Pharmacodynamics of Posaconazole in Experimental Invasive Pulmonary Aspergillosis: Utility of Serum Galactomannan as a Dynamic Endpoint of Antifungal Efficacy.

Aspergillus galactomannan antigenemia is an accepted tool for the diagnosis of invasive pulmonary aspergillosis (IPA) in neutropenic patients. Little is known, however, about the utility of this biomarker to assess the efficacy of antifungal therapies. The pharmacokinetics (PK) and pharmacodynamics (PD) of posaconazole in treatment and prophylaxis were investigated in the persistently neutropenic rabbit model of Aspergillus fumigatus IPA at doses between 2 and 20 mg/kg per day. Sparse plasma sampling was used to obtain PK data at steady state, and the serum galactomannan index (GMI), as a dynamic endpoint of antifungal response, was obtained every other day, in addition to conventional outcome parameters including survival and fungal tissue burden. Nonparametric PK/PD model building was performed using the Pmetrics package in R. A one-compartment model with linear elimination best described the PK of posaconazole. The PD effect of posaconazole exposure in plasma on the GMI in serum was best described by dynamic Hill functions reflecting growth and killing of the fungus. Through calculations of the area under the concentration-time curve from 0 to 24 h (AUC0-24) at steady state, the exposure-response relationship between posaconazole and the GMI for treatment followed a sigmoidal function with an asymptote forming above an AUC0-24 of 30 mg · h/liter. All prophylactic doses were able to control the fungal burden. A nonparametric population PK/PD model adequately described the effect of posaconazole in prophylaxis and treatment of experimental IPA. An AUC0-24 greater than 30 mg · h/liter was associated with adequate resolution of the GMI, which well supports previously suggested exposure-response relationships in humans.

Amphotericin B Penetrates into the Central Nervous System Through Focal Disruption of the Blood Brain Barrier in Experimental Hematogenous Candida Meningoencephalitis.

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.

Environmental remediation of sulfidic tailings with froth flotation: Reducing the consumption of additional resources by optimization of conditioning parameters and water recycling.

The removal of sulfidic species in tailings using froth flotation is a promising approach to prevent phenomena such as acid mine drainage. However, flotation requires the consumption of reagents and water that represent additional expenses. Despite the strong interest of scientists and industry alike on tailings remediation, there is no study on the minimization of resource consumption to promote the implementation of desulfurization with froth flotation. Following a systematic analysis based on Design of Experiments (DoE), this work aims to determine the implications of a decrease in the consumption of flotation reagents and fresh water. It was found that: i) recovery of sulfidic species is strongly influenced by collector concentration and the use of a preliminary re-dispersion step; ii) higher frother concentrations have a negative impact on sulfur grade in the concentrate; and iii) the interactions between the conditioning variables hereby explored have no significant impact on flotation performance. Composition analysis showed that flotation further aids in the removal of hazardous species, such as As, Co and Zn. Finally, the use of recycled water appears possible since flotation performance remained unchanged over 10 flotation cycles, despite the observed accumulation of metallic ions and organic species in the process water.

Microvascular Perfusion Changes following Transarterial Hepatic Tumor Embolization.

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.

Direct Quantification and Comparison of Intratumoral Hypoxia following Transcatheter Arterial Embolization of VX2 Liver Tumors with Different Diameter Microspheres.

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.

Effects of host response and antifungal therapy on serum and BAL levels of galactomannan and (1→3)-ß-D-glucan in experimental invasive pulmonary aspergillosis.

Galactomannan and (1→3)-ß-D-glucan are useful biomarkers of invasive pulmonary aspergillosis (IPA). However, the effects of immunosuppression on levels of galactomannan or (1→3)-ß-D-glucan in IPA are not well understood or quantified. We therefore studied the simultaneous levels of galactomannan and (1→3)-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 (1→3)-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 (1→3)-ß-D-glucan concentration-time-curves were higher in UC-AraC vs. UC-CsA+M (Mann-Whitney U-test, P < .05). Serum galactomannan and (1→3)-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 (1→3)-ß-D-glucan but did affect galactomannan and (1→3)-ß-D-glucan levels in serum. The higher serum GMI and (1→3)-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 (1→3)-ß-D-glucan in IPA depended upon immunosuppression, which also affected severity of infection and hyphal morphology, while BAL fluid galactomannan and (1→3)-ß-D-glucan were sensitive biomarkers not affected by host response.

Increased virulence of Cunninghamella bertholletiae in experimental pulmonary mucormycosis: correlation with circulating molecular biomarkers, sporangiospore germination and hyphal metabolism.

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.

