) cells may be responsible, at least in part, for the delayed platelet engraftment associated with CB transplantation. However, platelet engraftment is markedly improved in recipients of ex vivo-expanded CB. This may be a consequence of an increased proportion of CD34(+)MA6(+) cells present in the ex vivo-expanded product and also suggests that optimizing ex vivo culture conditions to generate CD34(+)MA6(+) cells might further improve platelet engraftment in CB recipients.

Multifaceted actions of 8-amino-adenosine kill BCR-ABL positive cells.

Survival of chronic myelogenous leukemia (CML) cells is dependent on BCR-ABL kinase, the activity of which is contingent on the level of BCR-ABL protein and the availability of adenosine triphosphate (ATP). We hypothesized that 8-amino-adenosine (8-amino-Ado)-mediated reduction in cellular ATP level and inhibition of mRNA synthesis leading to a decrease in protein level would result in a multifaceted targeting of BCR-ABL. Using K562 cells, we demonstrated that there was a dose- and time-dependent increase in 8-amino-ATP accompanied by a > 95% decline in the endogenous ATP pool. In parallel, 8-amino-Ado inhibited RNA synthesis and resulted in a depletion of BCR-ABL transcript. Consistent with this, BCR-ABL and ABL protein levels were also decreased. These effects were associated with the initiation of cell death as visualized by poly(ADP-ribose) polymerase (PARP) cleavage, decreased clonogenicity and greater than additive interaction with imatinib. In imatinib-sensitive and -resistant KBM5 cells, 8-amino-Ado treatment augmented the imatinib effect on growth inhibition.

Ex vivo fucosylation improves human cord blood engraftment in NOD-SCID IL-2Rγ(null) mice.

Delayed engraftment remains a major hurdle after cord blood (CB) transplantation. It may be due, at least in part, to low fucosylation of cell surface molecules important for homing to the bone marrow microenvironment. Because fucosylation of specific cell surface ligands is required before effective interaction with selectins expressed by the bone marrow microvasculature can occur, a simple 30-minute ex vivo incubation of CB hematopoietic progenitor cells with fucosyltransferase-VI and its substrate (GDP-fucose) was performed to increase levels of fucosylation. The physiologic impact of CB hematopoietic progenitor cell hypofucosylation was investigated in vivo in NOD-SCID interleukin (IL)-2Rγ(null) (NSG) mice. By isolating fucosylated and nonfucosylated CD34(+) cells from CB, we showed that only fucosylated CD34(+) cells are responsible for engraftment in NSG mice. In addition, because the proportion of CD34(+) cells that are fucosylated in CB is significantly less than in bone marrow and peripheral blood, we hypothesize that these combined observations might explain, at least in part, the delayed engraftment observed after CB transplantation. Because engraftment appears to be correlated with the fucosylation of CD34(+) cells, we hypothesized that increasing the proportion of CD34(+) cells that are fucosylated would improve CB engraftment. Ex vivo treatment with fucosyltransferase-VI significantly increases the levels of CD34(+) fucosylation and, as hypothesized, this was associated with improved engraftment. Ex vivo fucosylation did not alter the biodistribution of engrafting cells or pattern of long-term, multilineage, multi-tissue engraftment. We propose that ex vivo fucosylation will similarly improve the rate and magnitude of engraftment for CB transplant recipients in a clinical setting.

Ex vivo graft purging and expansion of autologous blood progenitor cell products from patients with multiple myeloma.

Autologous peripheral blood progenitor cell (PBPC) transplantation is the treatment of choice for selected myeloma patients. However, tumor cells contaminating the apheresis product are a potential source of relapse. Here we report a sequential purging strategy targeting mature and immature clonogenic myeloma cell populations in the autograft. Thawed PBPC products of myeloma patients were treated with rituximab to kill CD138(-)20(+) B cells (highly clonogenic immature cells), and bortezomib to target CD138(+) cells (normal and differentiated myeloma plasma cells), followed by coculture with allogeneic mesenchymal stem cells (MSC) from normal donors. After 7 days of coculture, nonadherent cells were removed and cultured in the absence of MSC for an additional 7 days. Then, efficacy of purging (removal of CD138(-)20(+) and CD138(+) cells) was assessed by flow cytometry and PCR. We used our ex vivo purging strategy to treat frozen aphereses from 16 patients. CD138(+) and CD138(-)20(+)(19(+)) cells present in the initial products were depleted more than 3 and 4 logs, respectively based on 10(6) flow-acquisition events, and to levels below the limit of detection by PCR. In contrast, total nucleated cell (TNC), CD34(+) cell, and colony-forming cell numbers were increased by approximately 12 to 20, 8-, and 23-fold, respectively. Overall, ex vivo treatment of apheresis products with rituximab, bortezomib, and coculture with normal donor MSC depleted mature and immature myeloma cells from clinical aphereses while expanding the normal hematopoietic progenitor cell compartment.

