Akinobu Nakano

Cathepsin B and H activities and cystatin C concentrations in cerebrospinal fluid from patients with leptomeningeal metastasis

BACKGROUND Cysteine proteases are involved in the extension of cancer into the subarachnoid space. The presence of cathepsins B and H along with their potent inhibitor cystatin C in the cerebrospinal fluid (CSF) was investigated in patients with leptomeningeal metastasis of cancer (LM). MATERIALS AND METHODS CSF samples were obtained in 16 cases of LM (10 solid tumors and 6 leukemia or lymphoma) and compared with 11 cancer cases without involvement of the central nervous system, 12 multiple sclerosis cases and 34 healthy volunteers. The activity of the enzymes was measured, their molecular forms were analyzed by the Western blotting, and the concentration of cystatin C was measured by ELISA. Immunohistochemistry of the leptomeningeal tissues was also performed in six autopsy cases of LM. RESULTS High activities of cathepsins B and H along with decreased cystatin C concentration were observed in CSF of LM as compared with three control groups. Western blot analysis revealed higher concentration of the enzyme protein as well as its active forms in samples with higher enzyme activity. Cells metastasizing leptomeningeal tissue were clearly positive in immunohistochemical staining of cathepsins, indicating active production by tumor cells. CONCLUSION Production of cathepsins B and H by tumor cells and their high activity along with concomitant decrease of their potent inhibitor, cystatin C, in the CSF might contribute in the process of metastasis and spread of the cancer cells in the leptomeningeal tissues. A high enzyme activity/cystatin C concentration ratio in the CSF could be useful when diagnosing LM in combination with other parameters.

Expression of Leukocyte Common Antigen (CD45) on Various Human Leukemia/Lymphoma Cell Lines

CD45 antigen (leukocyte common antigen), a unique and ubiquitous membrane glycoprotein with a molecular mass of about 200 kDa, is expressed on almost all hematopoietic cells except for mature erythrocytes. However, the biological function of this glycoprotein still remains to be resolved. In order to clarify the role of CD45 antigen in hematopoietic cell differentiation and function, its expression on human leukemia lymphoma cell lines was studied by membrane immunofluorescence. Thirty eight established cell lines were analyzed using T29–33, a monoclonal antibody (MoAb) that recognizes the common epitopes of this glycoprotein molecule. Conventional cell marker studies were also carried out on these cell lines to compare their CD45 expression. It was shown that CD45 expression varies among B lineage cells depending on cell differentiation, in contrast to its stable expression on leukemic T cell (6/6, positive) and myeloid (5/5, positive) lineage cell lines. On the other hand, only two out of six histiomonocytoid lineage cell lines were positive. Human T cell leukemia lymphoma virus type I (HTLV‐I) associated T cell lines derived from peripheral blood leukocytes of patients with adult T cell leukernia/ lymphoma (ATL/L) in Japan did not express CD45 on their cell surface. Taken together, these observations suggest that CD45 has a functional role in hematopoietic cell activation and differentiation. Acta Pathol Jpn 40: 107–115, 1990.

Establishment of a human preadipose cell line, HPB‐AML‐I: Refractory to PPARγ‐mediated adipogenic stimulation

In this study, we established a unique cell line, HPB‐AML‐I (AML‐I), from peripheral blood mononuclear cells collected from a patient with acute myeloid leukemia (AML: M1). Morphological and phenotypical analyses of the established AML‐I cells demonstrated that they belong to a preadipocyte cell line as indicated by their storage of lipid droplets and expression of surface antigens similar to those found on bone marrow stromal cells (MSC). Through cell culture under adipogenic conditions, effective differentiation of AML‐I cells into adipocytes was induced by an adipogenesis inducing cocktail (INC) made up of a mixture of methylisobutylxanthine, hydrocortisone, and indomethacin. By contrast, activation of peroxisome proliferator‐activated receptor (PPARγ), which plays a key role in lipids metabolism and is highly expressed in AML‐I cells, decreased the number of lipid droplets in AML‐I cells. Here we report the establishment of a unique human derived‐preadipocyte cell line, AML‐I, and its bi‐directional adipogenic response to different type stimulation, i.e., one is a refractory response to troglitazone, a well‐known adipogenic stimulator, and a positive response to INC, an adipogenesis induction cocktail. These results suggest that, based on the adipogenic response, there might be some distinct lineages in human adipocytes and that the unique differentiation of AML‐I cells should be useful for analyzing both the differentiation and regulation of human preadipocytes. J. Cell. Physiol. 197: 42–52, 2003© 2003 Wiley‐Liss, Inc.

