Masaru Fukuda

Quantitative studies on proliferative changes of reactive astrocytes in mouse cerebral cortex

Cell number and proliferation of reactive astrocytes were studied quantitatively in the stabbed cerebral cortex of adult mice, using immunohistochemistry for glial fibrillary acidic protein (GFAP) and [3H]thymidine autoradiography. GFAP-positive astrocytes increased in cell number gradually from 24 to 96 h after stabbing, and their immunoreactivity became intense. The maximum number of GFAP-positive cells was about 4.5 times normal in the layers II-VI of the cortex, whereas it was only 1.5 times normal in the layer I (molecular layer). In contrast to the gradual increase in cell number, no GFAP-positive astrocytes were labeled with [3H]thymidine prior to 48 h after stabbing, in either the layer I or the layers II-VI. Then 3-5% of them were labeled at 72 and 96 h, but very few again after 6 days. By injecting [3H]thymidine successively for 6 days after stabbing, only 17% of GFAP-positive astrocytes of the layer I or the layers II-VI were labeled. These results reveal that, in the cortical layers II-VI, many GFAP-negative source cells initially express much more GFAP-antigen without proliferation and change into GFAP-positive reactive astrocytes. Proliferation of reactive astrocytes is not the major factor for the marked increase in number of them. The cortical layer I would have few GFAP-negative source cells for reactive astrocytes. These source cells may be protoplasmic astrocytes.

Herniation of cervical intervertebral disc: immunohistochemical examination and measurement of nitric oxide production.

Study Design. Surgically obtained cervical herniated intervertebral discs were examined histologically and immunohistochemically. The production of nitric oxide (NO) in the local tissue was examined using the electron spin resonance (ESR) method. Objectives. To investigate the local histologic and immunohistochemical changes in cervical disc herniation, including NO production, and to compare such changes with those in autopsy cases. Summary of Background Data. Very little is known about the histopathologic processes of cervical disc herniation. In addition, no information is available on the level of in vivo NO production in cervical disc herniation. Methods. Thirty-six herniated cervical discs obtained from 31 patients were immunohistochemically examined for localization of blood vessels, matrix metalloproteinase (MMP)-3, and inducible NO synthetase (iNOS). We also compared the production of NO, measured by the ESR method, in eight specimens with that of five control discs obtained from fresh cadavers. Results. The presence of herniated discs correlated with the degeneration of cartilaginous endplate and torn anulus fibrosus. Formation of new blood vessels around the herniated discs was detected, using von Willebrand factor antibody, in seven uncontained hernias and 20 contained hernias. Immunohistochemical studies showed the presence of cells positive for MMP-3 (chondrocytes), iNOS (chondrocytes and granulation tissue) in cervical disc hernias. ESR analysis showed a significantly higher NO production in herniated cervical discs than in disc samples of fresh cadavers. Conclusions. Herniated cervical intervertebral disc is characterized by the presence of an inflammatory process associated with neovascularization and increased expression of MMP-3. Production of NO was markedly high in both contained- and uncontained-type hernias.

Cell proliferation and differentiation in intramucosal and advanced signet ring cell carcinomas of the human stomach

In order to study the progression of signet ring cell carcinomas in the human stomach, we compared cell proliferation and differentiation between small and large intramucosal cancers, and between intramucosal and advanced cancers. Fine-structurally, signet ring cells were differentiated to 3 cell types: a foveolar, a glandular and an intestinal type. In the mucosa, the foveolar-type cells and glandular-type cells were distributed at the superficial and the deep zone, respectively. In the small mucosal cancers, intestinal-type cells were rare and a layered structure was often seen. In this structure, the mode of cell production resembled that in the normal gastric mucosa; the foveolar-type signet ring cells in the superficial layer were not proliferative and the proliferating cells were small cells in the middle layer and a few glandular-type cells in the deep layer. In the large mucosal and advanced cancers, intestinal-type cells and proliferating small round cells were often distributed throughout the depth of the mucosa, and signet ring cells of the foveolar type were also proliferative. These findings indicated that large part of the signet ring cell carcinomas initially form the layered structure and that it becomes indistinct while intestinal-type cells appear as the tumour grows. However, we found several small advanced cancers, lacking both the layered structure and the intestinal-type cells. These cancers appear to start without the layered structure and progress very rapidly.

