Naoki Hosaka

Membrane and Soluble Forms of Fas (CD95) and Fas Ligand in Peripheral Blood Mononuclear Cells and in Plasma from Human Immunodeficiency Virus- Infected Persons

The expression of membrane-bound Fas ligand (FasL) and Fas in lymphocytes and monocytes and levels of soluble forms of FasL (sFasL) and Fas (sFas) in plasma from human immunodeficiency virus (HIV)-positive and -negative subjects was evaluated. Surface FasL was detectable on monocytes, but poorly so on lymphocytes, even in the presence of KB8301, a metalloproteinase inhibitor. Unexpectedly, monocytes of HIV-positive subjects expressed less FasL than those of HIV-negative volunteers. sFasL levels in plasma of HIV-positive persons were elevated and correlated with levels in plasma and with HIV RNA burden. sFas levels in plasma of HIV-positive subjects were also elevated and correlated with Fas expression in apoptotic lymphocytes. Finally, culture-induced lymphocyte apoptosis of HIV-positive subjects was enhanced by anti-Fas agonistic antibody but was not inhibited by anti-FasL blocking antibodies. These results suggest that significant dysregulation of both Fas and FasL occurs in HIV infection and contributes to increased sensitivity of lymphocytes to apoptosis.

Maternal Microchimerism in Underlying Pathogenesis of Biliary Atresia: Quantification and Phenotypes of Maternal Cells in the Liver

OBJECTIVE. The goal was to examine whether microchimerism plays a crucial role in the pathogenesis of biliary atresia; we analyzed the localization of maternal microchimeric cells and their phenotypes. METHODS. Liver biopsy specimens from 8 male infants with biliary atresia and 6 control subjects with other liver diseases were investigated for maternal chimeric cells and their phenotypes through double-staining fluorescence in situ hybridization and immunohistochemical analyses. RESULTS. Significantly larger numbers of maternal XX+ cells were found in the portal area and sinusoids of patients with biliary atresia, in comparison with control patients. In phenotypic analyses of XX+ cells, CD8+ T cells, CD45+ cells, and cytokeratin-positive cells were found, and the numbers and proportions among total CD8+ T cells were significantly higher than those in control patients. CONCLUSIONS. Significantly more maternal chimeric CD8+ T cells in the livers of patients with biliary atresia suggest that maternal immunologic insults represent the underlying pathogenesis in biliary atresia. The findings support the recently postulated mechanisms of alloautoimmune and/or autoalloimmune responses.

Prevention of graft-versus-host disease by intra-bone marrow injection of donor T cells.

We have recently found that intra‐bone marrow‐bone marrow transplantation (IBM‐BMT) can be used to prevent graft‐versus‐host disease (GvHD), even when intensive donor lymphocyte infusion (DLI) is carried out. In the present study, in conjunction with IBM‐BMT, allogeneic splenic T cells as DLI were also injected into the bone marrow cavity of lethally irradiated (8.5 Gy) recipients. The extent of GvHD was compared with that of recipients that had received allogeneic IBM‐BMT plus i.v. injection of allogeneic T cells (intravenous DLI [IV‐DLI]). GvHD in recipients treated with allogeneic IBM‐BMT plus IBM‐DLI was far milder than in those treated with allogeneic IBM‐BMT plus IV‐DLI. This was confirmed macroscopically and histopathologically. The frequency of regulatory T cells (Tregs) detected as CD4+CD25+ and CD4+Foxp3+ cells was significantly higher in recipients treated with IBM‐BMT plus IBM‐DLI than in those treated with IBM‐BMT plus IV‐DLI. Donor‐derived helper T (Th) cells polarized to Th2 type in recipients treated with IBM‐BMT plus IBM‐DLI, whereas Th1 cells were dominant in recipients treated with IBM‐BMT plus IV‐DLI. Furthermore, the production of transforming growth factor‐β and hepatocyte growth factor from bone marrow stromal cells was enhanced after IBM‐DLI. Thus, IBM‐BMT plus IBM‐DLI seem to preferentially induce Tregs and Th2, resulting in the prevention of GvHD.

Ectopic Pituitary Adenoma With Malignant Transformation

We report here a case of ectopic pituitary adenoma with malignant transformation after repeated relapses. First, an ectopic pituitary adenoma producing follicle-stimulating hormone was found in the nasal cavity extending to the frontal cranial fossa. Despite repeated surgical resections of the tumor, it recurred three times in 2 years. The tumor gradually showed cellular atypia, mitosis, and necrosis. Immunohistochemical analyses revealed that the expressions of proliferating cell nuclear antigen and MIB-1 increased progressively. Moreover, the expression of p53 was detected at the second recurrence. Finally, at the third recurrence the tumor showed dissemination to the subarachnoid space and multiple metastases in the brain. The patient died of the tumor 10 months after the last resection. These findings indicate that the ectopic pituitary adenoma became malignant. To our knowledge, this is the first report on malignant transformation of ectopic pituitary adenoma. It is important to know that ectopic pituitary adenomas show malignant transformation and that the above parameters (proliferating cell nuclear antigen, MIB-1, and p53) may be useful indicators of the malignant potential of both ectopic and sellar pituitary tumors.

