Rationale: Granulocyte/macrophage colony-stimulating factor (GM-CSF) autoantibodies (GMAb) are strongly associated with idiopathic pulmonary alveolar proteinosis (PAP) and are believed to be important in its pathogenesis. However, levels of GMAb do not correlate with disease severity and GMAb are also present at low levels in healthy individuals. Objectives: Our primary objective was to determine whether human GMAb would reproduce PAP in healthy primates. A secondary objective was to determine the concentration of GMAb resulting in loss of GM-CSF signaling in vivo (i.e., critical threshold). Methods: Nonhuman primates (Macaca fascicularis) were injected with highly purified, PAP patient-derived GMAb in dose-ranging (2.2-50 mg) single and multiple administration studies, and after blocking antihuman immunoglobulin immune responses, in chronic administration studies maintaining serum levels greater than 40 mg/ml for up to 11 months. Measurements and Main Results: GMAb blocked GM-CSF signaling causing (1) a milky-appearing bronchoalveolar lavage fluid containing increased surfactant lipids and proteins; (2) enlarged, foamy, surfactant-filled alveolar macrophages with reduced PU.1 and PPARg mRNA, and reduced tumor necrosis factor-a secretion; (3) pulmonary leukocytosis; (4) increased serum surfactant protein-D; and (5) impaired neutrophil functions. GM-CSF signaling varied inversely with GMAb concentration below a critical threshold of 5 mg/ml, which was similar in lungs and blood and to the value observed in patients with PAP. Conclusions: GMAb reproduced the molecular, cellular, and histopathologic features of PAP in healthy primates, demonstrating that GMAb directly cause PAP. These results have implications for therapy of PAP and help define the therapeutic window for potential use of GMAb to treat other disorders.
To define the toxicity of cystic fibrosis transmembrane conductance regulator gene (CFTR) gene therapy with a replication-deficient recombinant adenovirus (Av1Cf2) in a nonhuman primate model, 10(10) plaque forming units (pfu) were instilled directly through a bronchoscope into the right lung of 5 macaques, and a lower dose of 4 x 10(6) pfu was administered to the right lung of 1 macaque. One sham-treated control received phosphate-buffered saline (PBS). The macaques were evaluated sequentially by clinical examination, vital signs, weight, hematology, blood chemistry, chest radiography, pulse oximetry, and bronchoalveolar lavage (BAL) at baseline and 3-28 days post-treatment. After the period of observation, macaques were sacrificed for autopsy and histological examination. The macaques tolerated the experimental therapy clinically with no changes in body temperature, oxygen saturation, heart rate, body weight, or blood pressure. However, 1 macaque with visible evidence of aspiration at the time of initial bronchoscopy developed tachypnea with right lower lobe (RLL) pneumonia on chest radiograph and by histology. There were no changes in Hgb, Wbc, BUN, plasma electrolytes, bilirubin, or hepatic transaminases. In the macaques that received 10(10) pfu, there was a progressive increase in the number of CD8+ lymphocytes in BAL that was maximal at 28 days. Histological examination of the treated lungs of the high-dose macaques at 3 days showed marked peribronchial and perivascular cuffing by inflammatory cells and alveolar accumulation of neutrophils and macrophages. The alveolitis appeared to be resolving at 28 days, although the perivascular and peribronchial aggregates of mononuclear cells were still present. In the high-dose macaques, BAL interleukin-8 (IL-8) was increased at all time points (256-388 pg/ml versus 1-84 pg/ml at baseline and in control), whereas IL-1 beta was increased only at days 21 and 28 (341-852 pg/ml versus 30-92 pg/ml at baseline and in control). There were no increases in BAL cell counts, IL-1 beta or IL-8, and histological changes were mild in the macaque that received 4 x 10(6) pfu. Evaluation for Av1Cf2-derived human CFTR expression using RS-PCR demonstrated expression at 3, 10, and 21, but not 28 days in macaques treated with 10(10) pfu of Av1Cf2. In situ hybridization analysis demonstrated human CFTR mRNA in the alveolar regions of the lobes that received the vector at 10 and 21 days. There was no evidence of expression after treatment with 4 x 10(6) pfu. This study showed that high-dose adenoviral vector administration to the lung achieved CFTR gene transfer and expression but was associated with increased concentrations of cytokines in BAL and alveolar inflammation. A low dose, equivalent to the maximum clinical dose currently proposed for phase I trials in human subjects, was not associated with cellular or cytokine evidence of inflammation, and histological abnormalities were mild.
Pooled fecal specimens collected from striped field mice (Apodemus agrarius), yellow-necked mice (Apodemus flavicollis), and bank voles (Myodes glareolus) and individual stool samples collected from laboratory mice were tested for the presence of picornaviruses and astroviruses. Picornavirus RNA was detected only in one striped field mouse sample pool, while astrovirus RNA was detected in two yellow-necked mouse sample pools and in six of the 121 laboratory mouse samples. In a 234-amino acid (aa) fragment of the viral RNA dependent RNA polymerase (RdRp), the wild mouse picornavirus revealed the closest homology to the canyon mouse (Peromyscus crinitus) (93 % aa) and canine kobuviruses (92 % aa) and to Aichi virus (88 % aa). The two astroviruses detected in the yellow-necked mouse samples shared 77 % aa homology with each other in the partial (125 aa) RdRp region, 61-62 % aa homology with rat astroviruses and only 54-58 % aa homology with the house mouse (Mus musculus) astrovirus strain USA/2008/M52. The six laboratory mouse astroviruses displayed 97-100 % aa homology to each other, and shared 71-77 % aa homology with the yellow-necked mouse astroviruses, 58-59 % aa homology with rat astroviruses and 55-56 % aa homology with strain USA/2008/M52. The sequence of a 3,263 bp genome segment including the partial ORF1b (RdRp), complete ORF2 (capsid precursor), and 3' NTR of a research mouse astrovirus strain (TF18LM) was determined. The full-length ORF2 showed low identities (17-34 % aa) with other members of the Mamastrovirus genus and only 17 % aa homology with the house mouse astrovirus strain USA/2008/M52, indicating that AstVs described in this study represent a novel Mamastrovirus species. The relevance of astrovirus infection and its effect on biomedical research conducted in mice needs to be investigated.
Sexually mature domestic cats were hormonally stimulated to induce superovulation; embryo recovery was accomplished by laparotomy. Embryos were frozen by conventional embryo freezing methods used in the domestic cattle embryo transfer industry. Thawing was achieved in a 28 degrees C or 37 degrees C waterbath or in ambient air. Overnight culture of the frozen-thawed embryos in a supplemented Nutrient Mixture F-10 (Ham's) or Earle's Balanced Salt Solution with 20% heat-treated newborn calf serum resulted in five successful term litters from recipient queens. Embryo recipients who became pregnant had been treated with a subcutaneous injection of follicle-stimulating hormone (FSH-P) once every 24 hr for 6 days in the amount of 0.2 mg/day for the first 5 days and 0.1 mg on the sixth day, followed by two intramuscular 750 IU injections of human chorionic gonadotropin 24 hr apart, beginning on the same day as the sixth injection of FSH-P.
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