Takuro Sakagami

Pulmonary macrophage transplantation therapy

Bone-marrow transplantation is an effective cell therapy but requires myeloablation, which increases infection risk and mortality. Recent lineage-tracing studies documenting that resident macrophage populations self-maintain independently of haematological progenitors prompted us to consider organ-targeted, cell-specific therapy. Here, using granulocyte–macrophage colony-stimulating factor (GM-CSF) receptor-β-deficient (Csf2rb−/−) mice that develop a myeloid cell disorder identical to hereditary pulmonary alveolar proteinosis (hPAP) in children with CSF2RA or CSF2RB mutations, we show that pulmonary macrophage transplantation (PMT) of either wild-type or Csf2rb-gene-corrected macrophages without myeloablation was safe and well-tolerated and that one administration corrected the lung disease, secondary systemic manifestations and normalized disease-related biomarkers, and prevented disease-specific mortality. PMT-derived alveolar macrophages persisted for at least one year as did therapeutic effects. Our findings identify mechanisms regulating alveolar macrophage population size in health and disease, indicate that GM-CSF is required for phenotypic determination of alveolar macrophages, and support translation of PMT as the first specific therapy for children with hPAP.

Natural Selection and Population History in the Human Angiotensinogen Gene (AGT): 736 Complete AGT Sequences in Chromosomes from Around the World

Several lines of evidence suggest that patterns of genetic variability in the human angiotensinogen gene (AGT) contribute to phenotypic variability in human hypertension. The A(-6) promoter variant of AGT is associated with higher plasma angiotensinogen levels and increased risk of essential hypertension. The geographic distribution of the A(-6) variant leads to the intriguing hypothesis that the G(-6) promoter variant has been selectively advantageous outside Africa. To test these hypotheses, we investigated the roles of population history and natural selection in shaping patterns of genetic diversity in AGT, by sequencing the entire AGT gene (14400 bp) in 736 chromosomes from Africa, Asia, and Europe. We found that the A(-6) variant is present at higher frequency in African populations than in non-African populations. Neutrality tests found no evidence of a departure from selective neutrality, when whole AGT sequences were compared. However, tests restricted to sites in the vicinity of the A(-6)G polymorphism found evidence of a selective sweep. Sliding-window analyses showed that evidence of the sweep is restricted to sites in tight linkage disequilibrium (LD) with the A(-6)G polymorphism. Further, haplotypes carrying the G(-6) variant showed elevated levels of LD, suggesting that they have risen recently to high frequency. Departures from neutral expectation in some but not all regions of AGT indicate that patterns of diversity in the gene cannot be accounted for solely by population history, which would affect all regions equally. Taken together, patterns of genetic diversity in AGT suggest that natural selection has generally favored the G(-6) variant over the A(-6) variant in non-African populations. However, important localized effects may also be present.

Patient-derived Granulocyte/Macrophage Colony–Stimulating Factor Autoantibodies Reproduce Pulmonary Alveolar Proteinosis in Nonhuman Primates

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 microg/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 PPARgamma mRNA, and reduced tumor necrosis factor-alpha 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 microg/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.

ADAM33 polymorphisms are associated with aspirin-intolerant asthma in the Japanese population

AbstractMultiple single nucleotide polymorphisms (SNPs) within ADAM33 have been reported to be associated with asthma and bronchial hyper-responsiveness in Caucasian populations. We examined whether these SNPs contribute to a predisposition to asthma, especially aspirin-intolerant asthma (AIA), in the Japanese population. Ten polymorphic sites (ST+4, ST+7, T1, T2, T+1, V-3, V-2, V-1, V4, V5) were genotyped in 102 AIA patients, 282 aspirin-tolerant asthma (ATA) patients and 120 control (CTR) subjects by direct sequencing. Haplotype frequencies were estimated by the expectation-maximization method. Differences in allele and haplotype frequencies among phenotypes were analyzed by the chi-square and permutation tests. ST+7, V-1 and V5 sites in the AIA group were significantly different from those in the ATA group (P=0.034-0.004) and from those in the CTR group (P=0.019-0.002). Haplotypes at three sites (ST+7, V-1, and V5) were significantly different in frequency between the AIA and ATA (P=0.008) or CTR (P=0.001) groups. Sequence variations in ADAM33 are likely to correlate with susceptibility to AIA in the Japanese population.