Combination effect of photodynamic and sonodynamic therapy on experimental skin squamous cell carcinoma in C3H/HeN mice

We studied a combination of photodynamic therapy (PDT) and sonodynamic therapy (SDT) for improving tumoricidal effects in a transplantable mouse squamous cell carcinoma (SCC) model. Two sensitizers were utilized: the pheophorbide‐a derivative PH‐1126, which is a newly developed photosensitizer, and the gallium porphyrin analogue ATX‐70, a commonly used sonosensitizer. Mice were injected with either PH‐1126 or ATX‐70 i.p. at doses of 5 or 10 mg/kg.bw. At 24 (ATX‐70) or 36 hr (PH‐1126) (time of optimum drug concentration in the tumor) after injection, SCCs underwent laser light irradiation (88 J/cm2 of 575 nm for ATX‐70; 44 J/cm2 of 650 nm for PH‐1126) (PDT), ultrasound irradiation (0.51 W/cm2 at 1.0 MHz for 10 minutes) (SDT), or a combination of the two treatments. The combination of PDT and SDT using either PH‐1126 or ATX‐70 as a sensitizer resulted in significantly improved inhibition of tumor growth (92–98%) (additive effect) as compared to either single treatment (27–77%). The combination using PH‐1126 resulted in 25% of the treated mice being tumor free at 20 days after treatment. Moreover, the median survival period (from irradiation to death) of PDT + SDT‐treated mice (>120 days) was significantly greater than that in single treatment groups (77–95 days). Histological changes revealed that combination therapy could induce tumor necrosis 2–3 times as deep as in either of the single modalities. The combination of PDT and SDT could be very useful for treatment of non‐superficial or nodular tumors.

Reactions of S-100-positive glia after injury of mouse cerebral cortex

Reactions of glial cells after stab wounding of mouse cerebral cortex were studied by [3H]thymidine autoradiography combined with immunohistochemistry for S-100 protein. S-100-positive cells in the stabbed cortex had the light and electron microscopic characteristics of astrocytes, and they showed remarkable hypertrophic changes 4 to 5 days after stabbing. There were many cells labeled with [3H]thymidine in the stabbed cortex from 24 h to 8 days after stabbing, and the number of labeled cells was maximum at 48 h. A few of the labeled cells were S-100-positive, and the labeled S-100-positive cells were seen 24 h to 6 days after stabbing, mostly after 72-96 h. By successive injections of [3H]thymidine for 6 days after stabbing, about 90% of labeled cells were S-100-negative, and about 90% of S-100-positive cells were unlabeled with [3H]thymidine. The increase in number of S-100-positive cells by day 6 after stabbing was not statistically significant (P greater than 0.05). These results suggest that reactive proliferation of astrocytes is a minor phenomenon in gliosis of injured cerebral cortex, in contrast with their remarkable reactive hypertrophy.

Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer.

In order to identify the prognostic factors that significantly influence the disease-free survival rate after surgical resection of primary breast cancers, we determined tumour and lymph node grades, and immunohistochemical staining for estrogen and progesterone receptors (ER and PR), c-erbB-2, p53, bcl-2, bax and PCNA in 76 patients. Univariate analysis showed that increased grade of tumour and lymph nodes, negative immunostaining for ER, positive immunostaining for c-erbB-2, and a high PCNA index (> or = 30%) negatively influenced the disease-free survival rate, but PR, p53, bcl-2 and bax had no predictive value. Although p53 was not an independent prognostic factor by itself, the combination of p53, bcl-2, and bax proved to correlate with the disease-free survival, with the best prognosis noted in tumours negative for p53 and positive for both bcl-2 and bax, intermediate prognosis in tumours negative for p53 and positive for either bcl-2 or bax and worst prognosis in tumors negative for p53 as well as bcl-2 and bax. Tumour grade correlated positively with PCNA index, while positive staining for ER correlated negatively with tumour grade as well as with PCNA index, although this was statistically insignificant. Immunostaining of breast cancers for bcl-2 correlated negatively with tumour grade and PCNA index. Immunostaining for c-erbB-2 correlated positively with PCNA but not with tumour grade. Immunostaining for p53 tended to correlate positively with PCNA, but not with tumour grade. Immunostaining for PR and bax did not correlate with tumour grade and PCNA index. These results suggest that in addition to tumour size and lymph node involvement, immunostaining for ER, c-erbB-2, and a high PCNA index are important prognostic factors in human breast cancer. Wild-type p53 with preserved bcl-2 and bax gene products is also a favorable prognostic factor indicating breast cancer at an early stage of cancer progression.

