Granular Reasoning Using Zooming In & Out

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ネコカリシウイルスの分子疫学的研究 : カプシド遺伝子塩基配列の系統樹解析を中心にして

ネコカリシウイルスルfeline calicivirus(FCV)はネコの呼吸器病の原因ウイルスとして重要であり、プラス一直鎖RNAを持ち、粒子の直径は27～40nmとピコルナウイルスよりやや大きく表面に窪みのある形態学的特徴を有するウイルスである。最近、ネコにおいて致死性の兎ウイルス性出血病様症例がPedersenら(2000)により報告され注目を集めている。本疾患の予防にはワクチンが投与されているが、十分に防圧するに至っていない。この理由としてFCVの変異の激しさが指摘されている。FCVの血清型については中和試験、モノクローナル抗体により検討されてきたが血清多型性は認められないことから、各株は同じ血清型のvariantとみなされていることがあげられる。一方、遺伝子の系統解析も試みられているが未だに遺伝子型別がなされていないことから、Glennら(1999)はFCVが単一の遺伝子型からなるため血清学、病原性の生物学、地理および年代などと遺伝的関連性がないのではないかと考えた。　本論文はFCVの遺伝子型を検討し分子疫学的研究を行なったものであり、FCVの遺伝子型別を初めて明らかにし、流行の把握と新たなワクチン開発の必要性に関する成績を得た。　FCVの塩基配列に基づく系統樹を用いて遺伝子型別を行うため、日本株47株と外国株30株の合計77株についてカプシド領域(B-F)の1105から1739番目の塩基配列およびアミノ酸配列(307-580)を決定した。そしてこれによって作成した系統樹と年代、地域性、病原性、血清学的性状の関連について検討を行った。また、ワクチンブレイクを呈したネコから分離した一部の株について遺伝性型別とワクチン抵抗性について検討を試みた。1. 遺伝子型の検討1-1　遺伝子群の解析　系統樹の解析によりFCVは高い確率で2遺伝子に分かれることが明らかになった。用いた77株のうち53株(69%)が遺伝子群1(genogroup I)に、24株(31%)が遺伝子群II(genogroup II)に属することが明らかとなった。これらのブートストラップ値は96.5%と十分高かった。群IIはさらに99.7%のブートストラップ値で2遺伝子亜群(subgenogroup;IIa、IIb)を形成した。亜群IIaには日本株13株(17%)、亜群IIbには11株(14%)が含まれた。1-2　アミノ酸の系統樹解析　遺伝子の塩基配列により想定されるアミノ酸配列に基づく系統樹では遺伝子群1、IIに相当するIAとIIAの2群に大別されたが、IIAの亜群に関しては遺伝子亜群IIa、IIbと異なりブートストラップ値が53.6%と低く、アミノ酸配列の亜群として区別されなかった。このことから疫学的検討には塩基配列による系統樹を用いた。1-3　アミノ酸の比較　アミノ酸の多重解析により群Iと群IIに共通するアミノ酸について比較検討したところ、F領域の539番目のアミノ酸は、群Iのアラニンまたはプロリンが、群IIではバリンまたはフェニルアラニン、又、557番目のグルタミン(群I)がセリン(群II)と、2箇所でアミノ酸残基の置換があり、この置換を指標として型別を行える可能性が示唆された。2. 疫学的検討2-1　国別の分布比較　分離された国別にFCVを遺伝子型別に分けると、群Iにはドイツ4株、イギリス5株、カナダ5株、アメリカ9株、ニュージランド1株を含むオーストラリア6株と、外国で分離された株はすべて群Iに属し、群IIにはこれらの外国で分離されたFCVは含まれなかった。日本株のうち約半数の23／47(49%)が群Iに属し、群IIは24／47(51%)の日本株のみからなっていた。このことから群IのFCVは日本を含め広く世界中に分布しており、群IIは日本に限局して分布することが明らかとなった。2-2　日本で分離されたFCVの地域・県別分布の比較　群Iに属する日本株の分布は九州64%、近畿58%、関東33%で、地域間における有意な差は見られなかった。また、各都道府県における群I、IIa、IIbの各亜群の分布状況についても顕著な差は認められなかった。2-3　年代別による分布　日本株については分離された年代別に、I、II群の分布を比較したところ、特に遣出子群と年代の関連は確認されなかった。2-4　塩基配列の相同性　日本株20株と外国株の塩基配列について相同性を比較したところ、群Iあるいは群IIの中ではどちらの群でも約70%程度の群内相同性が認められ、群Iと群IIの間では大体60-70%の相同性がみられた。同様に、アミノ酸配列については群1あるいは群IIの中では約80%程度の相同性が認められたのに対し、群Iと群IIの間では70%程度であった。このような2群間における相同性の低さは遺伝子型の違いを裏付けている。また、ワクチン株F9およびFCV255はいずれも群Iに属し、群IIの日本株との相同性は約70%程度と低く、今回用いた日本株は群IIの日本株と遺伝的に隔たりがあることを示していた。2-5　近縁性　系統樹で群Iに属する7つのクラスター(C1～C7)に分けられた株間の相関性から近縁性を検討したところ、アメリカ、カナダ、オーストラリアにおいて分離された群1の株間では近縁性が高く、近縁株が分離年代や地域の隔たりを超えて流行していたことを示していたが、群Iの日本株間には高い相同性を持つ株は認められなかった。しかし、群IIのウイルスにおいて、近縁な複数の株が狭い地域内で流行していることもあり、又、異なる地域にまたがって流行していることもあることが明らかとなった。2-6　病原性との関連性　呼吸器病以外の疾病をもつネコから分離された株の塩基配列の近縁関係と病原性の相関性について検討した。上述のように分けられたクラスターと比較すると、下痢を起こす株は群IのC2とIIb亜群に属し、慢性胃炎を起こした株はC1とC4に、急性胃炎、健康な個体に由来する株もそれぞれ異なったクラスターに属し、跛行を起こした株は同じC3に属していたが離れた位置にあり、塩基配列に基づく系統的差異と病原性とには明らかな相関関係はなかった。　ウサギウイルス性出血病ウイルスと似た病原性を有するFCV-Ari株のE領域の塩基配列について、突然死したネコから分離したFCV-S株および実験感染により死亡を認めたH10株の塩基配列と比較したところ、相同性はそれぞれ70.4%、74.7%と、比較的低かった。2-7　血清学的性状　日本株21株の抗F9株免疫ネコ血清に対する中和抗体価と遺伝子型との関連について検討したところ、群Iの7株の抗体価の幾何学平均値は20.5±5.5(平均±SD)、群IIの14株の幾何平均値は11.3±2.6(平均±SD)で、2群間の抗体価には顕著な差はなく、従来の報告にあるように、中和抗体と遺伝子型との相関性は認められなかった。2-8　抗原性と関連のあるE超可変領域の系統解析と中和抗体の反応性　日本株21株の、抗F9株免疫ネコ血清の中和抗体に対する反応性が、カプシド遺伝子の超可変領域(E領域、アミノ酸配列426-458:抗原性と関連がある)のアミノ酸配列の変異と相関するかどうかを、Sealにより報告されたUPGMA法で作成された系統樹によって検討した。FCVはI-VIIIのクラスターに分別され、クラスターの構成メンバーの反応性を比較したところ、抗体の反応性と変異の型(近縁度)は必ずしも相関しないことが判明した。2-9　NJ法によるE領域の系統樹解析　これらのアミノ酸配列についてNJ法により系統樹を作成したところ、これらのクラスターのブートストラップ値は低い値を示して、明瞭な遺伝子型に分かれなかった。このことからE領域は地域性とも関連しないようである。3. ワクチンに対する抵抗性の検討　ワクチン(F9株)接種済みのネコから分離されたH10株とFCV-S株の2株について遺伝子型を決め、ワクチン抵抗性の株別の変異を検討した。H10株は遺伝子群1に、FCV-S株は遺伝子群IIに属し異なる遺伝子型を呈した。この株を市販ワクチン(F9株)で免役したネコに対して攻撃し感染防御能の有無について検討した。3-1　H10株に対する感染防御　H10株は、市販ワクチンF9株免疫ネコ血清に対してH10株の抗原性は低かった。そこでH10株に対する抗体価を上げるため市販ワクチンを3回投与した4頭とワクチン未接種のネコ3頭にH10株の経鼻・経口投与を行なった。この結果、ワクチンの投与により抗体価が上昇した場合にはウイルスの排泄は短期間で終わるが、低い場合には発症は抑制されたが、感染を阻止できず長期にウイルスを排泄することからワクチン抵抗株を産生する可能性がある。3-2　FCV-S株に対する感染防御　1回のワクチン投与を行なった4頭のネコ、市販ワクチンを投与していないネコ2頭に対してFCV-Sで攻撃した。