Galactomannan antigenemia after infusion of gluconate-containing Plasma-Lyte.

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.

Molecular detection and species-specific identification of medically important Aspergillus species by real-time PCR in experimental invasive pulmonary aspergillosis.

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.

The initial 96 hours of invasive pulmonary aspergillosis: histopathology, comparative kinetics of galactomannan and (1->3) ß-d-glucan and consequences of delayed antifungal therapy.

Acute invasive pulmonary aspergillosis is a rapidly progressive and frequently lethal infection. Relatively little is known about early events in the pathogenesis and relationship between the cell wall biomarkers galactomannan and (1→3)-ß-d-glucan. The consequences of delayed antifungal therapy are also poorly defined. A persistently neutropenic rabbit model of invasive pulmonary aspergillosis was used to describe the histopathology of early invasive pulmonary aspergillosis and the kinetics of galactomannan and (1→3)-ß-d-glucan. The time course of both molecules was mathematically modeled by using a population methodology, and Monte Carlo simulations were performed. The effect of progressive delay in the administration of amphotericin B deoxycholate 1 mg/kg at 24, 48, 72, and 96 h postinoculation on fungal burden, lung weight, pulmonary infarct score, and survival was determined. Histopathology showed phagocytosis of conidia by pulmonary alveolar macrophages at 4 h postinoculation. At 12 to 24 h, there was a progressive focal inflammatory response with conidial germination and hyphal extension. Subsequently, hyphae invaded into the contiguous lung. Galactomannan and (1→3)-ß-d-glucan had similar trajectories, and both exhibited considerable interindividual variability, which was reflected in Monte Carlo simulations. Concentrations of both molecules began to rise <24 h postinoculation before pulmonary hemorrhagic infarction was present. Delays of 72 and 96 h in the administration of amphotericin B resulted in fungal burdens and lung weights that were indistinguishable from those of controls, respectively. Galactomannan and (1→3)-ß-d-glucan have similar kinetics and are comparable biomarkers of early invasive pulmonary aspergillosis. Antifungal treatment at ≥48 h postinoculation is associated with suboptimal therapeutic outcomes.

Combination therapy in treatment of experimental pulmonary aspergillosis: in vitro and in vivo correlations of the concentration- and dose- dependent interactions between anidulafungin and voriconazole by Bliss independence drug interaction analysis.

We studied the antifungal activity of anidulafungin (AFG) in combination with voriconazole (VRC) against experimental invasive pulmonary aspergillosis (IPA) in persistently neutropenic rabbits and further explored the in vitro and in vivo correlations by using Bliss independence drug interaction analysis. Treatment groups consisted of those receiving AFG at 5 (AFG5 group) and 10 (AFG10 group) mg/kg of body weight/day, VRC at 10 mg/kg every 8 h (VRC group), AFG5 plus VRC (AFG5+VRC group), and AFG10 plus VRC (AFG10+VRC group) and untreated controls. Survival throughout the study was 60% for the AFG5+VRC group, 50% for the VRC group, 27% for the AFG10+VRC group, 22% for the AFG5 group, 18% for the AFG10 group, and 0% for control rabbits (P < 0.001). There was a significant reduction of organism-mediated pulmonary injury, measured by infarct scores, lung weights, residual fungal burdens, and galactomannan indexes, in AFG5+VRC-treated rabbits versus those treated with AFG5 and VRC alone (P < 0.05). In comparison, AFG10+VRC significantly lowered only infarct scores and lung weights in comparison to those of AFG10-treated animals (P < 0.05). AFG10+VRC showed no significant difference in other outcome variables. Significant Bliss synergy was found in vivo between AFG5 and VRC, with observed effects being 24 to 30% higher than expected levels if the drugs were acting independently. These synergistic interactions were also found between AFG and VRC in vitro. However, for AFG10+VRC, only independence and antagonism were observed among the outcome variables. We concluded that the combination of AFG with VRC in treatment of experimental IPA in persistently neutropenic rabbits was independent to synergistic at a dosage of 5 mg/kg/day but independent to antagonistic at 10 mg/kg/day, as assessed by Bliss independence analysis, suggesting that higher dosages of an echinocandin may be deleterious to the combination.

Diagnostic imaging of experimental invasive pulmonary aspergillosis.

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.

Cerebrospinal fluid and plasma (1-->3)-beta-D-glucan as surrogate markers for detection and monitoring of therapeutic response in experimental hematogenous Candida meningoencephalitis.

