ABSTRACT
Hematogenous Candida meningoencephalitis (HCME) is a life-threatening complication of neonates and immunocompromised children. Amphotericin B (AmB) shows poor permeability and low cerebrospinal fluid (CSF) concentrations, but is effective in treatment of HCME. In order to better understand the mechanism of CNS penetration of AmB, we hypothesized that AmB may achieve focally higher concentrations in infected CNS lesions. An in vitro BBB model was serially infected with C. albicans. Liposomal AmB (LAMB) or deoxycholate AmB (DAMB) at 5 µg/ml were then provided, vascular and CNS compartments were sampled 4h later. For in vivo correlation, rabbits with experimental HCME received a single dose of DAMB 1 mg/kg or LAMB 5 mg/kg, and were euthanized after 1, 3, 6 and 24h. Evans blue solution (2%) 2 ml/kg administered IV one hour prior to euthanasia stained infected regions of tissue but not histologically normal areas. AmB concentrations in stained and unstained tissue regions were measured using UPLC. For selected rabbits, MRI scans performed on days 1-7 postinoculation were acquired before and after IV bolus Gd-DTPA at 15min intervals through 2h post-injection. The greatest degree of penetration of DAMB and LAMB through the in vitro BBB occurred after 24h of exposure (P=0.0022). In vivo the concentrations of LAMB and DAMB in brain abscesses were 4.35±0.59 and 3.14±0.89-times higher vs. normal tissue (P≤0.019). MRI scans demonstrated that Gd-DTPA accumulated in infected areas with disrupted BBB. Localized BBB disruption in HCME allows high concentrations of AmB within infected tissues, despite the presence of low CSF concentrations.
ABSTRACT
PURPOSE: To quantify changes in tumor microvascular (< 1 mm) perfusion relative to commonly used angiographic endpoints. MATERIALS AND METHODS: Rabbit Vx2 liver tumors were embolized with 100-300-µm LC Bead particles to endpoints of substasis or complete stasis (controls were not embolized). Microvascular perfusion was evaluated by delivering two different fluorophore-conjugated perfusion markers (ie, lectins) through the catheter before embolization and 5 min after reaching the desired angiographic endpoint. Tumor microvasculature was labeled with an anti-CD31 antibody and analyzed with fluorescence microscopy for perfusion marker overlap/mismatch. Data were analyzed by analysis of variance and post hoc test (n = 3-5 per group; 18 total). RESULTS: Mean microvascular density was 70 vessels/mm(2) ± 17 (standard error of the mean), and 81% ± 1 of microvasculature (ie, CD31(+) structures) was functionally perfused within viable Vx2 tumor regions. Embolization to the extent of substasis eliminated perfusion in 37% ± 9 of perfused microvessels (P > .05 vs baseline), whereas embolization to the extent of angiographic stasis eliminated perfusion in 56% ± 8 of perfused microvessels. Persistent microvascular perfusion following embolization was predominantly found in the tumor periphery, adjacent to normal tissue. Newly perfused microvasculature was evident following embolization to substasis but not when embolization was performed to complete angiographic stasis. CONCLUSIONS: Nearly half of tumor microvasculature remained patent despite embolization to complete angiographic stasis. The observed preservation of tumor microvasculature perfusion with angiographic endpoints of substasis and stasis may have implications for tumor response to embolotherapy.