市販ワクチン未接種、接種のどちらの個体も同じ様に、体温の上昇、白血球数の増加、下痢が見られ、糞便におけるウイルスの排泄は6日間以内に限定された。FCV-Sについてはワクチンによる顕著な防御効果は見られなかったことから本ウイルスは市販ワクチンに抵抗性を示すものと思われる。本ウイルスの投与による呼吸器症状は見られず、下痢を呈した点については、下痢発症ネコ由来のFCVは酸性(pH3)に抵抗性を示すことが報告されているが、FCV-SもpH3に抵抗性を示したことから本ウイルス株と理化学的性状に共通性があるようである。今後FCVのワクチンの開発には現在使用されている群1に属するワクチン株(F9株、FCV255株)に加え、群IIのFCV株をワクチン株として併用する必要があると考えられる。　本研究によりFCVは2種類の遺伝子型からなることが明らかになった。群Iは広く世界に流行しているが、群IIのウイルスは日本においてのみ見られ、今後このウイルスの起源を探る手がかりを与えると考えられる。また、特に日本においては群IIのウイルスに対する抗体による治療、またはワクチンを開発することがFCV感染症の効果的防御に重要であると結論できる。Feline calicivirus(FCV) causes an acute, highly contagious disease in cats especially in kitten. That is called the FCV infection characterized by upper respiratory disease, pneumonia, ulcerative stomatitis or gingivitis, and occasionally, enteritis or arthritis. The FCV particle is recognized by nonenveloped icosahedral symmetry with a diameter of 27 to 40 nm. The virion consists of a positive single-strand of nonsegmented RNA with 32 cup-shaped depressions on the capsid. The capsid is composed of a single major polypeptide, and serologically, the FCV is regarded as belonging to one single serotype. The molecular epidemiological study of FCV was performed and induced a genotypic classfication for Japanese and foreign strains by phylogenetic analyses of the nucleotide sequences and amino acid sequence(370～580) of the capsid-coding regions(B through F) with examination of their isolations, ages, locality, pathogenicity and serological properties. These results suggest usefulness of developing a norvel vaccines directed against strains for which conventional vaccine has limited effectiveness.1. Genotyping1-1 Genotype grouping The construction of a phylogenetic tree for 77 strains divided FCVs into two distinct groups of genes. Of the 77 strains, 69% and 31% belonged to gene group I and II, respectively. Their bootstrap values were high at 96.5%, showing that both gene groups were divided into two genotypes with a high probability. Gene group I formed clusters consisting of many branches, but their low bootstrap values did not allow any of them to be distinguished from gene group I, whereas gene group II indicated a bootstrap value of 99.7% and formed two gene subgroups(IIa 17%, IIb 14%).1-2 Phylogenetic analysis of amino acid sequences A phylogenetic tree was constructed by converting nucleotide sequences into amino acid sequences, which were broadly divided into two gene groups(IA, IIA) consisting of similar members as in the phylogenetic tree of nucleotide sequences. Since the IIa and IIb subgroups could not be clearly distinguished bacause of a low bootstrap value of 53.6%, they were regarded as the same gene group. These results indicate that nucleotide sequence-based analysis is more sensitive in analyzing genotypes.1-3 Comparison of amino acids Multivariate analysis of the amino acids common to group I or II showed that gene groups I and II differed in two amino acid residues, that is, the amino acids at position 539 in the F region were alanine or proline in group I and valine or phenylalanine in group II, and those at position 557 were glutamine and serine, respectively.2. Epidemiological analysis2-1 Comparison of distribution by country Genotype classification of FCVs by country of origin showed that the foreign strains(German, English, Canadian, USA and Australian) and about half of the Japanese strains belonged to gene group I, while gene group II consisted only of Japanese strains without any foreign strains. There was thus a clear difference between the two groups(p<O.Ol). These results indicate that FCVs of group I are widely distributed throughout the world, while group II FCVs are restricted to Japan.2-2 Comparison of distribution of Japanese FCV by regions and prefectures of isolation Japanese strains