Review Autoimmune Diseases as ``Stem Cell Disorders''
Susumu Ikehara
First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570
Received for publication February 20, 1997
Using various animal models for autoimmune diseases, we have found that bone marrow transplantation (BMT) can be used to treat not only systemic autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), but also organ-specific autoimmune diseases such as immune thrombocytopenic purpura (ITP) and insulin-dependent diabetes mellitus (IDDM). We have also found that the transplantation of hemopoietic stem cell (HSC)-enriched populations from autoimmune-prone mice to normal mice induces autoimmune diseases in the recipients. These findings have recently been confirmed in humans; BMT can be used to treat autoimmune diseases such as RA, Crohn's disease and ulcerative colitis (UC), whereas autoimmune diseases such as Graves' disease and ITP have been transferred from donors to recipients by BMT. Based on these findings, we have proposed that autoimmune diseases are ``stem cell disorders''. To elucidate the difierences between normal and abnormal HSCs, we have very recently established a new method for purifying pluripotent HSCs (P-HSCs). Using P-HSCs purified by our method, we have compared normal and abnormal P-HSCs, and found that qualitative differences exist between them both in vivo and in vitro; although normal P-HSCs do not readily proliferate in major histocompatibility complex (MHC)-incompatible microenvironments, abnormal P-HSCs show a marked proliferative response even in the allogeneic environments. Abnormal P-HSCs are thus more ``resilient'' than normal P-HSCs.
Key words: Autoimmune disease, Stem cell disorder, Bone marrow transplantation
Expression of Basic Fibroblast Growth Factor, Epidermal Growth Factor, and Their Receptors in Castrated and Testosterone Injected Rat Prostates
Fang Guo, Toshiyuki Ishiwata, Munehiro Yokoyama and Goro Asano
Department of Pathology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113
Received for publication November 7, 1996 and in revised form March 1, 1997
To examine the role of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and their receptors in androgeninduced cell growth in the rat prostate, we divided 36 male wistar rats into 3 groups, consisting of an uncastrated and non-testosterone injected (C) group, a castrated and non-testosterone injected (CA) group, and a castrated and testosterone injected (CT) group. Each group was divided again into 2 groups and sacrificed at 4 and 8 weeks after the treatment, respectively. Injection with testosterone propionate was done subcutaneously at 4 mg/kg for a week. The epithelial cells in CT prostates exhibited much higher proliferative cell nuclear antigen (PCNA) labelling index compared with those in C prostates, and the PCNA index of the stromal cells of both groups was similar. bFGF expression increased mainly in epithelial and stromal cells in CA group. bFGF mRNA level also increased in the 4 weeks CA group. FGFR expression increased in all kind of cells of CA group, in which total FGFR mRNA level was also high. EGF immunoreactivity was intense in the proliferating epithelial cells of CT group, and EGF mRNA was also detected in the same cells. Epithelial cells in CA group were positive for EGFR, but not in CT group. However, total EGFR mRNA level increased in CT group. Taken together, bFGF synthesized by epithelial and stromal cells may participate in fibrosis and angiogenesis in the castrated rat prostates. On the other hand, androgen may induce the prostatic epithelial cell proliferation through, at least in part, autocrine or paracrine mechanism of bFGF and EGF.
Key words: Prostate, Androgen, Growth factor, Immunohistochemistry, In situ hybridization
Glycine-Containing Axon Terminals on Large Ventral Horn Cells Including Identified $\alpha$-Motoneurons in the Spinal Cord of the Chicken
Chun-Ying Yang, Hiroshi Sakamoto and Saoko Atsumi
Department of Anatomy, Yamanashi Medical University, Tamaho, Yamanashi 409-38
Received for publication November 7, 1996 and in revised form January 29, 1997
Glycine-like immunoreactive (LI) axon terminals were observed on large ventral horn cells by the indirect antibody peroxidaseantiperoxidase technique in the chicken. The glycine-LI terminals were also confirmed on identified $\alpha$-motoneurons by retrograde transport of horseradish peroxidase (HRP). Electron microscopically, glycine-LI terminals containing pleomorphic clear vesicles made axosomatic and axodendritic synapses with large ventral horn cells including identified $\alpha$-motoneurons. They formed mostly symmetrical synapses, but a few asymmetrical synapses were also seen. These glycine-LI terminals contained numerous mitochondria. The number of boutons per perimeter of a large ventral horn cell was 0.15$\pm$0.05/$\mu$m (n=90). The results indicate that $\alpha$-motoneurons receive glycinergic inputs. The origin of glycinergic inputs is discussed in the light of previous investigations.