Imaging long-term fate of intramyocardially implanted mesenchymal stem cells in a porcine myocardial infarction model.

The long-term fate of stem cells after intramyocardial delivery is unknown. We used noninvasive, repetitive PET/CT imaging with [(18)F]FEAU to monitor the long-term (up to 5 months) spatial-temporal dynamics of MSCs retrovirally transduced with the sr39HSV1-tk gene (sr39HSV1-tk-MSC) and implanted intramyocardially in pigs with induced acute myocardial infarction. Repetitive [(18)F]FEAU PET/CT revealed a biphasic pattern of sr39HSV1-tk-MSC dynamics; cell proliferation peaked at 33-35 days after injection, in periinfarct regions and the major cardiac lymphatic vessels and lymph nodes. The sr39HSV1-tk-MSC-associated [(18)F]FEAU signals gradually decreased thereafter. Cardiac lymphography studies using PG-Gd-NIRF813 contrast for MRI and near-infrared fluorescence imaging showed rapid clearance of the contrast from the site of intramyocardial injection through the subepicardial lymphatic network into the lymphatic vessels and periaortic lymph nodes. Immunohistochemical analysis of cardiac tissue obtained at 35 and 150 days demonstrated several types of sr39HSV1-tk expressing cells, including fibro-myoblasts, lymphovascular cells, and microvascular and arterial endothelium. In summary, this study demonstrated the feasibility and sensitivity of [(18)F]FEAU PET/CT imaging for long-term, in-vivo monitoring (up to 5 months) of the fate of intramyocardially injected sr39HSV1-tk-MSC cells. Intramyocardially transplanted MSCs appear to integrate into the lymphatic endothelium and may help improve myocardial lymphatic system function after MI.

Pre- and postoperative radiographic and computed tomographic evaluation of dogs with medial patellar luxation.

OBJECTIVE: To quantify, using radiographic and computed tomographic (CT) techniques, the effects of surgical procedures most commonly combined to treat dogs with medial patellar luxation (MPL). STUDY DESIGN: Prospective study. METHODS: Six dogs with 8 MPL were studied. Radiographs and CT of the pelvic limbs were obtained before and immediately after soft-tissue reconstruction, trochlear wedge recession, and tibial crest transposition. Radiographic measurements included angle of inclination, Norberg angle, quadriceps angle (QA), anteversion angle, ratio of the length of the patellar tendon (PT) to the length of the patella, and change in patella tendon angle. CT measurements included angle of inclination, Norberg angle, QA, anteversion angle, depth of the femoral trochlear groove, ratio of the middle femoral trochlear groove depth to the patella thickness, and tibial crest alignment. RESULTS: Conformation of the coxofemoral joint was not affected by surgery. Surgical treatment corrected the QA by 33-58%. Trochlear wedge recession was most effective in deepening the proximal trochlea by 103.5%. The ratio of the middle femoral trochlear groove depth to the thickness of the patella postoperatively resulted in 50% coverage of the patella. Tibial crest transposition resulted in caudalization of the PT by 8.5+/-3.0 degrees, with lateralization of the tibial tuberosity of 11.3 degrees. CONCLUSION: The effects of surgery for MPL can be quantified with radiographic and CT measurements. Surgical correction restored the alignment of the quadriceps and adequately deepened the femoral trochlear groove. Tibial crest transposition resulted in caudalization of the patella tendon and lateralization of the tibial tuberosity. CLINICAL RELEVANCE: These pilot data quantified the effects of surgical procedures most commonly combined to treat MPL. We hope to use these measurements to correlate surgical treatment with functional outcome and postoperative occurrence of luxation.