belonging to group I were distributed in the Kyushu, Kinki and Kanto regions, showing no significant differences among regions. No significant differences were noted in the distribution of group I and subgroups IIa and IIb eithes.2-3 Distribution by ages The distribution of gene groups I and II of Japanese strains did not differ significantly according to ages.2-4 Homology The Japanese and foreign strains showed approximately 70% nucleotide sequence homology whithin each of gene groups I and II. The highest homology of 99.8% was found between the FCV-2 and FCV-6 strains isolated in Kanagawa prefecture, while the lowest homology of 64.4% was observed between the USA strain FRI-NCI and the Kanagawa strain FCV-15. The homology between group I and II tended to be low at 60% to 70%. Strains whithin each of gene groups I and II had approximately 80% amino acid sequence homology, higher than nucleotide sequence homology. The FCV-2 and FCV-6 strains showed the perfect match of 100%, and the Japanese strains ML2 and FPL(Bolin) showed the lowest homology of 67.8%. The homology between gene groups I and II was low at about 70%, in agreement with the difference in genotypes. The vaccine strains F9 and FCV255 belonging to gene group I had low homology with the Japanese strains in gene group II, suggesting that the Japanese strains used in this study are genetically distinct.2-5 Relationship The strains classified as gene group I were divided into seven clusters(C1 through C7) to analyze their relationship. The nucleotide sequence of FCVs isolated in the same coutry, region or prefecture tended to be located close to each other. The FPL(Bolin) and FRI-NCI strains belong to C1 isolated in USA in different years shared a high homology(97.2% nucleotide homology and 98.9% amino acid homology), suggesting that they prevailed in those years in the same country. Such marked relationships were also found in the Austrakian strains V274 and V276 in C2 and three Canadian Strains in C4. These strains originated from prevalent disease in different years did not show a significantly high homology with the Japanese strains in gene group I. On the other hand, among gene group II viruses, the FCV-1, FCV-2, FCV-6 and FCV-11 strains isolated in Kanagawa showed high homology with one another(99.5% to 99.8% nucleotide homology and 99.5% to 100% amino acid homology). Similarly, among gene subgroup IIb, the FCV-15 and FCV-170 isolated in Kanagawa, the H1, ML2, ML5 and ML6 strains isolated in Tokyo, Osaka and Kanagawa, the S1 and S2 strains in Kanagawa, the ND35 strain in Ibaraki and the TOKYO 1 strain in Tokyo showed high homology with each other. Thus, gene group II strains isolated in Japan also prevail in a limited area, and closely related viruses prevail over different regions.2-6 Relation to pathogenicity The relationship was investigated between the nucleotide sequences of strains isolated from cats without any respiratory symptoms and their pathogenicity. Among strains causing diarrhea and showing resistance to pH 3.0, the FCV-43 and FCV-170 strains belonged to gene group I and gene subgroup IIb, respectively, clearly indicating different genotypes. Similarly, three strains causing chronic gastritis, two strains causing acute gastritis, and two strains from healthy individuals formed different clusters. Two of the three strains derived from cats with claudication belonged to the same cluster, but was located a little apart from each other. Thus, it became clear that nucleotide sequences did not correlate with pathogenicity. The FCV-Ari strain having simlar pathogenicity to rabbit hemorrhagies disease virus showed very low nucleotide sequence homology of 70.4% and 74.7%, with the FCV-S strain isolated from a cat dying suddenly and the H1O strain from cats dying of experimental