Increased expression of integrin β-4 in papillary thyroid carcinoma with gross lymph node metastasis

Although the prognosis is generally good for patients with papillary thyroid carcinoma, gross nodal metastasis of carcinoma has a poor prognosis. It is necessary to clarify how carcinoma progresses to gross nodal metastasis in order to establish a cure. The adhesion molecule integrin β‐4 is considered to be related to cell migration and metastasis in many carcinomas. In the present study, we examined integrin β‐4 expression in 65 cases of human papillary thyroid carcinoma using immunohistochemical methods. Expression of integrin β‐4 was found in all papillary carcinomas, but in few normal thyrocytes. Interestingly, integrin β‐4 expression in the carcinomas with gross (˘3 cm) lymph node metastasis was significantly higher than that in carcinomas with small (<3 cm) or no lymph node metastasis. These results suggest that integrin β‐4 expression in thyroid carcinoma may play a role in the development of gross lymph node metastasis of papillary carcinomas.

Presence of donor-derived thymic epithelial cells in [B6-->MRL/lpr] mice after allogeneic intra-bone marrow-bone marrow transplantation (IBM-BMT).

We have previously shown that allogeneic intra-bone marrow-bone marrow transplantation (IBM-BMT) can be used to treat autoimmune diseases in MRL/lpr (H-2(K)) mice with replacing not only hematolymphoid cells but also stromal cells by normal C57BL/6 (B6: H-2(b)) mouse cells. In the present study, we examined for existence of donor-derived thymic epithelial cells (TECs) in the host thymus using green fluorescent protein (GFP)-B6 (H-2(b)) mice. In [GFP-B6-->MRL/lpr] chimeric mice, splenocytes and thymocytes were completely replaced by donor-type cells, and levels of serum autoantibodies and proteinuria were significantly - reduced to those levels of normal donors. Interestingly, GFP-expressing TECs - not only medullary TECs, which express mouse thymus stromal (MTS)-10, but also cortical TECs, which express cytokeratin 18 - were found. Also, the number of autoimmune regulator (AIRE) expressing TECs, which regulates tissue-specific antigens to delete autoreactive cells, was reduced in the chimeric mice to that of the donor, whereas the number of forkhead box N1 (FOXN1) expressing TECs, which are crucial in the terminal differentiation of TECs, remained unchanged. These findings suggest that BMCs contain the precursors of functional TECs, and that they can differentiate into TECs, thereby correcting thymic function.

Extensive Studies on Perfusion Method Plus Intra‐Bone Marrow‐Bone Marrow Transplantation Using Cynomolgus Monkeys

The collection of bone marrow cells (BMCs) using a perfusion method has been advantageous not only because of the low contamination of BMCs with T cells from the peripheral blood but also the enrichment of stromal cells, which support hemopoiesis. Before the application of this new method to humans, its safety needed to be confirmed using cynomolgus monkeys. We therefore performed the perfusion method on more than 100 cynomolgus monkeys using the long bones (such as the humerus and femur) and also the iliac bones (for human application); in the more than 150 trials to date, there have been no accidental deaths. Furthermore, the technical safety of a new method for the intra‐bone marrow (IBM) injection of BMCs (termed IBM‐bone marrow transplantation) has also been confirmed using 30 monkeys.

Adult thymus transplantation with allogeneic intra-bone marrow-bone marrow transplantation from same donor induces high thymopoiesis, mild graft-versus-host reaction and strong graft-versus-tumour effects.