Key words: Glycine, Motoneuron, Synapse, Spinal cord, Electron microscopy
Actin and Cytokeratin in Superficial Transitional Epithelial Cells of the Rabbit Urinary Bladder: A Confocal and Electron Microscopic Study
Masayuki Kamada, Toshihiro Kobayashi*, Patrick C. Nahirney**, Teruhiko Okada*, Eva Garcia del Saz*, Taro Shuin and Harumichi Seguchi*
Department of Urology, and *Department of Anatomy and Cell Biology, Kochi Medical School, Kohasu, Okoh-cho, Nankoku, Kochi, 783, and **Department of Anatomy, University of British Columbia, Vancouver, B.C., Canada, V6T 1Z3
Received for publication December 28, 1996 and in revised form February 27, 1997
Transitional epithelial cells undergo dynamic morphological alterations during the contraction-expansion cycle in the mammalian urinary bladder. In this study, the localization of two major cytoskeletal elements, actin and cytokeratin, was examined by fluorescence and electron microscopic methods to elucidate the relationship of these components in the transitional epithelial cells of non-distended bladders. Using phalloidin-FITC and confocal laser scanning microscopy (CLSM), a strong staining for filamentous actin was observed at subplasmalemmal regions of the superficial cells. Cytoplasmic regions of these cells were characterized by punctate regions of staining that increased at sites of cell-cell contact. Ultrastructural observations using immunogold and heavy meromyosin (HMM) labelling techniques for detecting actin filaments revealed a rich actin filament network in superficial cells most notably in the subplasmalemmal regions. These filaments occurred in orientations that were both parallel and perpendicular relative to the surface membrane. Intermediate and basal cells contained a less dense network of filamentous actin in comparison to superficial cells. Immunolabelling for cytokeratin 18 revealed abundant thread-like filaments coursing throughout the cytoplasm and making contact with the plasmalemma in superficial cells. By electron microscopy, the cytokeratin filaments formed a dense interlacing network arranged into bundles that interconnected with membrane-bound structures. These observations indicate that actin and cytokeratin are major cytoskeletal elements of transitional epithelial cells and that they are related to the configurational alternations of transitional epithelial cell borders and their contents. It is postulated that these proteins stabilize and support the membrane of superficial cells from hydrostatic pressure within the bladder.
Adenosine 5'-Triphosphate Decreases the Permeability of a Mesothelial Cell Monolayer
Taichi Takeda, Tetsuo Yukioka, Hiroharu Matsuda and Syuji Shimazaki
Department of Traumatology and Critical Care Medicine, Kyorin University School of Medicine, Tokyo
Received for publication January 3, 1997 and in revised form March 3, 1997
We have developed an in vitro assay system to evaluate permeability of a rat mesothelial cell monolayer and examined the direct effect of ATP. Rat peritoneal mesothelial cells formed a confluent monolayer on the membrane of a co-culture chamber. ATP at the concentration between 10-6-10-4 M decreased the permeability of the monolayer to dextran in 4 hr. Since our assay system doesn't contain inflammatory cells, it is suggested that ATP directly modulates the cells and decreases the permeability.