Local adaptation and population differentiation at the interleukin 13 and interleukin 4 loci

A 25.6 kb region at chromosome 5q31, covering the entire human interleukin 13 (IL-13) and interleukin 4 (IL-4) genes, has been reported to be associated with bronchial asthma. We have examined nucleotide variations at this locus in African, European American, and Japanese populations, using 120 diallelic variants. A block of strong linkage disequilibrium (LD) (∣D′∣>0.7) spans a 10 kb region containing IL-4 in European American and Japanese populations, and is present but less clear in African samples. Two major haplotypes at IL-4 account for >80% of haplotypes in European Americans and Japanese. These haplotypes are common and quite diverged from each other and the ancestral haplotype, resulting in highly significant deviations from neutrality. FST statistics show that European American and Japanese populations are unusually distinct at the IL-4 locus. The most common haplotype in the European American population is much less common in the Japanese population, and vice versa. This implies that natural selection has acted on IL-4 haplotypes differently in different populations. This selected variation at IL-4 may account for some genetic variance underlying susceptibility to asthma and other allergic diseases. The strong LD observed in the IL-4 region may allow more efficient disease-association studies using this locus.

A Novel Prostacyclin Agonist Protects against Airway Hyperresponsiveness and Remodeling in Mice

Airway remodeling in bronchial asthma results from chronic, persistent airway inflammation. The effects of the reversal of airway remodeling by drug interventions remain to be elucidated. We investigated the effects of ONO-1301, a novel prostacyclin agonist with thromboxane inhibitory activity, on the prevention and reversibility of airway remodeling in an experimental chronic asthma model. Mice sensitized and challenged to ovalbumin (OVA) three times a week for 5 consecutive weeks were administered ONO-1301 or vehicle twice a day from the fourth week of OVA challenges. Twenty-four hours after the final OVA challenge, airway hyperresponsiveness (AHR) was assessed, and bronchoalveolar lavage was performed. Lung specimens were excised for staining to detect goblet-cell metaplasia, airway smooth muscle, and submucosal fibrosis. Mice administered ONO-1301 showed limited increases in AHR compared with mice administered the vehicle. The histological findings of airway remodeling were improved in ONO-1301-treated mice compared with vehicle-treated mice. Presumably, these therapeutic effects of ONO-1301 are attributable to the up-regulation of production of hepatocyte growth factor (HGF) in lung tissue, because the neutralization of HGF by antibodies prevented the effects of ONO-1301 on AHR and airway remodeling. Mice administered ONO-1301 showed similar levels of AHR and airway remodeling as mice administered montelukast, a cysteinyl-leukotriene-1 receptor antagonist, and lower levels were observed in mice administered dexamethasone. These data suggest that ONO-1301 exerts the effect of reversing airway remodeling, at least in part through an elevation of HGF in the lungs, and may be effective as an anti-remodeling drug in the treatment of asthma.

Novel assay to detect increased level of neutralizing anti-interferon gamma autoantibodies in non-tuberculous mycobacterial patients

Subjects exposed to non-tuberculous mycobacterium (NTM) species do not always develop an active disease, which likely reflects underlying host susceptibility factors. Recent reports have shown that anti interferon gamma (IFN-γ) neutralizing autoantibodies (IFN-γ Ab) are associated with the development of disseminated NTM in patients without known evidence of immunodeficiency. The purpose of this study is to establish the screening method if subjects have IFN-γ Ab. Whole blood was obtained from patients with disseminated NTM, those with pulmonary NTM, and healthy controls. The neutralizing capacity to IFN-γ activity was assessed as an inhibition of Signal Transducer and Activation of Transcription 1 (STAT-1) phosphorylation in leukocyte after stimulation with exogenous IFN-γ by flow cytometer. The strength of phosphorylation was described as STAT1 phosphorylation index. Antigen capture assay was performed to measure the relative titer of Immunoglobulin-G fraction of IFN-γ Ab. STAT1 phosphorylation by IFN-γ was significantly inhibited in the leukocytes from patients with disseminated NTM compared to that in healthy subjects, while this inhibition was not observed in patients with pulmonary NTM. All subjects with inhibited STAT1 phosphorylation had high titer of Immunoglobulin-G that reacted with IFN-γ in the antigen capture assay. The measurement of STAT1 phosphorylation index in whole blood leukocytes and antigen capture assay are simple and useful method for detection of anti-IFN-γ neutralizing autoantibodies, and is valuable in the pathophysiological diagnosis of disseminated NTM patients without obvious immunodeficiency.

