変形性膝関節炎患者膝関節液中の細胞外マトリックス成分と可溶性VCAM-1

It is generally accepted that the number of patients with osteoarthritis (OA) of the knee must increase in the future because of the increase of the aged. The pathogenic mechanisms of OA haven't been elucidated yet, but it is supposed that mediate pannus may cause the factors for irreversible cartilage damage in the inflammatory process. Therefore it is very important to find and predict OA at the early stage. Now there were many indices to determine joint damage with OA, for example, cytokines including IL, TNF, TGF, matrix metalloproteinase (MMPs), tissue inhibitor of metalloproteinase (TIMPs), proteoglycan (PG), hyaluronate (HA), super oxide, adhesion molecules including, ICAM-1, VCAM-1 and PECAM. In this study, we measured that the soluble VCAM-1 (sVCAM-1), soluble collagen (sCOLL), heparansulfate proteoglycan (HSPG), uronic acis and CRP in synovial fluids (SF) with OA. A positive correlation between the level of sVCAM-1 and sCOLL (ρ = 0.652) was noted. However there was no factor showing a correlation with CRP. It was suggested that the levels of sVCAM-1 and sCOLL in the synovia fluid were active inflammation indices of knee damage, as the different aspects from CRP. The level of HSPG also showed a unique trend because HSPG on the cell surface was quickly digested endocytosis, so that it was not suitable for the inflammatory index in synovial fluid with OA.変形性膝関節炎(OA)患者は,高齢化社会の到来とともに今後ますます増加するものと考えられる｡OAの発症メカニズムについては,関節軟骨の破壊･修復やパンヌス形成に関わる多くの関連因子(サイトカイン,マトリックス分解酵素,分解酵素抑制因子,プロテオグリカン,活性酸素,細胞接着因子など)について,多面的に研究が進んでいる｡しかし,膝関節の炎症初期の病態のメカニズムについては,なお詳細に解明されておらず,関節炎初期の炎症指標の検索が今後さらに必要と考えられる｡本研究で私たちは, OA患者膝関節液中の細胞外マトリックス構成成分であるコラーゲン,へパラン硫酸プロテオグリカン,ウロン酸および炎症の最も初期に血管内皮細胞に出現するVCAM-1等の可溶化成分濃度を測定した｡患者膝関節液(SF)中の可溶性コラーゲン量と可溶性VCAM-1(sVCAM-1)量とは有意な正の相関関係が(ρ=0.652)観察された｡しかし,ヘパラン硫酸プロテオグリカン(HSPG)と可溶性コラーゲン(sCOLL)値間には有意な相関関係は確認されなかった｡また,一般に炎症の指標として用いられるCRP値とも比戟検討したが,CRP値と他の測定項目値とは一定の相関関係は存在しなかった。以上の結果より,OA患者のSF中の炎症マーカ-として,急性炎症時には従来の炎症指標蛋白質であるCRP値が有力であるというSipe JDの報告から推測するとsCOLLとsVCAM-1はCRP値と一定の相関を示さないことより,CRPの動態とは異なった炎症指標としての有用性が考えられる｡しかし,HSPGはエンドサイトシスにより速やかに代謝されるためSF中への解離が少なく,炎症指標としての可能性は低いと考えられる

An experimental study on the relation of T2-signal high intensity in MRI to histopathological changes in the kainic acid model of temporal lobe epilepsy in rats.