Prognostic Factors and Proliferative Activities in Breast Cancer. A Comparative Study between Recurrent and Non-Recurrent Cases of Human Breast Carcinomas
Yoshiaki Imamura, Makoto Ishida, Kohjiro Horita and Masaru Fukuda
Department of Pathology, Fukui Medical School, Matsuoka, Yoshida-gun, Fukui, 910-11
Received for publication February 1, 1997
Twenty-one recurrent and twenty-six non-recurrent cases of breast carcinoma were compared with respect to prognostic factors and proliferative activities. The mean number of lymph nodes involved (lymph node score) and tumor stage in recurrent and non-recurrent cases were 10.0$\pm$14.5/ 2.05$\pm$0.59 and 0.96$\pm$2.75/1.42$\pm$0.50, respectively. Expression of p53/c-erbB-2 was demonstrated immunohistochemically in 28.6/52.4% of recurrent cases, and in 15.4/34.6% of non-recurrent cases, respectively. PCNA indices in the two groups were 47.8$\pm$14.2 and 39.5$\pm$18.2, respectively. Fluorescent nucleolar organizer regions (FlNORs) scores determined by confocal laser scanning microscope in the two groups were 3.03$\pm$0.78 and 2.32$\pm$ 0.54, respectively. There were significant differences between the two groups in lymph node score, tumor stage, and FlNORs score, but not in the expression of p53 or c-erbB-2 or in PCNA indices. These findings indicate that lymph node score, tumor stage, and FlNORs score may be useful as prognostic factors in patients with breast carcinoma.
Key words: Breast carcinoma, Prognosis, p53, PCNA, Fluorescent nucleolar organizer regions
Endothelin-Induced c-fos Expression in the Rat Forebrain
Kiyoshi Kurokawa, Hisao Yamada and Junzo Ochi
Department of Anatomy, Shiga University of Medical Science, Otsu, 520-21
Received for publication February 3, 1997
The expression of c-fos was analyzed immunohistochemically after injection of endothelin isopeptide into cerebroventricle of the rat. The c-Fos immunoreactivity increased and reached the peak at 1 hr-1.5 hr after the administration of endothelin. Endothelin-1 and endothelin-2 specifically induced c-fos expression in the neurons of paraventricular, supraoptic and periventricular nuclei and zona incerta. Endothelin-3 induced c-Fos in the organum vasculosum laminae terminalis in addition to those regions. In these areas, the number of c-Fospositive neurons increased with a dose (0 to 100 pg of ET) dependent manner. We presume that they are the targeting neurons of central nervous endothelins (ETs).
Key words: Rat, Hypothalamus, Endothelin, c-Fos, Immunohistochemistry
1 Department of Anesthesiology, 2 First Dept. of Internal Medicine and 3 First Dept. of Pathology Kyoto Prefectural University of Medicine, 465 Kajiicho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602
Received for publication February 5, 1997
To evaluate the role of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in acute respiratory distress syndrome (ARDS), we immunostained alveolar macrophages (AMs) in the bronchoalveolar lavage fluid (BALF) collected from patients with ARDS, patients in the operating room whose lung function were normal, and non-ARDS patients who were intubated and artificially ventilated for one week in the intensive care unit. We observed the significant expression of iNOS, interleukin-6 (IL6) and interleukin-8 (IL-8), in AMs obtained from BAL in ARDS patients by the immunocytochemical approach. By comparison, statistically significant elevation was detected in IL-8 levels in BALF supernatant from the ARDS patients compared with the control and the non-ARDS patients. These results suggest that the expression of proinflammatory cytokines and iNOS in AMs as determined by immunocytochemical technique may be an important predictor of the early stage of lung injury in ARDS. We suggest that NO and proinflammatory cytokines produced by AMs might play a pivotal role in acute lung injury. The immunocytochemical approach could be the most sensitive predictor for activated AMs in ARDS patients.
Department of Pathology, Saga Medical School, Nabeshima 5-1-1, Saga-City 849
Received for publication February 7, 1997
The proliferative ability of fat cells has been studied both in the adipose tissue in vivo and in culture. We studied the proliferation of unilocular fat cells of young rats in a three-dimensional collagen gel matrix culture, which provides a physiological environment for fat cells. In this setting, the unilocular fat cells were able to maintain their cellular functions and actively proliferate. In this study using cells derived from 3 to 6-week-old rats, we observed the appearance of very small fat cells at the surface of unilocular fat cells following the division of the nucleus. We concluded that small fat cells were created mostly in a ``budding'' manner, and then proliferated. Insulin accelerated both the budding and proliferation process. In other unilocular fat cells, lipid droplets divided after the division of the nucleus, and very small fat cells appeared within the unilocular fat cells in a ``sharing'' manner. Adenosine deaminase, a potent lipolytic factor, accelerated this process. An analysis of the cellular volume by a Coulter counter confirmed the proliferation of small fat cells derived from the unilocular fat cells. These small fat cells grew and developed into unilocular fat cells. It is supposed that not all unilocular fat cells are terminally differentiated, nor do they lose their proliferative activity.