infection, respectively. This is indicating a great genotype difference.2-7 Serological properties The relationship between the neutralizing antibody titers of feline anti-F9 serum against 21 Japanese strains and the gene groups was analyzed. The mean antibody titers against gene groups I and II were 20.5 and 11.3, respectively, with no significant intergroup difference. These indicate no association between antibody titer and genotype as shown to date.2-8 Phylogenetic analysis of antigenicity-related E hypervariable region and neutralizing antibody reactivity Using a phylogenetic tree constructed by the UPGMA method, the correlation was investigated between the reactivity of the 21 Japanese strains for the neutralizing antibody in feline anti-F9 serum and the antigenicity-related E hypervariable region(amino acid sequences 426-458). FCVs were divided into clusters EI through EVIII to compare the reactivity of the cluster-composing members. In gene group I, the F9 strain and the Japanese strain S21 were close to each other, and the S21 strain had a high antibody-neutralizing titer(1:89.6), but the reactivity differed according to groups. Gene group II included the low-reactive H1O and FCV-43 strains and moderately reactive ML4 strain; group III, the high-reactive FCV-5 and moderately-reactive FPL-NC1 strain; group IV, the high reactive ML-7 strain and moderately-reactive ML-3 strain; group V, the FCV-2, FCV-6 and FCV-11 strains with the same amino acid sequence and similar reactivity; and group VI consisted of 11 Japanese strains(three low reactive strains and eight moderately-reactive strains) including the FCV-B strain. The FCV-15 strain(Kanagawa, 1990) and the FCV-170 strain(Kanagawa, 1996) had the identical amino acid sequences, and the H1 strain(Tokyo, 1998), the ML2 strain(Osaka, 1997), the ML5 strain(Kanagawa, 1997), and ML6 strain(Osaka,1997) also had the identical amino acid sequence and similar antibody titers. However, this group included the high-reactive foreign CFI/68 strain. Thus, the reactivity to antibody did not agree with clusters.The S21 and F9 strains differed in amino acid sequence at seven positions. The CFI/68 strain had the same amino acid sequence as some of the 11 Japanese strains, but differed in seven amino acids from the S1 strain located close to CFI/68 strain and in 13/33(39%) from the FCV-15 strain located a little apart from the CFI/68 strain. Thus, strains in the same cluster did not show high homology and this supported non-correlation between E hypervariable region amino acid sequences and serological properties.2-9 Phylogenetic analysis of genetic E hypervariable region A phylogenetic tree of the above described amino acid sequences was constructed by the NJ method. These clusters were regarded as the same group because of their low bootstrap values, and did not fall into distinct genotypes.3. Evaluation of resistance to vaccines The H10 and FCV strains isolated from vaccinated cats were genotyped and evaluated for resistance to vaccines. The H10 strain and FCV-S belonged to gene group I and II, respectively, showing different genotypes. SPF cats immunized with a commercially available vaccine(F9 strain) were challenged with these two strains, and evaluated for anti- infection ability.3-1 Protection against H1O infection Since the F9-immunized cat\u27s sera were detected only a low antibody titer against the H1O strain, four cats were inoculated theree times with a commercially available vaccine(F9 strain) to raise antibody titers. These four SPF cats and three unvaccinated SPF cats were administred the H1O strain nasally and orally. Of the unvaccinated cats challenged with the H10 strain, one died with high fever, and two had diarrea in the early stage, recovering eight to 14 days later with respiratory symptoms. Vaccinated