側頭葉てんかんでは，てんかん焦点に一致してMRI T2高信号領域が見られ，FLAIR法でこれがより明瞭になるが，このMRI所見と病理組織学的変化との関係は必ずしもはっきりしていない。そこで，Sprague－Dawleyラットにカイニン酸（KA）でけいれん発作重積状態を起こし，経時的にMRIを記録するとともに，ニッスル染色，GFAP免疫染色での病理組織学的変化を調べて両者の関係について検討した。KA群では，MRIで1～8週間後のいずれにおいてもpiriform cortexからentorhinal cortexにかけて不整形のT2高信号領域がみられたが，stage3のけいれん発作しか出現しなかったラットではstage4，5が出現したラットに比べて程度が弱かった。組織学的には，CA1，subiculum，piriform cortex，entorhinal cortexで神経細胞の消失，濃染細胞の増加と萎縮，GFAP免疫反応の増強が見られたが，piriform cortex，entorhinal cortexでの神経細胞消失の程度はT2信 号の程度と相関せず，GFAP免疫反応が増強した領域に一致して高信号がみられた。しかし，海馬のGFAP免疫反応増強はMRI所見に反映されず，これはMRIの解像度の限界にもよると考えられた。The relation of T2-signal high intensity areas observed in temporal lobe epilepsy to histopathological changes in limbic structures was examined in the rat kainic acid (KA) model of epilepsy. Male 8-week-old Sprague-Dawley rats were injected with 10mg/kg(i.p.) of KA or saline (control). Repetitive generalized convulsions (stage 3, or stage 4,5 seizures of amygdala kindled seizures) lasted for 3 to 4 hrs. were induced by KA injection in all rats. MRI was recorded on the day before the KA injection and 1, 2, 4, 8 weeks after the injection by fluid-attenuated inversion recovery method under deep pentobarbital anesthesia. Following the last MRI recording, rats were perfused with 4% paraformaldehyde (PFA) from left cardiac ventricle, post-fixed overnight in 4% PFA and brains were embedded in parafin. Coronal brain sections (6μm) were stained with cresyl violet, or mouse anti-GFAP antibody followed by biotinylated goat anti-mouse IgG and avidin-biotin-peroxidase (vectastain ABC kit). Irregularshaped moderate to severe high T2-signal areas were observed in bilateral piriform and entorhinal cortex in MRI. These high T2-signal areas were ovserved from 1 week after the KA injection to 8 weeks after the injection, and were more prominent in rats elicited stage 4 or 5 seizures than in rats elicited stage 3 seizures. Loss of pyramidal neurons and increased GFAP immunoreactivity were observed in piriform cortex, entorhinal cortex, CA1, subiculum, and hilus of dentate gyrus. The increase of GFAP immunoreactivity, but not the intensity of neuronal loss, in piriform and entorhinal cortex was almost correponded to the size and intensity of T2-signal. However, the increase of GFAP immunoreactivity in hippocampus was not detected as the increase of T2-signal in MRI. These findings indicate that astroglial reactions in piriform and entorhinal cortex are more sensitive to T2-weighted MRI than those in hippocampus

Robot-assisted endoscopic surgery experience

In Japan,１２ new operative robot-assisted endoscopic surgery methods were officially approved by the ２０１８ revision of the medical payment system, and robot-assisted endoscopic surgery is using in each field. We have used the pneumatically driven endoscope manipulator robot（EMAROⓇ）in three video-assisted thoracoscopic surgery（VATS）lobectomy cases : right upper lobe lung cancer in a ５３-year-old male, right lower lobe lung cancer in a ７０-year-old male, and right lower lobe lung cancer in an ８１-year-old female. The average operative time was ２０９ min. The average bleeding volume was ４６．７ml. The grades ware nearly equal to those obtained in a standard VATS lobectomy. The head sensor that monitors the movement of the operator is connected to the endoscope holder. The operator can thus move the endoscope smoothly from the operator’s point of view, and endoscope-user skill is not needed. A VATS lobectomy can be performed by only two operators （no scope user is needed）, and this is a major advantage of the new manipulator robot. We expect to see further advances in the use of robot assistance in surgeries

The Units of IgM, IgA and IgG Rheumatoid Factors by Enzyme Linked Immunosorbent Assay in Human Sera

The measurement of Rheumatoid Factor (RF) has been a valuable tool in diagnosing Rheumatoid Arthritis (RA) for many years. Since Waaler's discovery of the RF, many studies have been carried out. In the recent years studies have been shown that there is no such correlation between the level of the RF titers and disease activity. In this report we have measured the IgM-RF, IgA-RF and IgG-RF by an Enzyme linked immunosorbent assay (ELISA) for 144 normal human sera and 50 patients sera. At the same time we have measured the titers of gelatin particle agglutination (RAPA) and the Latex agglutination (RA-test). The value of IgG-RF have been most correlated to the titers of RAPA (r=0.9037). Using this ELISA technic to detect IgM-, IgA- and IgG-RF, have been aviable more clearly the activity and the stage of Rheumatoid disease

A study on the correlation between the results of entrance examination and the academic record of the students in the department of laboratory technology