Decreased expression of Bax is correlated with poor prognosis in oral and oropharyngeal carcinoma

We investigated the expression of apoptosis-related factors, p53, Bax, Bcl-2, and spontaneous apoptosis in 57 cases of oral and oropharyngeal squamous cell carcinoma (SCC) by immunochemical staining and ApopTag kit. Positive expression of Bax was inversely associated with advanced tumor stage (P = 0.0225), lymph node metastasis (P = 0.0225), clinical stage (P = 0.0083) and poor prognosis (P = 0.0478). Positive expression of p53 was related to poor prognosis (P = 0.0445) and was associated with negative expression of Bax (P = 0.0439). The apoptosis index did not correlate with clinical outcome. These results suggest that abnormality of Bax expression plays an important role in tumor progression in oral and oropharyngeal SCC.

Leiomyomatosis peritonealis disseminata with malignant change in a man

Leiomyomatosis peritonealis disseminata (LPD) is a rare clinicopathological entity typically observed in women of reproductive age. We report a case of LPD with malignant change in a man. A 77‐year‐old man presented with a mass measuring 10 cm in diameter at the terminal ileum and numerous peritoneal small nodules that were revealed by abdominal computed tomography. Right hemicolectomy with lymph node dissection was performed. Macroscopically, a tumor of the terminal ileum consisted of aggregates of small nodular lesions with calcification and necrosis. The wall of the ileum and colon was intact. Microscopically, some of the nodular lesions consisted of neoplastic growths of atypical spindle cells with cellular atypism and abnormal mitoses. A few of these lesions were completely surrounded by smooth muscle bundles. Hemorrhages and necroses were found within the tumor nodules. Immunohistochemically, the tumor cells were positive for vimentin, desmin, muscle actin, α‐smooth muscle actin, cytokeratin and p53. The remaining nodular lesions, including small peritoneal lesions, were composed of hypocellular hyalinizing nodules. This case was thought to be LPD with malignant change, although the pathogenesis was uncertain because the tumor cells were negative for estrogen and progesterone receptors.

Regional ploidy variations in signet ring cell carcinomas of the stomach

Regional ploidy variations within individual tumors were analyzed by in‐situ cytofluorometry of metaphase cells in Feulgen‐stained paraffin sections, using 45 resected stomachs with early and advanced signet ring cell carcinomas. Aneuploid cells were found in one of 30 early cancers and in eight of 15 advanced cancers, and were almost always accompanied by diploid cancer cells in the mucosal part of the cancers. The diploid and the aneuploid cells were generally found to be distributed in different territories in the mucosa, and aneuploid foci were often included in the diploid area. These findings suggest the diploid origin of signet ring cell carcinomas and the occurrence of aneuploidy during the tumor development. Moreover, the aneuploid cells appeared to infiltrate beyond the mucosa more readily than the diploid cells; most of the aneuploid populations already invaded the extramucosal tissue, and the cancer cells infiltrating in the extramucosal tissue were predominantly aneuploid in six of the nine cancers with aneuploidy. Thus, it appears that the occurrence of aneuploid clones may accelerate the progression of signet ring cell carcinomas from early to advanced stages.

Reciprocal expression of bcl-2 and p53 oncoproteins in urothelial dysplasia and carcinoma of the urinary bladder

Abstract In order to investigate if and when the bcl-2 oncoprotein is activated in bladder tumorigenesis and its relationship with p53 overexpression and patient survival, we studied bcl-2 and p53 expression immunohistochemically in matched normal urothelium, dysplasia and cancer specimens selected by step-sectioning from 54 radically resected bladders for non-metastatic transitional cell carcinoma (TCC). In normal urothelium and mild dysplasia, bcl-2 was restricted to the basal cell compartment, while in moderate and severe dysplasia its expression was detectable also in the upper regions. Excess bcl-2 immunoreactivity was found in 27 (50%) of carcinomas, and a larger proportion of high-grade TCCs showed bcl-2 expression compared with that of low-grade TCCs (P < 0.05). Overexpression of p53 protein showed a increasing trend toward the progression of bladder tumorigenesis (P < 0.01) and a significant reciprocal correlation was found between bcl-2 and p53 expression in either various dysplasias (P < 0.01) or carcinoma (P < 0.05). With the evolution from mild dysplasia to carcinoma in individual cases, loss of bcl-2 expression was more frequently observed in superficial (P < 0.02) or low-grade carcinoma (P < 0.05) than in muscle-invasive or high-grade carcinoma. Furthermore, patients with negative immunostaining for both bcl-2 and p53 in cancer lesions had a significantly more favorable prognosis compared with those with positive immunostaining for the oncoproteins (P < 0.05), although bcl-2 by itself did not predict patient survival. We suggest that aberrant activated bcl-2, which is seen earlier than p53, appears to facilitate bladder tumorigenesis and to enhance tumor aggression in some extent.