Akihiro Tsukahara

Circadian rhythm of leucocytes and lymphocyte subsets and its possible correlation with the function of the autonomic nervous system

There are physiological variations in the levels of leucocytes. Among these, the circadian rhythm is very important in terms of the magnitude. Since newly identified lymphocyte subsets (i.e. extrathymic T cells) have recently been detected, a comprehensive study of the circadian rhythm was conducted. All leucocytes were found to vary in number or proportion with a circadian rhythm and were classified into two groups. One group—granulocytes, macrophages, natural killer (NK) cells, extrathymic T cells, γδ T cells, and CD8+ subset—showed an increase in the daytime (i.e. daytime rhythm). The other group—T cells, B cells, αβ T cells, and CD4+ subset—showed an increase at night. Humans are active and show sympathetic nerve dominance in the daytime. Interestingly, granulocytes and lymphocyte subsets with the daytime rhythm were found to carry a high density of adrenergic receptors. On the other hand, lymphocyte subsets with the night rhythm carried a high proportion of cholinergic receptors. Reflecting this situation, exercise prominently increased the number of cells with the daytime rhythm. These results suggest that the levels of leucocytes may be under the regulation of the autonomic nervous system.

Autologous killing by a population of intermediate T-cell receptor cells and its NK1.1+ and NK1.1− subsets, using Fas ligand/Fas molecules

Self‐reactive clones, estimated by anti‐Vβ monoclonal antibodies (mAb) in conjunction with the Mls system, are confined to a population of intermediate (int) T‐cell receptor (TCR) (or CD3) cells (i.e. TCRint cells), but are not found among TCRhigh cells. The next questions to be answered are whether autologous killing is confined to TCRint cells and how such killing is mediated. In this study, 51Cr‐labelled thymocytes of syngeneic or allogeneic origin were used as target cells (4‐hr assay). When liver and splenic mononuclear cells (MNC) obtained from B6 mice were used as effector cells, prominent autologous killing was seen in liver MNC, but not splenic MNC. Such killing was not seen when thymocytes from B6‐lpr/lpr mice (i.e. Fas−) were used as target cells, nor when liver MNC from MRL‐gld/gld mice (i.e. Fas ligand−) were used as effector cells (target thymocytes of MRL‐+/+ mice). Cell separation experiments using a cell sorter revealed that autologous killing was mediated for the most part by CD3int cells, while allogeneic killing was mediated entirely by natural killer (NK) cells, TCRint cells and TCRhigh cells. Among CD3int cells, the NK1.1+ subset (i.e. NK1.1+ T cells) manifested a higher level of autologous killing than did the NK1.1− subset. Consistent with the results of a functional assay, it was found by reverse‐transcription–polymerase chain reaction (RT‐PCR) assay that CD3int cells among liver MNC showed the expression of Fas ligand mRNA, while thymocytes expressed Fas mRNA. When class I major histocompatibility complex (MHC)− thymocytes (from β2‐microglobulin‐deficient mice) were used as target cells, NK cells, but not CD3int cells, showed potent cytotoxicity. These results suggest that autologous killing is a major function of TCRint cells with self‐reactivity, and that such killing is mediated by means of Fas ligand/Fas molecules.

Self-reactive forbidden clones are confined to pathways of intermediate T-cell receptor cell differentiation even under immunosuppressive conditions

It is believed that self‐reactive forbidden T‐cell clones are generated by ‘failure’ of the pathway of T‐cell differentiation in the thymus, if it is disturbed. We examined how such forbidden clones are generated under immunosuppressive conditions. Mice were treated with an injection of deoxyspergualin, FK506, or cycloporin A. From day 3, the number of cells yielded by various organs decreased. Because of the resistance of intermediate (int) T‐cell receptor (TCR) cells (i.e. TCRint cells), they became more prominent in proportion than TCRhigh cells. TCRhigh cells are conventional T cells generated through the mainstream in the thymus, whereas TCRint cells are primordial T cells generated by the extrathymic pathway or an alternative intrathymic pathway. Similar to untreated mice, forbidden Vβ3+ and Vβ11+ clones in C3H/He (Mls‐1b2a) mice were confined to TCRint cells after treatment; there was no leakage of forbidden clones into TCRhigh cells in the thymus and periphery. In parallel with the increase in the proportion of TCRint cells, the proportion of forbidden clones also increased under immunosuppressive states, especially in the liver. Liver mononuclear cells isolated from treated mice still had the potential to mediate autologous killing. The present results suggest that the generation of self‐reactive clones is highly restricted to the pathways of TCRint cell differentiation even under immunosuppressive conditions.

Quick recovery in the generation of self-reactive CD4low natural killer (NK) T cells by an alternative intrathymic pathway when restored from acute thymic atrophy.

The thymus comprises the mainstream of T cell differentiation which produces conventional T cells and an alternative pathway which produces primordial T cells with intermediate density of T cell receptor (TCR)–CD3 complex on the surface (i.e. intermediate TCR cells or TCRint cells). We induced acute thymic atrophy in mice by an administration of hydrocortisone (10 mg) or irradiation (6.5 Gy). It was demonstrated that CD3intCD4lowNK1.1+ T cells were immediately generated by an alternative intrathymic pathway without passing through the double‐positive CD4+8+ stage, when restored from thymic atrophy (days 3–14). These CD3intCD4lowNK1.1+ T cells mediated self‐reactivity and appeared even in the periphery. mRNA of an invariant chain of TCR Vα14Jα281 gene product was detected in these CD4low T cells, but not remaining CD4high T cells. The mainstream of T cell differentiation in the thymus was not restored up to day 14 and there was no leakage of self‐reactive clones into the population generated through the mainstream. These results reveal that an alternative intrathymic pathway is associated with the generation of self‐reactive T cells, in an early restoration phase after thymic atrophy.