Functional analysis of clonally expanded CD8,TCRγδ T cells in a patient with chronic T-Gamma lymphoproliferative disease

Abstract Leukemic cells from a patient with T-gamma lymphocytosis were found to have the surface phenotype, CD3 + , CD4 − , CD8 + , Leu19 + , TCRδ1 + , WT31. The clonal nature of the TCRγδ T cell proliferation was documented by flow cytometry and Southern blot analysis. Morphologically, they were large to medium-sized mature lymphocytes with cytoplasmic granules. Functionally, the cells revealed strong cytotoxic activities against NK-sensitive target cells, but had neither killer T cell activity nor suppressive activity on PWM-driven immunoglobulin synthesis by B cells. Interestingly, both suppressive and cytotoxic T cell activities were recovered with the depletion of CD8 + T cells. These studies may suggest some functions of the CD8 + population of human TCRγδ T cells in a normal immune system.

Ataxia and male sterility (AMS) mouse. A new genetic variant exhibiting degeneration and loss of cerebellar Purkinje cells and spermatic cells

We describe a novel genetic variant mouse that exhibited ataxia and male sterility , named the AMS mouse. It arose in autoimmune‐prone MRL/lpr strain and putative ams mutation showed an autosomal recessive inheritance pattern. Clinical symptoms were first discernible at approximately 21 days of age and consisting of subtle sway of the trunk followed by failure to maintain still posture and appearance of abnormal walk, but no further worsening was noted with advancement of age. The abnormal motor coordination was ascribed to almost complete loss of Purkinje cells of the cerebellum. The cell loss in the Purkinje cell layer began before onset of ataxia and rapidly progressed towards near‐complete loss by 6 weeks of age. Another symptom was male sterility due to severe oligozoospermia associated with cellular degeneration during spermatic differentiation in the seminiferous tubules. Thus, the effects of the genetic variation were apparent in two different organs after the development of their basic histological structures, and degeneration and loss of particular cell types in these two tissues produced overt clinical symptoms. Genetic pleiotropism, provided that the nature of genetic variation is of a single gene mutation, is discussed.

Effects of chronic bromocriptine-induced hypoprolactinemia on plasma testosterone responses to human chorionic gonadotropin stimulation in normal men

To study the role played by normal levels of plasma prolactin (PRL) in the secretion of testosterone (T) in the testes, we induced hypoprolactinemia with a daily dose of 5 mg bromocriptine administered orally in five normal men 20 to 35 years of age for 8 weeks. The basal PRL, T, luteinizing hormone, follicle-stimulating hormone, and maximum responses of plasma T to human chorionic gonadotropin (hCG) stimulation were measured every 2 weeks. Basal levels of plasma T were reduced in the 1st 2-week-long period of hypoprolactinemia. In the 4-week-long period of hypoprolactinemia, the maximal response of plasma T to hCG stimulation was significantly reduced. The findings suggest that normal levels of plasma PRL may play an important role in the secretion of T in the human testes in vivo.