1987年4月から1995年4月の9年間に衛生技術学科へ入学した361名を対象に,高校評点,入学試験(学力試験)成績と,入学後の学業成績,退学,留年,臨床検査技師国家試験との関係を調べた｡ 1)入学者総数は361名で,このうち留年者が39名(10,8%),退学者が18名(5%)であった｡退学者の中には,実質入学辞退者が6名,4年制大学へ進路を変更した者が4名いたが,こうした退学者は1992年度までの入学者に多かった｡ 2)入学者全体では,予定卒業者,留年者,退学者の間で高校評点,入学試験成績には差がなかったが,入学年度ごとにみると,1987,1988年度は留年者が予定卒業者に比べて入学試験の総得点が高い傾向があり,逆に1991年,1993年度は予定卒業者に比べて有意に低かった｡ 3)高校評点は,データが入手できた1991年度以降一貫して留年者の評点が予定卒業者より低く, とくに1991年,1993年度は留年者の評点が有意に低かった｡ 4)留年者も含めて,一般教育科目の成績と専門科目の成績との間に有意を正の相関がみられた｡ 5)高校評点と入学試験成績との間には軽度の相関しかみられなかったが,高校評点および入学試験総合点は入学後の一般教育科目,専門科目の成績と高い正の相関を示した｡ 6)しかし,個々の入学試験科目の得点は,入学後の成績と相関しないか,軽度の相関しかみられなかった｡ 7)国家試験不合格者は,高校評点,入学試験総合点,一般科目平均点,専門科目平均点のいずれも合格者に比べて得点が低かったが,有意差はなかった｡以上の結果から,短期大学部開設当初は留年者,退学者の中に予定卒業者より学力のある学生がいたが,年を追うごとに入学者の学力が均質化し,学力が不十分なために留年する学生が多くなってきていると言える。一般教育科目の成績と専門科目の成績が強い正の相関を示すことは,1年次の成績がそのまま2,3年次の成績に反映されることを示しており,入学当初の動機付けと勉学意欲の喚起が重要なことを示している｡高校評点や学力試験の成績は入学後の成績とよく相関しており,学生選抜の有効な指標であると考えられたが,各入試科目との相関は乏しかったことから,個々の試験科目の問題内容については改善すべき課題があると思われた｡The correlation between the school record in high school, result of entrance examination and the academic record, failure in promotion and withdrawal from school was examined in 361 students entered into department of laboratory technology from 1987 to 1995. 1) Thirty-nine students (10.8%) had failed in promotion, and 18 students (5%) had leaved from the school. The reason of withdrawal was refusal in 6 cases, and 4 students had changed their course to other universities. These cases were more frequent in the period until 1990 than in the period after 1991. 2) There were no differences in the school record in high school and the result of entrance examination among students who graduated scheduled period of attendance (regular students), those who failed in promotion, and those who had leaved the school. However, the students who failed in promotion had got higher score in entrance examination than regular students in 1987 and 1988. In contrast, regular students had got higher score in entrance examination than the students failed in promotion in 1991 and 1993. 3) The school record in high school could be cheked during the period from 1991 to1995. In this period, the students failed in promotion had got lower score in high school than regular students. Especially, the difference was significant in 1991 and 1993. 4) There was a significant positive correlation between the results in examination of general subjects and those in examination of technical and proffesional education. 5) The school records in high school had relatively weak positive correlation with the results of entrance examination. In contrast, both the school record in high school and result of entrance examination had clear positve correlations with both the results in examination of general subjects and technical and proffesional education. 6) However, the result of each subject of entrance examination, i.e. Japanese, Mathematics, English, and Science, had no or weak positive correlation with both the results in examination of general subjects and technical and proffesional education. 7) The students who had not passed the national examination of laboratory technologist had lower score in school record in high school, entrance examination, examination of general subjects and technical and proffesional education, but the diffenreces were not statistically significant. The results of the study indicate that there were students who failed in promotion or leaved school despite their excellent ability during four years following the establishment of School of Health Sciences, but thereafter the ability of students gradually became homogeneous and increased the students who failed in promotion because of insufficient ability. The highly positive correlation between the results in examination of general subjects and those in examination of technical and proffesional education indicate that the results of examination in the first year were directly reflected in the results of examination in second and third year, and that it is important to encourage the students's motivation and volition to study with ardor immediately after the entrance. The positive correlation of school record in high school and result of entrance examination with academic record after the entrance implicate the potential usefulness of school record in high school and result of entrance examination in determining the eligibility to enter the school. However, it is desirable to improve the subjects and problems of entrance examination because the result of each subject of entrance examination was poorly reflected in the academic record