Cluster analysis identifies characteristic phenotypes of asthma with accelerated lung function decline

Abstract Objective: While the majority of individuals with asthma retain normal lung function over time, some exhibit accelerated lung function decline. Preservation of lung function is an important aspect of asthma management. Whether the asthma guidelines can prevent lung function decline remains controversial. This study was performed to determine the distribution of asthmatic subjects with greater lung function decline and to identify characteristic clinical features of such subjects treated in accordance with clinical guidelines by using hierarchical cluster analysis. Methods: Eighty-six asthmatic subjects without a history of smoking were assessed with respect to eight variables selected from clinical phenotypes by using step-wise multiple regression analysis. Hierarchical cluster analysis using Ward’s method generated a dendrogram for estimation of the number of clusters within the population and the differences between them. Results: Three distinct clusters were identified. Cluster 1 (n = 40) comprised women with late-onset asthma. Cluster 2 (n = 17) comprised subjects with early-onset asthma, atopy and long disease duration. Cluster 3 (n = 29) predominantly comprised older men who had late-onset asthma, a lower prevalence of exacerbation and a lower predicted % forced expiratory volume in 1 s (FEV1) at baseline. Subjects in cluster 3 showed a mean decline in FEV1 of 69 mL/year, which was the greatest lung function decline among the three clusters. Conclusion: We identified a subgroup of patients with accelerated lung function decline despite appropriate asthma treatment based on guidelines constructed by using subjective symptoms.

Preventive effect of irbesartan on bleomycin-induced lung injury in mice

BACKGROUND Idiopathic pulmonary fibrosis is a specific form of chronic fibrosing interstitial pneumonia that is limited to the lung. Angiotensin receptor blockers (ARBs) and peroxisome proliferator-activated receptor (PPAR) γ ligands have anti-inflammatory and anti-fibrotic effects. We investigated the effects of irbesartan-an ARB with PPAR γ activity-on the development of bleomycin-induced pulmonary fibrosis in mice. METHODS Lung injury was induced in imprinting control region (ICR) mice by intratracheal instillation of 2mg/kg of bleomycin. The treatment group orally received 20mg/kg of irbesartan for 5 consecutive days before instillation. The mice were sacrificed and were evaluated 14 days after bleomycin instillation. RESULTS Irbesartan reduced the fluid content and hydroxyproline level in the lung and improved the pathological findings as indicated by the Ashcroft score. Total cell counts, the numbers of macrophages, neutrophils, and lymphocytes, and the levels of transforming growth factor (TGF) β1 and monocyte chemotactic protein (MCP) 1 in the bronchoalveolar lavage fluid (BALF) were decreased. Treatment with a PPARγ antagonist GW9662 reversed some of the effects of irbesartan. CONCLUSIONS The results of this study indicated that irbesartan attenuated the development of bleomycin-induced pulmonary fibrosis in mice by decreasing TGF-β1 and MCP-1 via blocking of ATI, by binding to CCR2b, and by PPARγ-mediated inhibition of inflammation.

Branch Retinal Vein Occlusion in the Right Eye and Retinal Hemorrhage in the Left in a Patient with Classical Tsutsugamushi Disease

PURPOSE To report branch retinal vein occlusion and retinal hemorrhages associated with tsutsugamushi disease. METHODS Case report of a 60-year-old woman who complained of fever, chills, headache, lymphadenopathy, and blurred vision in the right eye following an insect bite to the lower right forehead. RESULTS Serological findings showed elevated titers for the strains of Rickettsia tsutsugamushi. Ophthalmologic examination disclosed bilateral conjunctival injection, flame-shaped hemorrhage in her right fundus, and scattered hemorrhage in her left fundus. Fluorescein angiography demonstrated dye leakage and dilation of capillaries. CONCLUSIONS Branch retinal vein occlusion associated with classical tsutsugamushi disease, as demonstrated in our patient, may be rare.