Subject(s)
Embolization, Therapeutic , Liver Neoplasms, Experimental/blood supply , Liver Neoplasms, Experimental/therapy , Microvessels , Analysis of Variance , Animals , Microscopy, Fluorescence , RabbitsABSTRACT
PURPOSE: To evaluate the effect of embolic diameter on achievement of hypoxia after embolization in an animal model of liver tumors. MATERIALS AND METHODS: Inoculation of VX2 tumors in the left liver lobe was performed successfully in 12 New Zealand white rabbits weighing 3.7 kg ± 0.5 (mean ± SD). Tumors were deemed eligible for oxygen measurements when the maximum transverse diameter measured 15 mm or more by ultrasound examination. Direct monitoring of oxygenation of implanted rabbit hepatic VX2 tumors was performed with a fiberoptic electrode during and after transarterial embolization of the proper hepatic artery to angiographic flow stasis with microspheres measuring 70-150 µm, 100-300 µm, or 300-500 µm in diameter. RESULTS: Failure to achieve tumor hypoxia as defined despite angiographic flow stasis was observed in 10 of 11 animals. Embolization microsphere size effect failed to demonstrate a significant trend on hypoxia outcome among the diameters tested, and pair-wise comparisons of different embolic diameter treatment groups showed no difference in hypoxia outcome. All microsphere diameters tested resulted in similar absolute reduction (24.3 mm Hg ± 18.3, 29.1 mm Hg ± 1.8, and 19.9 mm Hg ± 9.3, P = .66) and percentage decrease in oxygen (56.0 mm Hg ± 23.9, 56.0 mm Hg ± 6.4, and 35.8 mm Hg ± 20.6, P = .65). Pair-wise comparisons for percent tumor area occupied by embolic agents showed a significantly reduced fraction for 300-500 µm diameters compared with 70-150 µm diameters (P < .05). CONCLUSIONS: In the rabbit VX2 liver tumor model, three tested microsphere diameters failed to cause tumor hypoxia as measured by a fiberoptic probe sensor according to the adopted hypoxia definitions.
Subject(s)
Chemoembolization, Therapeutic/methods , Hemostatics/administration & dosage , Hemostatics/chemistry , Liver Neoplasms/metabolism , Liver Neoplasms/therapy , Oxygen/metabolism , Animals , Cell Hypoxia , Cell Line, Tumor , Cell Survival/drug effects , Female , Liver Neoplasms/diagnostic imaging , Microspheres , Particle Size , Rabbits , Treatment Outcome , UltrasonographyABSTRACT
Galactomannan and (1â3)-ß-D-glucan are useful biomarkers of invasive pulmonary aspergillosis (IPA). However, the effects of immunosuppression on levels of galactomannan or (1â3)-ß-D-glucan in IPA are not well understood or quantified. We therefore studied the simultaneous levels of galactomannan and (1â3)-ß-D-glucan in two rabbit models of experimental IPA: (1) AraC-induced neutropenia in untreated (UC-AraC) and liposomal amphotericin B-treated (LAMB-AraC) rabbits; and (2) nonneutropenic cyclosporine-methylprednisolone immunosuppression in untreated (UC-CsA+M) and LAMB-treated (LAMB-CsA+M) rabbits. Simultaneous levels of galactomannan and (1â3)-ß-D-glucan were determined in bronchoalveolar lavage (BAL) fluid and serial serum specimens and correlated with pulmonary host response. Serum galactomannan index (GMI) and (1â3)-ß-D-glucan concentration-time-curves were higher in UC-AraC vs. UC-CsA+M (Mann-Whitney U-test, P < .05). Serum galactomannan and (1â3)-ß-D-glucan in treatment groups demonstrated therapeutic responses with similarly lower levels in comparison to UC (P < .01) in both models. Host differences did not affect BAL fluid GMI or (1â3)-ß-D-glucan but did affect galactomannan and (1â3)-ß-D-glucan levels in serum. The higher serum GMI and (1â3)-ß-D-glucan concentration-time-curves in UC-AraC correlated with extensive pulmonary infiltration by angioinvasive hyphae and minimal inflammation, while the lower concentration-time-curves in UC-CsA+M were associated with shorter and fewer hyphae in lung tissue and an intensive neutrophil response to Aspergillus hyphae. Thus, serum levels of galactomannan and (1â3)-ß-D-glucan in IPA depended upon immunosuppression, which also affected severity of infection and hyphal morphology, while BAL fluid galactomannan and (1â3)-ß-D-glucan were sensitive biomarkers not affected by host response.