Participation of NK1.1+ T cells in the rejection of lpr αβT cells when bone marrow cells of lpr mice are transplanted into B6 mice

When C57BL/6 (B6) mice were irradiated (9 Gy) and received bone marrow (BM) cells of B6‐lpr/lpr mouse origin (i.e., lpr→B6), all mice died within 6 days. In the irradiated B6 mice, radioresistant CD3− IL‐2Rβ+ NK cells and IL‐2Rβ CD3int cells (i.e., CD3int cells of extrathymic origin) remained, especially in the liver. There were two subsets, NK1.1+ and NK1.1−, among the IL‐2Rβ+ CD3int cells. However, the NK1.1+ subset (i.e., NK1.1+ T cells) was much more radioresistant, and the majority of CD3int cells belonged to this subset in irradiated mice. The expansion of lymphocytes from injected BM cells did not occur in the irradiated B6 mice. However, such expansion did take place in irradiated B6‐lpr/lpr mice injected with both BM cells of B6‐lpr/lpr and B6 origin. As a result, the mice subjected to BM cells survived. Irradiated B6 mice were treated in vivo with anti‐NK1.1 mAb or anti‐asialoGM1 antibody to eliminate NK cells alone or both NK cells and NK1.1+ T cells. When irradiated B6 mice were pretreated with anti‐NK1.1 mAb, the mice could survive. These results suggest that intact NK1.1+ T cells of extrathymic origin may recognize abnormal BM cells with the lpr gene and inhibit the expansion of lymphocytes, including abnormal double‐negative CD4−8− cells, in B6‐lpr/lpr mice. To inhibit the expansion of lymphocytes, mechanisms other than Fas ligand/Fas molecules on extrathymic T cells may be responsible.

Laparoscopic right hemicolectomy for a patient with idiopathic retroperitoneal fibrosis: A case report.

A 62‐year‐old man with abdominal pain and lumbago was admitted to our hospital. Blood examination revealed renal insufficiency, and CT revealed retroperitoneal fibrosis causing bilateral hydrocele and ureteral compression. A colonoscopy was performed to rule out secondary retroperitoneal fibrosis due to malignancies, and this imaging revealed an ascending colon cancer. Laparoscopic right hemicolectomy with lymphadenectomy and retroperitoneal biopsy were performed. The retroperitoneum was filled with hard, white fibrous tissue, which made it difficult to mobilize the right mesocolon from the retroperitoneum. Devascularization performed before mobilization allowed for a safe and oncologically feasible procedure. Histologically, there were no malignant cells in the retroperitoneal tissue. The patient has been without colon cancer reoccurrence for 4 years. When the surgical challenges that distinguish these patients from ordinary cases are recognized preoperatively, laparoscopic colectomy may be a feasible option for patients with colorectal cancer with idiopathic retroperitoneal fibrosis.

An allogeneic microenvironment influences the phenotype of intermediate T-cell receptor cells expanding in MRL-lpr/lpr mice.

MRL‐lpr/lpr (lpr) mice fall victim to autoimmune disease owing to a lymphoproliferative disorder mainly of double‐negative (DN) CD4− CD8−αβT cells expressing a low density of interleukin‐2 receptor β‐chain (IL‐2Rβ). It was previously revealed that the lpr gene is a defective Fas gene, into which an early transposon (ETn) of retrovirus is transfected. As a result of the failure of apoptosis, intermediate T‐cell receptor (TCR) cells (i.e. TCRint cells) with DN phenotype abnormally accumulate in the periphery of lpr mice. We investigated herein how these TCRint cells are selected in terms of CD4, CD8 and TCR in lpr mice. When a whole fraction of mononuclear cells (MNC) in various immune organs of lpr mice was injected into scid mice (allogeneic circumstance), CD8+ TCRint cells mainly expanded. They had a high density of IL‐2Rβ. This was true when bone marrow cells of lpr mice were injected into scid mice. On the other hand, when MNC of the spleen and bone marrow in lpr mice were injected into irradiated (9 Gy) lpr mice (syngeneic circumstance), the major expanding cells were DN TCRint cells expressing a low density of IL‐2Rβ. A cell‐sorting experiment for purified fractions demonstrated that only CD8+ cells reconstituted TCRint cells in scid mice. Namely, DN CD4− CD8− cells as well as CD4+ cells which once acquired the mature phenotype, no longer switched their phenotype. These results suggest that the phenotype of TCRint cells is influenced by the surrounding microenvironment.

Benefits of Endoscopic Nasobiliary Drainage for Bile Leakage after Laparoscopic Cholecystectomy

腹腔鏡下胆嚢摘出術の術後合併症の1つに胆汁漏がある. 今回, 腹腔鏡下胆嚢摘出術を施行した293例中6例 (2.0%) に術後胆汁漏を認めた. この6例すべてに内視鏡的逆行性胆管造影を施行し, 胆管損傷形態を検索した. 引き続き内視鏡的経鼻胆管ドレナージを行い, 全例保存的に治療しえた. 胆管ドレナージ施行後, 平均2.3日で胆汁排出を認めなくなり, 平均9.8日でドレナージチューブの抜去が可能であった. 腹腔鏡下胆嚢摘出術後の胆汁漏に対する治療法として内視鏡的経鼻胆管ドレナージは侵襲が少なく, 胆汁漏の早期改善に有効であった. 本法は, 治療期間の短縮と手術的治療の回避に効果的な処置であることから, 第1選択と考えて積極的に施行すべき治療法と考えられた.