The treatment, diagnosis and therapeutic monitoring of hematogenous Candida meningoencephalitis (HCME) are not well understood. We therefore studied the expression of (1-->3)-beta-D-glucan (beta-glucan) in cerebrospinal fluid (CSF) and plasma in a nonneutropenic rabbit model of experimental HCME treated with micafungin and amphotericin B. Groups studied consisted of micafungin (0.5 to 32 mg/kg) and amphotericin B (1 mg/kg) treatment groups and the untreated controls (UC). Despite well-established infection in the cerebrum, cerebellum, choroid, vitreous humor (10(2) to 10(3) CFU/ml), spinal cord, and meninges (10 to 10(2) CFU/g), only 8.1% of UC CSF cultures were positive. By comparison, all 25 UC CSF samples tested for beta-glucan were positive (755 to 7,750 pg/ml) (P < 0.001). The therapeutic response in CNS tissue was site dependent, with significant decreases of the fungal burden in the cerebrum and cerebellum starting at 8 mg/kg, in the meninges at 2 mg/kg, and in the vitreous humor at 4 mg/kg. A dosage of 24 mg/kg was required to achieve a significant effect in the spinal cord and choroid. Clearance of Candida albicans from blood cultures was not predictive of eradication of organisms from the CNS; conversely, beta-glucan levels in CSF were predictive of the therapeutic response. A significant decrease of beta-glucan concentrations in CSF, in comparison to that for UC, started at 0.5 mg/kg (P < 0.001). Levels of plasma beta-glucan were lower than levels in simultaneously obtained CSF (P < 0.05). CSF beta-glucan levels correlated in a dose-dependent pattern with therapeutic responses and with Candida infection in cerebral tissue (r = 0.842). Micafungin demonstrated dose-dependent and site-dependent activity against HCME. CSF beta-glucan may be a useful biomarker for detection and monitoring of therapeutic response in HCME.

Detection of a molecular biomarker for zygomycetes by quantitative PCR assays of plasma, bronchoalveolar lavage, and lung tissue in a rabbit model of experimental pulmonary zygomycosis.

We developed two real-time quantitative PCR (qPCR) assays, targeting the 28S rRNA gene, for the diagnosis of zygomycosis caused by the most common, clinically significant Zygomycetes. The amplicons of the first qPCR assay (qPCR-1) from Rhizopus, Mucor, and Rhizomucor species were distinguished through melt curve analysis. The second qPCR assay (qPCR-2) detected Cunninghamella species using a different primer/probe set. For both assays, the analytic sensitivity for the detection of hyphal elements from germinating sporangiospores in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates from rabbits was 1 to 10 sporangiospores/ml. Four unique and clinically applicable models of invasive pulmonary zygomycosis served as surrogates of human infections, facilitating the validation of these assays for potential diagnostic utility. For qPCR-1, 5 of 98 infarcted lung specimens were positive by qPCR and negative by quantitative culture (qCx). None were qCx positive only. Among 23 BAL fluid samples, all were positive by qPCR, while 22 were positive by qCx. qPCR-1 detected Rhizopus and Mucor DNA in 20 (39%) of 51 serial plasma samples as early as day 1 postinoculation. Similar properties were observed for qPCR-2, which showed greater sensitivity than qCx for BAL fluid (100% versus 67%; P = 0.04; n = 15). The assay detected Cunninghamella DNA in 18 (58%) of 31 serial plasma samples as early as day 1 postinoculation. These qPCR assays are sensitive and specific for the detection of Rhizopus, Mucor, Rhizomucor, and Cunninghamella species and can be used for the study and detection of infections caused by these life-threatening pathogens.

Quantification of cortical GABA-glutamine cycling rate using in vivo magnetic resonance signal of [2-13C]GABA derived from glia-specific substrate [2-13C]acetate.

Brain [2-(13)C]gamma-aminobutyric acid (GABA) signal derived from the glia-specific substrate [2-(13)C]acetate reflects the extent of the GABA-glutamine neurotransmitter cycling between GABAergic neurons and glial cells. We report, for the first time, in vivo quantification of the GABA-glutamine cycling flux. The GABA-glutamine cycling flux rate was determined to be 1.8+/-0.4 micromol/(gh) (mean+/-S.D., n=6, approximately 6% of total tricarboxylic acid cycle rate) in the neocortex of vigabatrin-treated rats. The relatively small magnitude of glial contribution to the clearance of extracellular GABA measured in this study provided in vivo evidence to support the concept of a significant neuronal reuptake of GABA, which short-circuits the GABA-glutamine cycling pathway for repletion of released neurotransmitter GABA.

Nonmyeloablative hematopoietic stem cell transplantation corrects the disease phenotype in the canine model of leukocyte adhesion deficiency.

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.