Subject(s)
Antifungal Agents/therapeutic use , Bronchoalveolar Lavage Fluid/microbiology , Invasive Pulmonary Aspergillosis/drug therapy , Mannans/analysis , beta-Glucans/analysis , Animals , Bronchoalveolar Lavage Fluid/chemistry , Disease Models, Animal , Female , Galactose/analogs & derivatives , Host-Pathogen Interactions , Invasive Pulmonary Aspergillosis/microbiology , Invasive Pulmonary Aspergillosis/mortality , Lung/microbiology , Lung/pathology , Mannans/blood , Proteoglycans , Rabbits , beta-Glucans/bloodABSTRACT
Members of the order Mucorales are emerging invasive molds that cause infections in immunocompromised patients. However, little is known about the relation between different species of Mucorales and their virulence in invasive pulmonary mucormycosis. Based upon our earlier epidemiological studies, we hypothesized that Cunninghamella bertholletiae would demonstrate increased virulence. Therefore, we studied the relative virulence of C. bertholletiae (CB), Rhizopus oryzae (RO), R. microsporus (RM), and Mucor circinelloides (MC) in experimental invasive pulmonary mucormycosis in persistently neutropenic rabbits in relation to the fungi in vitro sporangiospore germination rate and hyphal metabolic activity. Rabbits infected with CB demonstrated (1) higher lung weights in comparison to RM (P ≤ 0.05), RO and MC (P ≤ 0.001), (2) pulmonary infarcts in comparison to RO and MC (P ≤ 0.001), (3) tissue fungal burden (CFU/g) vs. MC (P ≤ 0.001), and (4) the lowest survival of 0% (0/18), in comparison to 16% (3/18, P ≤ 0.01) of RM, 81% (21/26) of RO, and 83% (15/18) of MC-infected rabbits (P ≤ 0.001). Serum PCR concentration-time-curve showed the greatest amplitude for CB. Virulence correlated directly with sporangiospore germination rate at 4 h among species, i.e., CB (67-85%) > RM (14-56%) > RO (4-30%) > MC (0%), and hyphal metabolic activity, i.e., CB (1.22-1.51) > MC (0.54-0.64) = RM (0.38-0.41) = RO (0.37-0.59). C. bertholletiae was significantly more virulent in experimental invasive pulmonary mucormycosis than R. microsporus, R. oryzae, and M. circinelloides. In vivo virulence correlated with species-dependent differences of in vitro germination rate and hyphal metabolic activity.
Subject(s)
Cunninghamella/pathogenicity , Lung Diseases, Fungal/microbiology , Mucorales/pathogenicity , Mucormycosis/microbiology , Animals , Biomarkers , Cunninghamella/genetics , Cunninghamella/isolation & purification , Cunninghamella/metabolism , DNA, Fungal/blood , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA, Ribosomal/blood , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Female , Humans , Hyphae , Immunosuppression Therapy , Lung Diseases, Fungal/pathology , Mucor/genetics , Mucor/isolation & purification , Mucor/metabolism , Mucor/pathogenicity , Mucorales/genetics , Mucorales/isolation & purification , Mucorales/metabolism , Mucormycosis/pathology , Rabbits , Rhizopus/genetics , Rhizopus/isolation & purification , Rhizopus/metabolism , Rhizopus/pathogenicity , Species Specificity , Sporangia , Spores, Fungal , Survival Analysis , VirulenceABSTRACT
We demonstrated that sodium gluconate was the factor causing false-positive galactomannan (GM) antigenemia of Plasma-Lyte hydration solution. Infusion of sodium gluconate-containing solution but not gluconate-free Plasma-Lyte resulted in positive serum GM antigenemia. Serum GM concentrations also correlated with the volume and in vitro concentrations of GM within gluconate-containing solutions of infused Plasma-Lyte.