Key words: Small fat cells, Unilocular fat cells, Collagen
Freeze-Fracture Enzyme Cytochemistry Reveals the Distribution of Enzymes in Biological Membranes: Enzyme Cytochemical Label-Fracture and Fracture-Label
Toshihiro Takizawa, Eiko Nakazawa* and Takuma Saito
Department of Anatomy, Jichi Medical School, 3311 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi, 329-04 and *Application Technology Department, Techno Research Laboratory, Hitachi Instruments Engineering Co., Ltd., 882 Ichige, Hitachinaka-shi, Ibaraki, 312
Received for publication February 10, 1997
During the past decade, freeze-fracture cytochemistry, the combination of freeze-fracture electron microscopy with cytochemistry, has greatly contributed to the investigation of the macromolecular architecture of biological membranes. The application of enzyme cytochemical labeling technique to freeze-fracture cytochemistry (i.e., freeze-fracture enzyme cytochemistry) has not received considerable attention although by enzyme cytochemical labeling technique many enzyme molecules can be labeled with metal cations and enzyme cytochemistry has been of great utility in cell biology studies. In this study, we report freeze-fracture enzyme cytochemistry: ``label-fracture method'' and ``fracture-label method''. Freeze-fracture enzyme cytochemistry was applied to the study of acid phosphatase, 5'-nucleotidase in the proximal tubular epithelium of the rat kidney, ecto-adenosine triphosphatase in human neutrophils and alkaline phosphatase in rat neutrophils, and was shown to be a technique that visualized these enzyme activities on replicas. Cerium as the capture agent was a useful enzyme cytochemical probe in this technique. Lead was applicable for labeling of plasma membrane-associated enzyme molecules. This technique leads to new applications that may extend the usefulness of freeze-fracture cytochemistry for the analysis of biomembrane structure.
1 Department of Animal Sciences, 2 First Department of Pathology, School of Medicine and 3 Institute for Virus Research, Kyoto University, Kyoto 606-01
Received for publication February 28, 1997
The process of apoptosis in granulosa and cumulus cells of ovarian follicles during follicular atresia was investigated in multiparous pigs. In healthy follicles, no apoptotic cells were observed histochemically, and no DNA ladder formation or Ca2+/ Mg2+-dependent endonuclease activity were detected biochemically in granulosa or cumulus cells. In the follicles in the early stage of atresia, apoptosis occurred in granulosa cells located on the inner surface of the follicular wall but not cumulus cells. DNA ladder formation was observed in granulosa but not cumulus cells, and high levels of endonuclease activity were demonstrated only in the granulosa cells. No endonuclease activity was detected in cumulus cells obtained from the same atretic follicles. These findings suggest a lack of apoptotic cell death in cumulus cells in atretic follicles in multiparous pig ovaries.
Department of Orthopedic Surgery, and Anatomy, Jichi Medical School, Kawachi-gun, Tochigi, 329-04
Received for publication March 4, 1997
A cytochemical study of glucose-6-phosphatase (G-6-Pase) was made to see the changes in mucopolysaccharide metabolism in the synovial membrane during osteoarthritis, because the tissue is thought to play an important role in synovial fluid production by providing lubrication to the wear-and-tear surface of the synovial cartilage in osteoarthritis. In normal and osteoarthritic synovial membranes, a high G-6-Pase activity was clearly demonstrated in the cisternal spaces of the rough endoplasmic reticula, especially in the B cells of the thickened synovial membrane. The existence of an AB cells intermediate in character between A and B, was noted in having a high G-6-Pase activity in the accumulated rough endoplasmic reticula in the cytoplasm as B cells do located at the deeper part of the membrane, but also whose cell surface extends to the top of the membrane surface reaching into the synovial cavity, and protrude microvilli of a ruffled border indicating a high phagocytotic activity as A cells do. The existence of the AB cell is clearly one of intermediate structure and function, contributive to the compensatory and regenerative action of the synovial
membrane.