リウマチ因子陽性ヒト血清中の免疫グロブリンGクラスリウマチ因子と免疫複合体ならびにCRPとの相関性

We measured the concentration of three immunoglobulin classes of rheumatoid factors (Ig-RFs), C3d binding IgG immune complex (C3d-IC), C1q binding IgG immune complex (C1q-IC) and C-reactive protein (CRP) in 74 samples of human sera with high levels of RF (24.0~2350.0IU/ml). In sera with high levels of C3d-IC (>15.0μg/ml), there was a positive correlation between the levels of CRP and the IgG-RF, but there was no correlation between the levels of CRP and the immune complexes (C3d-IC and C1q-IC). And then, there was a positive correlation between the levels of CRP and IgG-RF or C3d-IC and IgG-RF when the levels of C1q-IC in patients sera were higher than 80.0μg/ml. However, there was no correlation between the levels of CRP and C1q-IC in these patients sera containing high levels of both C3d-IC and C1q-IC. These results indicated that the determination of C3d-IC, C1q-IC, IgG-RF and CRP in human sera containing RF denote different implications as inflammatory indexes on progression of rheumatoid arthritis and other autoimmune diseases.一般に慢性関節リウマチの診断ならびに活動性の一指標として、日常臨床検査では患者血清中のリウマチ因子の測定が実施されている。本論文ではRF検査依頼が有り、高RF値（24.0～2350.0IU/ml）を示した患者血清74検体について、免疫グロブリンクラス別RF値を測定するとともに自己IgGと補体との免疫複合体（IC）であるC1q-IgG・IC（C1q-IC）、C3d-IgG・IC（C3d-IC）ならびにCRP値を測定し、それらの測定値間の相関性について統計学的検討を行った。C3d-IC値が15.0μg/ml以上の高値を示す患者血清では、CRP値とIgGクラスのRF（IgG-RF）値間については正の相関結果がが得られた。また、C1q-IC値が80.0μg/ml以上の場合にもCRP値とIgG-RF値間およびC3d-IC値とIgG-RF値間では正の相関結果が得られた。しかし、C3d-ICならびにC1q-ICともに高値例の患者血清中のCRP値とC1q-IC値間には有意な相関は観察されなかった。以上の結果より、慢性関節リウマチの活動度を判定する上で、従来から炎症マーカーとしてCRP値が利用されているが、血清中に免疫複合体が高レベルに検出される患者については、炎症の指標としてCRP以外にもC1q-ICやC3d-ICおよびIgG-RF等を加えた総合的な判断が必要であることが示唆される

長期ホルマリン固定により失活したProliferating Cell Nuclear Antigen (PCNA) の免疫反応性回復条件の基礎的検討 ―マイクロウェーブ、オートクレーブの影響について―

Using paraffin-embedded tissue sections of liver cancer obtained from autopsy which had been preserved in 10% buffered formalin solution for 6 months while PCNA immunoreactivity was lost, we examined the effects of heat processing by either microwave(MW) and autoclave(AC) in the presence of various processing solution. It appeared that AC processing took shorter time period than MW irradiation to restore equal immunoreactivity. With regard to immunoreactivity retrieval by MW irradiation,however, variation of the degree of retrieval depending on processing time was smaller than in AC, and so the stable consequences were obtained. Although AC processed tissues tended to be stained deep, prolonged processing time presented strong background staining and blurred nuclear margins which made it difficult to estimate the positive cell count. As for the effects of processing solution, there was little difference in retrieval of PCNA among 0.01 M citrate buffer (pH 6.0), saturated solution of lead thiocyanate and distilled water, but the least background staining was observed with distilled water. These observations suggest that MW irradiation of which effect of retrieval is less dependent of processing time and with the least background stainability, is superior to AC processing for PCNA immunoreactivity retrieval on formalin-fixed tissues.10％緩衝ホルマリンに6ヶ月間浸漬していた剖検材料（肝臓癌）のパラフィン包埋後の組織切片を用いて、ホルマリンの固定作用により失活したPCNAの免疫反応性の回復にマイクロウェーブ（MW）及びオートクレーブ（AC）による熱処理とその時用いる処理溶液が、どのような影響を与えるかについて検討した。その結果、同等の免疫反応性を回復するのには、AC処理の方がMW照射より短時間でよいことが解った。しかし、MW照射による免疫反応性回復では、処理時間による影響がAC処理に比べて少なく、安定した結果が得られた。また、PCNAの染色所見については、AC処理の方が濃く染まる傾向が見られた。しかし、処理時間が長くなるとバックグラウンドの染色性が高くなる、核の周囲ににじみ現象が見られる等の所見があり判定に困難をきたした。一方、処理溶液についてはクエン酸（0.01M pH6.0）、チオシアン酸鉛飽和溶液、蒸留水について検討を行った結果、PCNAの回復には差は認められなかったが、バックグラウンドの染色については蒸留水が最も少なかった。以上の結果により、ホルマリン固定により失活したPCNAの免疫反応性を回復するには、MW照射の方がAC処理より処理時間に関係なく安定した染色性が得られ、なおかつバックグラウンドの染色性が少ない等の点で優れていることが示唆された