Subject(s)
False Positive Reactions , Fungemia/diagnosis , Gluconates/administration & dosage , Mannans/blood , Galactose/analogs & derivatives , Humans , Infusions, Intravenous , Magnesium Chloride/administration & dosage , Potassium Chloride/administration & dosage , Sodium Acetate/administration & dosage , Sodium Chloride/administration & dosageABSTRACT
Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential diagnostic utility, we then performed a blinded study of bronchoalveolar lavage (BAL) fluid specimens from well-characterized models of IPA with the four medically important species. A set of real-time qPCR primers and probes was developed by utilizing unique ITS1 regions for genus- and species-specific detection of the four most common medically important Aspergillus species (Aspergillus fumigatus, A. flavus, A. niger, and A. terreus). Pan-Aspergillus and species-specific qPCRs with BAL fluid were more sensitive than culture for detection of IPA caused by A. fumigatus in untreated (P < 0.0007) and treated (P ≤ 0.008) animals, respectively. For infections caused by A. terreus and A. niger, culture and PCR amplification from BAL fluid yielded similar sensitivities for untreated and treated animals. Pan-Aspergillus PCR was more sensitive than culture for detection of A. flavus in treated animals (P = 0.002). BAL fluid pan-Aspergillus and species-specific PCRs were comparable in sensitivity to BAL fluid galactomannan (GM) assay. The copy numbers from the qPCR assays correlated with quantitative cultures to determine the pulmonary residual fungal burdens in lung tissue. Pan-Aspergillus and species-specific qPCR assays may improve the rapid and accurate identification of IPA in immunocompromised patients.
Subject(s)
Aspergillus/isolation & purification , Invasive Pulmonary Aspergillosis/diagnosis , Invasive Pulmonary Aspergillosis/microbiology , Molecular Diagnostic Techniques/methods , Mycology/methods , Real-Time Polymerase Chain Reaction/methods , Animals , Aspergillus/genetics , Bacterial Load , Bronchoalveolar Lavage Fluid/microbiology , DNA Primers/genetics , DNA, Ribosomal Spacer/genetics , Disease Models, Animal , Female , Oligonucleotide Probes/genetics , Rabbits , Sensitivity and Specificity , Time FactorsABSTRACT
Pulmonary infiltrates in neutropenic hosts with invasive aspergillosis are caused by organism-mediated tissue injury, vascular invasion, and hemorrhagic infarction. Ultrafast computed tomography (UFCT) scanning reproducibly measures these lesions in experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits. The pulmonary lesion score from UFCT scanning is a useful outcome variable for measuring differences in efficacy of antifungal compounds alone and in combination, as well as the virulence of different strains and species of Aspergillus. Several studies demonstrate that the course of pulmonary lesions treated with amphotericin B, lipid formulations of amphotericin B, triazoles, echinocandins, and combination therapy measured by serial UFCT scans correlate with those measured by survival, histopathological resolution of lesions, microbiological clearance of Aspergillus fumigatus, and resolution of galactomannan index. We further developed a multidimensional volumetric imaging (MDVI) method for analysis of the volume of pulmonary infiltrates over time in response to antifungal therapy. Volumetric data by MDVI correlate with UFCT pulmonary lesion scores and validated biological endpoints. A recent pilot clinical study demonstrated the applicability of MDVI to human pulmonary fungal infections. MDVI also improves objectivity of radiological assessment of therapeutic response to antifungal therapy and merits more extensive evaluation in patients with invasive aspergillosis, as well as other fungal and bacterial pneumonias.
Subject(s)
Diagnostic Imaging , Invasive Pulmonary Aspergillosis/diagnosis , Radiography, Thoracic , Animals , Antifungal Agents/therapeutic use , Aspergillus fumigatus/isolation & purification , Drug Monitoring , Humans , Invasive Pulmonary Aspergillosis/drug therapy , Longitudinal Studies , Rabbits , TomographyABSTRACT
OBJECTIVE: The aim of this study was to test a nonmyeloablative hematopoietic stem cell transplant regimen applicable to children with leukocyte adhesion deficiency (LAD) who have a histocompatible sibling donor by using the canine model of LAD, namely canine leukocyte adhesion deficiency or CLAD. METHODS: Thirteen CLAD pups received a hematopoietic stem cell transplant from a dog leukocyte antigen (DLA)-matched littermate donor after pretransplant nonmyeloablative conditioning with 200 cGy total-body irradiation and posttransplant immunosuppression with cyclosporine and mycophenolate mofetil. Donor chimerism following transplant was assessed by flow cytometry for the presence of donor CD18 peripheral blood leukocytes and leukocyte subsets. RESULTS: Eleven of the 13 transplanted animals achieved stable mixed donor chimerism and reversal of the severe CLAD phenotype without graft-vs-host disease. The level of donor chimerism ranged from 3.9 to 95.5% at 1 year following transplant. There was one early death 3 weeks after transplant from thrombocytopenia and hemorrhage, and one dog with donor microchimerism (0.5% CD18+ donor leukocytes) who had attenuation of the CLAD phenotype. CONCLUSION: These results demonstrate that a nonmyeloablative transplant regimen from a DLA-matched littermate donor leads to mixed chimerism and reversal of the severe disease phenotype in dogs with CLAD, and provides support for the use of this approach in children with LAD who possess a histocompatible sibling donor.