Although allogeneic bone marrow transplantation (BMT) plus donor lymphocyte infusion (DLI) is performed for solid tumours to enhance graft‐versus‐tumour (GVT) effects, a graft‐versus‐host reaction (GVHR) is also elicited. We carried out intra‐bone marrow–bone marrow transplantation (IBM‐BMT) plus adult thymus transplantation (ATT) from the same donor to supply alloreactive T cells continually. Normal mice treated with IBM‐BMT + ATT survived for a long time with high donor‐derived thymopoiesis and mild GVHR. The percentage of CD4+ FoxP3+ regulatory T cells in the spleen of the mice treated with IBM‐BMT + ATT was lower than in normal B6 mice or mice treated with IBM‐BMT alone, but higher than in mice treated with IBM‐BMT + DLI; the mice treated with IBM‐BMT + DLI showed severe GVHR. In tumour‐bearing mice, tumour growth was more strongly inhibited by IBM‐BMT + ATT than by IBM‐BMT alone. Mice treated with IBM‐BMT + a high dose of DLI also showed tumour regression comparable to that of mice treated with IBM‐BMT + ATT but died early of GVHD. By contrast, mice treated with IBM‐BMT + a low dose of DLI showed longer survival but less tumour regression than the mice treated with IBM‐BMT + ATT. Histologically, significant numbers of CD8+ T cells were found to have infiltrated the tumour in the mice treated with IBM‐BMT + ATT. The number of terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end‐labelling (TUNEL)‐positive apoptotic tumour cells also significantly increased in the mice treated with IBM‐BMT + ATT. Allogeneic IBM‐BMT + ATT thus can induce high thymopoiesis, preserving strong GVT effects without severe GVHR.

Preferential expansion of human umbilical cord blood-derived CD34-positive cells on major histocompatibility complex-matched amnion-derived mesenchymal stem cells

In vitro expansion of human hematopoietic stem cells has remained cumbersome. This paper demonstrates that umbilical cord blood-derived lineage negative/CD34-positive cells can selectively expand in vitro when cultured on autologous amnion-derived adherent cells. Background We previously found in a murine hematopoietic system that hematopoietic stem cells show high differentiation and proliferation capacity on bone marrow-derived mesenchymal stem cells/stromal cells (microenvironment) with “self” major histocompatibility complex (MHC). Design and Methods We examined whether amnion-derived adherent cells have the characteristics of mesenchymal stem cells, and whether these adherent cells can support the proliferation of umbilical cord blood-derived lineage-negative and CD34-positive cells (Lin–CD34+ cells) obtained from the same fetus to a greater extent than those derived from other fetuses. Results Culture-expanded amnion-derived adherent cells expressed mesenchymal stem cell markers and HLA-ABC molecules and could differentiate into osteoblasts, adipocytes and chondrocyte-like cells, indicating that the cells have the characteristics of mesenchymal stem cells. The Lin–CD34+ cells purified from the frozen umbilical cord blood were strongly positive for HLA-ABC, and contained a large number of hematopoietic stem cells. When the Lin–CD34+ cells were cultured on the autologous (MHC-matched) or MHC-mismatched amnion-derived adherent cells in short-term assays (hematopoietic stem cell-proliferation) and long-term culture-initiating cell assays, greater expansion of the Lin–CD34+ cells was observed in the MHC-matched combination than in MHC-mismatched combinations. The concentration of granulocyte-macrophage colony-stimulating factor in the culture supernatants of the long-term culture-initiating cell assays was significantly higher in the MHC-matched combination than in MHC-mismatched combinations. Conclusions It is likely that a MHC restriction exists between hematopoietic stem cells and mesenchymal stem cells/stromal cells in the human hematopoietic system and that granulocute-macropage colony-stimulating factor contributes to some extent to the preferential hematopoiesis-supporting ability of the MHC-matched amnion-derived adherent cells.

Contribution of neural cell adhesion molecule (NCAM) to hemopoietic system in monkeys.

Neural cell adhesion molecules (CD56) are important adhesion molecules that are mainly expressed on neural cells and natural killer cells. Although freshly isolated cynomolgus monkey bone marrow cells (BMCs) contained only a few CD56-positive cells, almost all the BM adherent cells (obtained after a 2- to 3-week culture of the BMCs) were stained positively with anti-CD56 monoclonal antibody (mAb). The BM adherent cells showed uniformly fibroblastic morphology and were negative for hematolymphoid markers (CD4, CD8, CD11b, CD14, CD34, and CD45). Adipogenesis and osteogenesis were observed under inductive culture conditions. The BM adherent cells had the ability to support hemopoiesis of hemopoietic stem cells (HSCs) in vitro, and the proliferation of HSCs was significantly inhibited by the addition of anti-CD56 mAb to the coculture system. CD56 molecules were also expressed on HSCs because about 20% of an HSC-enriched population (lineage-negative and blast-gated cells) was positive for CD56. In addition, the immunostaining of monkey BM sections revealed that many stromal cells were CD56-positive, and some CD56-positive stromal cells came into direct contact with CD56-positive hemopoietic cells. These results indicate that the CD56 molecule is expressed on both HSCs and BM stromal cells (containing MSCs) in monkeys, and therefore it can be speculated that CD56 also contributes to the human hematopoietic system.