ヒト血清リポタンパク質とシクロデキストリンのアガロースゲル内における相互作用

Cyclodextrins (CDs) are cyclic oligosaccharides with low molecular weight. They have been known to bind to some serum lipoproteins and to form complexes. To elucidate whether each serum lipoprotein subclass could be separated by electrophoresis using CDs, we performed preliminary experiment, in which lipoprotein-CDs interaction was examined on electrophoresis with agarose gel. When the supporting agarose gel containing both α-CD and β-CD was prepared and was applied to isoelectric focusing for fractionating serum lipoproteins, apoB lipoproteins were found to be clearly separated into several fractions on this electrophoresis. This finding suggested that apoB lipoprotein may be detected as isolated form by arranging amounts of added CDs.シクロデキストリンは低分子量の環状オリゴ糖で, リポタンパク質と複合体を形成する｡シクロデキストリンを使用した電気泳動による血清リポタンパク質サブクラスの分離の可能性を明らかにするために, リポタンパク質とシクロデキストリンとの泳動用ゲル内での相互作用について検討をおこなった｡その結果,α-CDとβ-CDの2種類のCDをアガロースゲルに添加し,このゲルを泳動用支持体として等電点電気泳動をおこなったところ,apoB含有リポタンパク質が数分画分離された｡したがって,ゲル内へのシクロデキストリンの適正な添加条件が設定できるならば,apoB含有リポタンパク質サブクラスの分離が可能となることが示唆された

IgG Heavy-Chain Subclass Typing of Human Serum M-Protein -Optimal condition for the separation and the detection with high resolution of agarose gel isoelectrofocusing-

IgG Heavy-Chain subclass typing of human serum M-protein were isoelectrofocussed (IEF) in agarose gel, and then the bands of IgG were detected with peroxidase-conjugated anti-human IgG1-4 monoclonal antibodies on the same isoelectrofocussed agarose gel plate. This IEF enzyme immunodetection patterns were composed of four to eight discrete bands (The range of pI was 6.0 to 9.0). These bands were dependent on immunofixationbands. It was very specific and clear to detect the subclass of IgG Type M-protein in human serum using this method

Modified Method of Kluver-Barrera's Staining with Microwave Irradiation

クリューバー･バレラ(KB)染色は中枢神経系の白質における重要成分である髄鞘を選択的に染色する方法として日常的に広く使われている｡しかし現在の所,ルクソール･ファスト青(LFB)染色後の0.05%炭酸リチウム及び70%アルコールによる分別時間が確定されていないので,分別の良否を経験的にしか判断せざるをえないという欠点がある｡そこで,本研究において,マイクロウェーブ照射をLFB染色に応用することで,その後の分別について検討した結果,従来の0.05%炭酸リチウムでは良好な結果が得られなかったが分別液として,0.05%炭酸水素ナトリウムおよび70%アルコールを用いることで分別時間の確定が可能となった。Kluver-Barrera's (KB) staining is the well-known and widely used method for staining the Schwann's sheath of the central nervous systems. On the contrary, the optimum time is not established yet, which incubated with 0.05% lithium carbonate and 70% alcohol after luxol fast blue staining. The modified method was studied that was stained with the microwave irradiation and used 0.05% sodium bicarbonate in stead of 0.05% lithium carbonate. This method was simple and useful to differentiate Schwann's sheath from other components