Subject(s)
Disease Models, Animal , Hematopoietic Stem Cell Transplantation , Leukocyte-Adhesion Deficiency Syndrome/therapy , Animals , Dogs , PhenotypeABSTRACT
The confounding effect of recycling of amino acids derived from tissue protein breakdown into the precursor pool for protein synthesis has been an obstacle to adapting in vivo methods for determination of regional rates of cerebral protein synthesis (rCPS) to positron emission tomography (PET). We used a kinetic modeling approach to estimate lambda, the fraction of the precursor pool for protein synthesis derived from arterial plasma, and to measure rCPS in three anesthetized adult monkeys dynamically scanned after a bolus injection of L-[1-11C]leucine. In the same animals, lambda was directly measured in a steady-state terminal experiment, and values showed excellent agreement with those estimated in the PET studies. In three additional monkeys rCPS was determined with the quantitative autoradiographic L-[1-14C]leucine method. In whole brain and cerebellum, rates of protein synthesis determined with the autoradiographic method were in excellent agreement with those determined with PET, and regional values were in good agreement when differences in spatial resolution of the two methods were taken into account. Low intrasubject variability was found on repeated PET studies. Our results in anesthetized monkey indicate that, by using a kinetic modeling approach to correct for recycling of tissue amino acids, quantitatively accurate and reproducible measurement of rCPS is possible with L-[1-11C]leucine and PET.
Subject(s)
Amino Acids/metabolism , Brain/metabolism , Leucine/metabolism , Models, Neurological , Protein Biosynthesis/physiology , Animals , Carbon Radioisotopes/analysis , Carbon Radioisotopes/metabolism , Female , Kinetics , Leucine/analysis , Macaca mulatta , Male , Positron-Emission TomographyABSTRACT
In mammals, growth of long bones occurs at the growth plate, a cartilage structure that contains three principal layers: the resting, proliferative, and hypertrophic zones. The function of the resting zone is not well understood. We removed the proliferative and hypertrophic zones from the rabbit distal ulnar growth plate in vivo, leaving only the resting zone. Within 1 wk, a complete proliferative and hypertrophic zone often regenerated. Next, we manipulated growth plates in vivo to place resting zone cartilage ectopically alongside the proliferative columns. Ectopic resting zone cartilage induced a 90-degree shift in the orientation of nearby proliferative zone chondrocytes and seemed to inhibit their hypertrophic differentiation. Our findings suggest that resting zone cartilage makes important contributions to endochondral bone formation at the growth plate: 1) it contains stem-like cells that give rise to clones of proliferative chondrocytes; 2) it produces a growth plate-orienting factor, a morphogen, that directs the alignment of the proliferative clones into columns parallel to the long axis of the bone; and 3) it may also produce a morphogen that inhibits terminal differentiation of nearby proliferative zone chondrocytes and thus may be partially responsible for the organization of the growth plate into distinct zones of proliferation and hypertrophy.