Key words: Osteoarthritis, Glucose-6-phosphatase, Enzyme histochemistry, Synovial membrane, Electron microscopy
Immunoelectron Microscopy of Protein Kinase C in Resting and Phagocytosing Macrophages
Kazushi Fujimoto, Tohru Nakano and Kazuo Ogawa
Department of Anatomy, Faculty of Medicine, Kyoto University, Kyoto 606-01
Received for publication March 5, 1997
We report here the intracellular dynamics of protein kinase C (PKC) in rabbit alveolar macrophages during phagocytosis using immunoelectron microscopy. Pulmonary alveolar macrophages were obtained by tracheal lavage from rabbits. The harvested cells were exposed to latex beads (1 $\mu$m in diameter) for 20 min at 25$\circ$C, and then fixed for 1 hr in 4% formaldehyde and 0.1% glutaraldehyde. Ultrathin frozen sections were processed for immunolabelling with anti-PKC monoclonal antibodies (mAbs) against rabbit Type 1 ($\gamma$), 2 ($\beta$) and 3 ($\alpha$) PKC. Specific immunostaining was observed in macrophages incubated with anti-Type 2 or 3 mAb, and no apparent staining was observed with anti-Type 1 mAb. Type 2 and 3 mAbs labelling revealed a diffuse cytosolic distribution of the PKC in resting macrophages (without phagocytic pulse using latex beads). In phagocytosing macrophages, the most striking feature was an immunolabelling on the plasma membrane binding to latex beads and the phagosomal membrane. These results demonstrate that binding of latex beads causes a rapid translocation of cytosolic Type 2 and 3 PKC to the latex bead-attached plasma membrane, and imply that these isozymes have a crucial role in phagocytosis of foreign particles.
Key words: Protein kinase C, Macrophages, Phagocytosis, Immunoelectron microscopy
Third Department of Internal Medicine, Hyogo College of Medicine, Mukogawa-cho 1-1, Nishinomiya, Hyogo 663
Received for publication July 4, 1996 and in revised form February 12, 1997
The enzymatic histochemical localization of aldehyde oxidase in the rat liver lobule was investigated by use of a tissue protectant, polyvinyl alcohol, with tetra-nitro BT as the final electron acceptor. This method demonstrated the presence of aldehyde oxidase activity in the cytoplasm of liver cells. Distribution of aldehyde oxidase activity in the liver was uneven, being seen mainly in the pericentral rather than the periportal area as with xanthine oxidase. The reaction was not inhibited by TEI-6720 but was markedly inhibited by benzamidine. Results were consistent with those of immunohistochemical localization studies of this enzyme.
Key words: Aldehyde oxidase, Enzyme-histochemistry, Rat liver
Note Computer-Assisted Three-Dimensional Reconstruction of the Vestibular End-Organs of the Gerbil Using the Serial Paraffin Sections
Noriko Kawamura, Akiko Seto-Ohshima and Muneyuki Ito
Institute for Developmental Research, Aichi Human Service Center, Kasugai 480-03
Received for publication November 11, 1996 and in revised form February 7, 1997
To obtain spatial information regarding gerbil vestibular end-organs and surrounding components, serial paraffin sections of the decalcified heads, including inner ear, were prepared. Photographs of the sections were traced onto OHP papers, on which two reference points were plotted for the orientation. Sets comprised of a photograph and an OHP sheet with two reference points were digitized using a scanner and the computer program, ``Photoshop''. The figures were cropped to the same size using two reference points and after the color density of the objects was increased, they were superimposed using the computer program, ``VoxelView'' for three-dimensional reconstruction. This was an effective method of clarifying the spatial relationship of the components included. Using the results obtained, precise experiments for electrical vestibular stimulation were successfully performed.