Bestatin, an inhibitor of aminopeptidase B, suppresses the proliferation and differentiation of human C-cells in vitro

Bestatin, an inhibitor of aminopeptidase B, was examined for its effect on B-cell activation. Small, dense B-cells from human tonsil samples were isolated by Percoll density gradients from non-rosetted (E-) cells and were used as target cells. Although bestatin was not cytotoxic towards B-cells, it inhibited the proliferative response of B-cells induced by SAC- or PMA-stimulation. The inhibition of cell proliferation by bestatin was manifested as cell arrest caused by the selective block of G1b to S phase transition. This inhibitory effect was prevented by the addition of B-cell growth factor (BCGF) or interleukin-2 (IL-2). The presence of BCGF or IL-2 at the initiation of the culture prevented the bestatin-mediated suppressive effect on B-cell proliferation. Bestatin also has a direct inhibitory effect on the differentiation of B-cells independent of its suppressive effect on B-cell proliferation, which was not relieved by T-cell help. Conversely, bestatin suppressed neither proliferation nor Ig secretion of human B lymphoblastoid cell lines, although aminopeptidase activities on the membrane of these cell lines were strongly inhibited by bestatin. These results indicated that bestatin selectively suppressed normal B-cell proliferation and differentiation. Although several studies have demonstrated that bestatin has immunopotentiating effects in tumor-bearing subjects, the above results indicated that the mechanism of immunopotentiation by bestatin is not a direct stimulatory effect on B-cells.

Diffuse Large B-cell Lymphoma in the Ampulla of Vater Causing Obstructive Jaundice: Report of a Case

We report a case of diffuse large B-cell lymphoma (DLBCL) in the ampulla of Vater, causing painless obstructive jaundice in a 78-year-old woman. Duodenal endoscopy revealed a mass in the ampulla of Vater and narrowing of the second portion of the duodenum, although diagnosing DLBCL from an endoscopic biopsy was impossible because there were several kinds of leukocytes in the infiltrate. We performed pylorus-preserving pancreatoduodenectomy to establish a histological diagnosis, relieve the obstructive jaundice, and remove the narrowed second portion of the duodenum. Histological and immunohistochemical examination of the surgically resected specimen confirmed a diagnosis of DLBCL. Chemotherapy is the mainstay of treatment for DLBCL; however, surgery still plays an important role when the histological diagnosis cannot be established preoperatively and when complications are not amenable to nonsurgical therapy.

A new mutation, ataxia and male sterility (ams), of autoimmune-prone MRL/lpr mouse is not linked to lpr gene but associated with reduction of spleen size and alteration of lymphocyte subpopulations.

We describe changes in the immune system of the newly established mutant line, ataxia and male sterility (AMS) mouse, and that the putative ams mutation is independent of lpr but seemed to affect lymphoproliferation in its mother strain, MRL/lpr. The mean weights of the spleen and lymph nodes of ams‐lpr double‐homozygous mouse were reduced compared with lpr single‐homozygous mouse. Comparison between ams single‐homozygous and control mice revealed 45–50% reduction of the spleen weight in the former for which reduction of the number of nucleated cells contributed greatly. In the lymphocyte/monocyte fraction of the spleen, there were significant changes in the proportion of lymphocyte subpopulations, with a reduction of B cells, an increase in CD4 and CD8 T cells, and a decrease in the CD4 : CD8 ratio. In vitro response of splenocytes to concanavalin A showed inconspicuous dose‐ and time‐dependent responses in ams homozygous spleen, suggesting functional alteration of the immunological response. Our results indicate that ams mutation affects the immune system in addition to its two other major effects on the central nervous system and male reproductive system.

Brain stem glioblastoma with multiple large cyst formation and leptomeningeal dissemination in a 4-year-old girl

The authors report a 4-year-old girl who developed brain stem glioblastoma. Meningeal irritation was present at onset. Magnetic resonance imaging revealed intracranial and intraspinal leptomeningeal dissemination, which progressed faster than the original tumor. Multiple large cysts developed at the interhemispheric and prepontine cisterns, resulting in progressive obstructive hydrocephalus. The patient survived only 5 months after presentation. Histology was verified by autopsy.