Subject(s)
Chondrocytes/physiology , Chondrogenesis/physiology , Growth Plate/physiology , Animals , Cell Differentiation/physiology , Cell Division/physiology , Cell Size/physiology , Clone Cells , Growth Plate/cytology , Male , Rabbits , Stem Cells/physiology , Ulna/cytology , Ulna/growth & developmentABSTRACT
We investigated, in a well-established canine model of human sepsis, the effects of two different techniques of sympathetic blockade during bacterial peritonitis on pain relief, hemodynamics, and survival rate. Twenty-two purpose-bred beagles (12-28 months old, weighing 10-12 kg) were studied. Fourteen animals received an epidural infusion of bupivicaine and morphine, and the other eight received either a celiac plexus block (n = 4) or a sham block (n = 4). Eighteen of the 22 animals received an intraperitoneal challenge of Escherichia coli (1-10 x 10(9) CFU kg(-1) body weight). At comparable doses of intraperitoneal-implanted E. coli (2.5-5 x 10(9) CFU kg(-1) body weight), the addition of sympathetic blockade produced a synergistic decrease in survival times (P = 0.002) and mean left ventricular ejection fraction (P = 0.008), and increase in creatinine levels (P = 0.02). There was also a significant increase in tumor necrosis factor (TNF) levels (P = 0.004) and decrease in blood endotoxin clearance (P = 0.006) associated with sympathetic blockade during sepsis. The celiac plexus-blocked animals had no improvement in pain scores, and subjectively looked clinically worse than animals with sepsis without a celiac plexus block. In contrast, the epidural block was effective in blocking the pain and discomfort associated with low lethality doses of intraperitoneal bacteria reflected by no increase in pain scores compared with animals not receiving bacterial challenge. This study shows that during severe bacterial peritonitis, maintenance of sympathetic tone irrespective of pain relief provided is necessary for clearance of bacterial toxins, control of proinflammatory mediator release, hemodynamic stability, and survival.
Subject(s)
Escherichia coli Infections/physiopathology , Gram-Negative Bacterial Infections/physiopathology , Hemodynamics/physiology , Pain/physiopathology , Peritonitis/physiopathology , Sympathetic Nervous System/physiopathology , Animals , Blood Pressure , Bupivacaine/pharmacology , Cardiac Output/drug effects , Disease Models, Animal , Dogs , Escherichia coli , Gram-Negative Bacterial Infections/pathology , Hemodynamics/drug effects , Morphine/pharmacology , Pain Measurement , Peritonitis/microbiology , Peritonitis/pathology , Pulmonary Artery/drug effects , Pulmonary Artery/physiopathology , Reference Values , Sympathetic Nervous System/drug effects , Sympathetic Nervous System/pathologyABSTRACT
To exploit the fact that IL-2 receptors are expressed by T-cells responding to foreign antigens but not by resting T-cells, humanized anti-Tac (HAT) armed with alpha-emitting radionuclides (212)Bi and (211)At was evaluated in a cynomolgus cardiac allograft model. Control graft survival was 8.2+/- 0.5 days compared with 14.0+/-1.3 days (p<0.01) survival for monkeys treated with (212)Bi labeled HAT and 26.7+/-2.4 days survival (p<0.001 versus controls) with (211)At labeled HAT. Thus, (211)At labeled HAT may have application in organ transplantation and in treatment of IL-2 receptor expressing T-cell leukemia.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Astatine/therapeutic use , Bismuth/therapeutic use , Graft Rejection/immunology , Graft Rejection/radiotherapy , Radioimmunotherapy/methods , Animals , Astatine/immunology , Bismuth/immunology , Female , Graft Rejection/prevention & control , Graft Survival/immunology , Macaca fascicularis , Mice , Mice, Nude , Protein Subunits , Radioisotopes/therapeutic use , Radiopharmaceuticals/immunology , Radiopharmaceuticals/therapeutic use , Receptors, Interleukin-2/immunology , Treatment OutcomeABSTRACT
BACKGROUND: The purpose of the present study was to determine whether longitudinal growth of the cortex occurs through intramembranous bone formation involving the periosteum or through endochondral bone formation involving the growth plate and to explore the cellular and biochemical mechanisms responsible for this process. METHODS: Cortical bone formation was studied in the metaphyses of growing New Zealand White rabbits by means of (1) oxytetracycline labeling and fluorescence microscopy, (2) computer-assisted histomorphometry, (3) osteoblast culture and [(3) H]-thymidine incorporation in the presence of periosteum or periosteum-conditioned medium, and (4) surgical insertion of membranes between the periosteum and the underlying spongiosa. RESULTS: Within the metaphyseal cortex, oxytetracycline labeling produced fluorescent closed curves outlining enlarging trabeculae derived from coalescing endochondral trabecular bone. In this region of coalescing trabeculae close to the periosteum, osteoblast surface was increased compared with trabeculae farther from the periosteum (p < 0.001). The osteoclast surface did not differ. In vitro, osteoblast proliferation was increased in the presence of periosteum (p < 0.001) or periosteum-conditioned medium (p < 0.001). Surgical insertion of permeable or impermeable membranes between the periosteum and the spongiosa did not prevent cortex formation. CONCLUSIONS: These observations demonstrate that metaphyseal cortical bone is formed by coalescence of endochondral trabecular bone. This coalescence is associated with increased osteoblast surface in the peripheral spongiosa. The increased osteoblast surface could be due to inductive effects of periosteum; in the present study, periosteum stimulated osteoblast proliferation in vitro but was not required for metaphyseal cortical bone formation in vivo. CLINICAL RELEVANCE: Understanding metaphyseal cortical growth may help to elucidate the pathophysiology of osseous growth disorders in children.