cats with low antibody titers showed a marked increase in antibody titers on the challenge with the H10 strain. The cats with high antibody titers did not show a marked increase in antibody titers, but tended to have higher antibody titers than the cats that showed low antibody titers. All four cats developed transient fever, but survived without any respiratory symptoms. Similar to the unvaccinated cats, the cats with low antibody titers tended to show an increase in white blood cell count 1 day after the challenge, excreting viruses for as long as 13 days. These results indicate that vaccine administration suppresses the onset in cats with low antibody titers, but can not inhibit the infection, secreting viruses for long periods, and suggest the possibility of producing a vaccine-resistant strain.3-2. Protection against FCV-S infection FCV-S was a strain of FCV isolated from the spinal cord of two cats(of five vaccinated cats) dying suddenly, and showed resistance to pH 3.0. Since the neutralizing antibody titer was about 10 times lower than that in F9-immunized cats, the FCV-S strain was administred nasally and orally to four cats that had been vaccinated once and two unvaccinated cats. The antibody titers were low at 1:80～640 against the F9 strain and 1:40 against the FCV-S strain, and markedly increased against the F9 and FCV-S strains after challenge with the FCV-S strain. The four cats did not show a marked antibody response on the second administration, whereas the unvaccinated cats showed a marked secondary response. Both the vaccinated and unvaccinated cats had an elevated temperature and elevated white blood cells and diarrhea, and excreted viruses for six days. Three control cats that had been administered only culture medium containing no viruses had no diarrhea. Challenge with the FCV-S strain did not produce respiratory symptoms but caused diarrhea. This appears to be related to the fact that this strain has the physicochemical property of being resistant to pH 3.0. Since no protective effect of vaccination was observed, the FCV-S strain seems to have a resistance to the vaccine. However, the second admmistration did not cause diarrhea or other clinical symptoms, suggesting that the administration of appropriate vaccines prevents infection. It is considered that in the future, it is important to develop vaccines against FCVs in gene group II so that they can be used in combination with vaccines against FCVs in gene group I.博士(獣医学)麻布大

Post-traumatic diaphragmatic herniation of the liver, examined by positron emission tomography: case report

We present a case of post-traumatic diaphragmatic herniation of the liver, which mimicked an intrathoracic tumor. After an automobile accident, the patient underwent thoracotomies for hemothorax and lung cancer in the right chest. Seven months later, computed tomography (CT) demonstrated a round tumor in the thorax adjacent to the right diaphragm with a higher density than the liver parenchyma. An intrathoracic tumor including a primary or metastatic lung cancer was suspected. However, positron emission tomography (PET) showed that the uptake of fluorine-18-fluorodeoxyglucose (FDG) was identical to that in the liver, and the tumor appeared to be contiguous with the liver. Thus, we suspected liver herniation. Core needle biopsy revealed liver cells without neoplastic tissue. Upon surgical exploration, herniation of the liver was found and repaired. PET was helpful in providing morphological and functional information leading to accurate diagnosis of liver herniation in this unusual case

An Agent Control Method based on Rough-Set-based Granularity

Proceedings of Joint 4th International Conference on Soft Computing and Intelligent Systems and 9th International Symposium on advanced Intelligent Systems in Nagoya on September 17-21, 2008 (SCIS & ISIS 2008