Minimally Invasive Lumbar Port System for the Collection of Cerebrospinal Fluid from Rhesus Macaques (Macaca mulatta).

Biomedical translational research frequently incorporates collection of CSF from NHP, because CSF drug levels are used as a surrogate for CNS tissue penetration in pharmacokinetic and dynamic studies. Surgical placement of a CNS ventricular catheter reservoir for CSF collection is an intensive model to create and maintain and thus may not be feasible or practical for short-term studies. Furthermore, previous NHP lumbar port models require laminectomy for catheter placement. The new model uses a minimally invasive technique for percutaneous placement of a lumbar catheter to create a closed, subcutaneous system for effective, repeated CSF sample collection. None of the rhesus macaques (Macaca mulatta; n = 10) implanted with our minimally invasive lumbar port (MILP) system experienced neurologic deficits, postoperative infection of the surgical site, or skin erosion around the port throughout the 21.7-mo study. Functional MILP systems were maintained in 70% of the macaques, with multiple, high-quality, 0.5- to 1.0-mL samples of CSF collected for an average of 3 mo by using aspiration or gravitational flow. Among these macaques, 57% had continuous functionality for a mean of 19.2 mo; 50% of the cohort required surgical repair for port repositioning and replacement during the study. The MILP was unsuccessful in 2 macaques, at an average of 9.5 d after surgery. Nonpatency in these animals was attributed to the position of the lumbar catheter. The MILP system is an appropriate replacement for temporary catheterization and previous models requiring laminectomy and is a short-term alternative for ventricular CSF collection systems in NHP.

Measurement of regional rates of cerebral protein synthesis with L-[1-11C]leucine and PET with correction for recycling of tissue amino acids: II. Validation in rhesus monkeys.

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.

Composite tissue allotransplantation: development of a preclinical model in nonhuman primates.

BACKGROUND: Composite tissue allotransplantation (CTA) has been recently introduced as a potential treatment for tissue loss secondary to burns, injuries, or resections. However, the optimal strategies to prevent CTA rejection remain undefined. Presently, no CTA model exists to evaluate human-specific immunosuppressants or the relative immunogenicity of all CTA tissues. METHODS: We established a NHP CTA model utilizing a sensate osteomyocutaneous radial forearm flap that avoids functional impairment even in the case of graft loss. The model was evaluated in19 monkeys that underwent auto- or allotransplantation, with or without subtherapeutic immunosuppression to temporarily characterize rejection. RESULTS: Autografts showed no evidence of rejection. Nonimmunosuppressed allografts were rapidly rejected showing a perivenular T-cell infiltrate. This was associated with subsequent alloantibody formation and led to graft thrombosis without prominent dermal infiltration. Subtherapeutically immunosuppressed animals also developed alloantibody and rejected in a delayed fashion exhibiting a marked dermal lymphocytic infiltrate similar in magnitude and distribution to previously reported human cases. CONCLUSION: Our NHP model for CTA is well tolerated by NHPs, results in allosensitization, is responsive to immunosuppression, allows for the evaluation of CTA histology and can be used for the systematic preclinical evaluation of therapeutic maneuvers to improve allograft survival.

Development of a cerebrospinal fluid lateral reservoir model in rhesus monkeys (Macaca mulatta).

Rapid, serial, and humane collection of cerebrospinal fluid (CSF) in nonhuman primates (NHP) is an essential element of numerous research studies and is currently accomplished via two different models. The CSF reservoir model (FR) combines a catheter in the 4th ventricle with a flexible silastic reservoir to permit circulating CSF flow. The CSF lateral port model (LP) consists of a lateral ventricular catheter and an IV port that provides static access to CSF and volume restrictions on sample collection. The FR model is associated with an intensive, prolonged recovery and frequent postsurgical hydrocephalus and nonpatency, whereas the LP model is associated with an easier recovery. To maximize the advantages of both systems, we developed the CSF lateral reservoir model (LR), which combines the beneficial features of the 2 previous models but avoids their limitations by using a reservoir for circulating CSF flow combined with catheter placement in the lateral ventricle. Nine adult male rhesus monkeys were utilized in this study. Pre-surgical MRI was performed to determine the coordinates of the lateral ventricle and location of choroid plexus (CP). The coordinates were determined to avoid the CP and major blood vessels. The predetermined coordinates were 100% accurate, according to MRI validation. The LR system functioned successfully in 67% of cases for 221 d, and 44% remain functional at 426 to 510 d postoperatively. Compared with established models, our LR model markedly reduced postoperative complications and recovery time. Development of the LR model was successful in rhesus macaques and is a useful alternative to the FR and LP methods of CSF collection from nonhuman primates.