Subject(s)
Bone Development/physiology , Osteoblasts/physiology , Periosteum/physiology , Analysis of Variance , Animals , Coculture Techniques , Male , Microscopy, Fluorescence , Osteogenesis/physiology , Oxytetracycline/pharmacology , Photomicrography , RabbitsABSTRACT
The genetic disease canine leukocyte adhesion deficiency (CLAD) is characterized by recurrent, severe bacterial infections, typically culminating in death by 6 months of age. CLAD is due to a mutation in the leukocyte integrin CD18 subunit, which prevents surface expression of the CD11/CD18 leukocyte integrin complex. We demonstrate that stable mixed donor:host hematopoietic chimerism, achieved by a non-myeloablative bone marrow transplant from a histocompatible littermate, reverses the disease phenotype in CLAD. Donor chimerism following the transplant was demonstrated both by flow cytometric detection of donor-derived CD18-positive leukocytes in the peripheral blood of the recipient, and by the demonstration of donor-derived DNA microsatellite repeats in the peripheral blood leukocytes of the recipient. These results indicate that mixed hematopoietic chimerism reverses the clinical phenotype in CLAD and represents a potential therapeutic approach for the human disease leukocyte adhesion deficiency.
Subject(s)
Dog Diseases/therapy , Hematopoietic Stem Cell Transplantation/veterinary , Leukocyte-Adhesion Deficiency Syndrome/veterinary , Transplantation Chimera/immunology , Animals , Antigens, CD34/immunology , CD11 Antigens/immunology , CD18 Antigens/immunology , DNA/chemistry , DNA/genetics , Dog Diseases/immunology , Dog Diseases/pathology , Dogs , Flow Cytometry/veterinary , Hematopoietic Stem Cell Transplantation/methods , Leukocyte Count/veterinary , Leukocyte-Adhesion Deficiency Syndrome/immunology , Leukocyte-Adhesion Deficiency Syndrome/pathology , Leukocyte-Adhesion Deficiency Syndrome/therapy , Polymerase Chain Reaction/veterinaryABSTRACT
In this study we compared rat (n = 16) responses to euthanasia with either gradual-fill CO(2) or rapid induction argon gas by evaluating the animals' heart rate via radiotelemetry, behavior, and vocalizations. We also evaluated the histologic effects of the gases. Rats were placed in an open test chamber 24 h before the start of the experiment. During baseline tests, rats were exposed to oxygen to evaluate the effects of the noise and movement of gas entering the chamber; 1 wk later, rats were euthanized by gas displacement with either 10%/min CO(2) or 50%/min argon gas. Rats tended to have higher heart rats and were more active during the baseline test, but these parameters were normal before the euthanasia experiment, suggesting that the rats had acclimated to the equipment. Heart rate, behavior, and ultrasonic vocalizations were recorded for 2 min after gas introduction in both groups. All rats appeared conscious throughout the test interval. The heart rates of rats exposed to argon did not change, whereas those of rats exposed to CO(2) declined significantly. Unlike those exposed to CO(2), rats euthanized with argon gas gasped and demonstrated seizure-like activity. There were no differences in the pulmonary lesions resulting from death by either gas. Our results suggest that argon as a sole euthanasia agent is aversive to rats. CO(2) using a 10%/min displacement may be less aversive than more rapid displacements. Future research investigating methods of euthanasia should allow sufficient time for the rats to acclimate to the test apparatus.