Risk factors associated with late aneurysmal sac expansion after endovascular abdominal aortic aneurysm repair

PURPOSEWe aimed to identify the risk factors associated with late aneurysmal sac expansion after endovascular abdominal aortic aneurysm repair (EVAR).METHODSWe retrospectively reviewed contrast-enhanced computed tomography (CT) images of 143 patients who were followed for ≥6 months after EVAR. Sac expansion was defined as an increase in sac diameter of 5 mm relative to the preoperative diameter. Univariate and multivariate analyses were performed to identify associated risk factors for late sac expansion after EVAR from the following variables: age, gender, device, endoleak, antiplatelet therapy, internal iliac artery embolization, and preprocedural variables (aneurysm diameter, proximal neck diameter, proximal neck length, suprarenal neck angulation, and infrarenal neck angulation).RESULTSUnivariate analysis revealed female gender, endoleak, aneurysm diameter ≥60 mm, suprarenal neck angulation >45°, and infrarenal neck angulation >60° as factors associated with sac expansion. Multivariate analysis revealed endoleak, aneurysm diameter ≥60 mm, and infrarenal neck angulation >60° as independent predictors of sac expansion (P < 0.05, for all).CONCLUSIONOur results suggest that patients with small abdominal aortic aneurysms (<60 mm) and infrarenal neck angulation ≤60° are more favorable candidates for EVAR. Intraprocedural treatments, such as prophylactic embolization of aortic branches or intrasac embolization, may reduce the risk of sac expansion in patients with larger abdominal aortic aneurysms or greater infrarenal neck angulation

Analysis of H2O Masers in Sharpless 269 using VERA Archival data --- Effect of maser structures on astrometric accuracy

Astrometry using H2O maser sources in star forming regions is expected to be a powerful tool to study the structures and dynamics of our Galaxy. Honma et al. (2007) (hereafter H2007) claimed that the annual parallax of S269 is determined within an error of 0.008 milliarcsec (mas), concluding that S269 is located at 5.3 kpc +- 0.2 kpc from the sun, and R= 13.1 kpc. They claimed that the rotational velocity of S269 is equal to that of the sun within a 3% error. This small error, however, is hardly understood when taking into account the results of other observations and theoretical studies of galactic dynamics. We here reanalyzed the VERA archival data using the self-calibration method (hybrid mapping), and found that clusters of maser features of S269 are distributed in much wider area than that investigated in H2007. We confirmed that, if we make a narrow region image without considering the presence of multiple maser spots, and only the phase calibration is applied, we can reproduce the same maser structures in H2007. The distribution extent of maser spots in the feature differs 0.2 mas from east to west between our results and H2007. Moreover, we found that change of relative positions of maser spots in the cluster reaches 0.1 mas or larger between observational epochs. This suggests that if one simply assumes the time-dependent, widely distributed maser sources as a stable single point source, it could cause errors of up to 0.1 mas in the annual parallax of S269. Taking into account the internal motions of maser spot clusters, the proper motion of S269 cannot be determined precisely. We estimated that the peculiar motion of S269 with respect to a Galactic circular rotation is ~20 km/s. These results imply that the observed kinematics of maser emissions in S269 cannot give a strong constraint on dynamics of the outer part of the Galaxy, in contrast to the claim by H2007.Comment: 33 pages, 11 figures, to be published in New Astronom