Subject(s)
Argon/pharmacology , Behavior, Animal/drug effects , Carbon Dioxide/pharmacology , Euthanasia, Animal/methods , Heart Rate/drug effects , Lung/drug effects , Animals , Animals, Laboratory , Laboratory Animal Science/methods , Lung/pathology , Male , Rats , Rats, Sprague-DawleyABSTRACT
[11C](R)-(-)-RWAY ([11C]2, 3, 4, 5, 6, 7-hexahydro-1{4-[1[4-(2-methoxyphenyl)-piperazinyl]]-2-phenylbutyry}-1H-azepine) is a new radioligand for imaging brain 5-HT1A receptors with positron emission tomography. In [11C](R)-(-)-RWAY, the direction of the amide bond is expected to reduce metabolism by hydrolysis while allowing easy 11C-labeling at the methoxy position. The purposes of this study were to evaluate different tracer kinetic models in nonhuman primates to quantify 5-HT1A receptors with [11C](R)-(-)-RWAY and to test for the possible action of P-glycoprotein (P-gp), one of the known efflux pumps at the blood-brain barrier. The brain uptake of radioactivity from [11C](R)-(-)-RWAY into 5-HT1A receptor-rich brain regions was severalfold greater than for its antipode ([11C](S)-(+)-RWAY) and could be displaced by receptor saturating doses of the selective 5-HT1A antagonist, WAY-100635. Pretreatment with tariquidar, a potent inhibitor of P-gp, increased brain uptake of [11C](R)-(-)-RWAY about 1.5-fold and the plasma free fraction about 1.8-fold. Thus, the effect of tariquidar on brain uptake may have been caused by displacement of the radioligand binding to plasma proteins. Mathematical modeling showed that the estimated values of regional binding potential were correlated strongly between two-tissue compartment model and multilinear reference tissue model, and thus, supported the use of the cerebellum as a reference region.
Subject(s)
Azepines/pharmacology , Brain/diagnostic imaging , Brain/metabolism , Piperazines/metabolism , Piperazines/pharmacokinetics , Receptor, Serotonin, 5-HT1A/analysis , Receptor, Serotonin, 5-HT1A/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Binding, Competitive/drug effects , Binding, Competitive/physiology , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Brain/anatomy & histology , Brain Mapping/methods , Carbon Radioisotopes , Macaca mulatta , Male , Metabolic Clearance Rate/drug effects , Metabolic Clearance Rate/physiology , Models, Biological , Molecular Structure , Piperazines/chemical synthesis , Piperazines/pharmacology , Positron-Emission Tomography/methods , Pyridines/metabolism , Pyridines/pharmacokinetics , Quinolines/pharmacology , Radioligand Assay , Serotonin/metabolism , Serotonin Antagonists/metabolism , Serotonin Antagonists/pharmacokineticsABSTRACT
Children with the severe phenotype of the genetic immunodeficiency disease leukocyte adhesion deficiency or LAD experience life-threatening bacterial infections because of molecular defects in the leukocyte integrin CD18 molecule and the resultant failure to express the CD11/CD18 adhesion molecules on the leukocyte surface. Hematopoietic stem cell transplantation remains the only definitive therapy for LAD; however, the degree of donor chimerism and particularly the number of CD18(+) donor-derived neutrophils required to reverse the disease phenotype are not known. We performed nonmyeloablative hematopoietic stem cell transplantations from healthy matched littermates in 9 dogs with the canine form of LAD known as CLAD and demonstrate that in the 3 dogs with the lowest level of donor chimerism, less than 500 CD18(+) donor-derived neutrophils/microL in the peripheral blood of the CLAD recipients resulted in reversal of the CLAD disease phenotype. These results demonstrate the value of a disease-specific, large-animal model for identifying the lowest therapeutic level required for successful cellular and gene therapy.