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Macrophages and microglia are implicated in spinal cord injury, but their precise role is not clear. In the present study, activation of these cells was examined in a spinal cord injury model using 2 different antibodies against ED1 clone and ionized calcium binding adaptor molecule 1 (Iba1). Activation was observed at 1, 4, 8, and 12 weeks after contusion injury and was compared with sham operated controls. Our results indicate that activation could be observed in both the dorsal funiculus and the ventral white matter area in the spinal cord at 5 mm rostral to the epicenter of injury. For both cells, there was a gradual increase in activation from 1-4 weeks, followed by down-regulation for up to 12 weeks. As a result, we could stain macrophages by ED1 and microglia by Iba1. We concluded that macrophages may play a role in the phagocytosis of denatured dendrites after spinal cord injury, while microglia may have some cooperative functions, as they were found scattered near the macrophages.

The effects of irradiation on different cell compartments in the submandibular gland were analyzed in adult C57BL/6 mice exposed to X-ray irradiation and followed up for 10 days. Apoptosis was quantified using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labeling method (TUNEL). Cell proliferation was detected using immunohistochemistry for proliferating cell nuclear antigen (PCNA). Radiation-induced apoptosis occurred rapidly, reaching a maximum 3 days post-irradiation. The percentage of apoptotic cells increased with the irradiation dose. At day 1 post-irradiation, cell proliferation was significantly reduced in comparison to sham-irradiated controls. After post-irradiation arrest of the cell cycle, proliferation increased in all gland compartments, reaching a maximum at day 6 post-irradiation. The proliferation response corresponded to the dose of irradiation. We suggest that the reason for gland dysfunction could be the coexistence of high apoptotic and proliferative activity in the irradiated gland.

The ultrastructure of the serotonin (5HT) system in the spinal cord of rats was studied by an immunohistochemical peroxidase-antiperoxidase (PAP) method. Under the light microscope, 5HT immunoreactive staining was observed as brown-colored dots in the anterior horn, lateral horn, posterior horn and pericentral canal region. These positively staining dots were probably indicative of 5HT immunoreactive varicosities and nerve terminals. At the ultrastructural level, 5HT immunoreactive nerve fibers appeared as darkly stained varicosities with PAP positive large electron dense vesicles (80-100 nm), as well as small clear vesicles (30-40 nm) finely coated with PAP immunoreactive products. In the anterior horn, some of the 5HT immunoreactive structures were clearly nerve terminals forming asymmetric synaptic contact with soma or dendrites of the anterior horn cells. In the lateral horn, posterior horn and pericentral canal region, however, only 5HT positive varicosities were detected.

Cellular immunity against human bile proteins was investigated by the leukocyte migration inhibition test (LMIT) with 13 primary biliary cirrhosis (PBC) patients, 10 chronic aggressive hepatitis (CAH) patients and 21 healthy adults. Hepatic bile taken from patients operated on for lithiasis of the biliary tract was fractionated into five fractions with Sepharose 6B gel. A subtoxic dose of each fraction was determined in the healthy adults, and used as the antigen for LMIT. Out of the 5 fractions, only the third fraction led to an LMIT positive response in 8 out of 11 (73%) PBC patients and in 1 out of 10 (10%) CAH patients. The difference between PBC and CAH was significant (p less than 0.005). The remaining 3 PBC patients with LMIT negative responses were all under D-penicillamine treatment. Antibody to each fraction was prepared in rabbits. Using the antibodies after absorption with human serum, the localization of the antigens which were present in each fraction was investigated immunohistochemically using human liver sections. The antigen to the anti-first fraction antibody was detected specifically in the epithelial cells of the bile ducts and the ductules, and the antigen to the anti-third fraction antibody was detected specifically on the membrane of the bile canalicules. The third fraction was fractionated into three fractions by Sephadex G-200 gel. Only the first of the 3 fractions showed an LMIT positive response in 3 PBC patients, and its molecular weight was determined to be about 500,000. It is concluded that PBC patients develop cellular immunity against canalicular-antigen-containing fractions but not ductal-antigen-containing ones.

We investigated the organ distribution of four types of red blood cells (RBC) preparations: native RBC, asialo-RBC, native ghosts and asialo-ghosts. Intravenously injected asialo-ghosts were rapidly removed from the blood stream and accumulated mainly in the liver 120 min after the injection. Our results suggest that asialo-ghosts are a simple and effective carrier for targeting of drugs to the liver.

Spleen cells from tumor-bearing mice showed decreased natural killer (NK) activity and decreased binding to target cells with progression of the tumor. Treatment of spleen cells from tumor-bearing mice with vibrio cholerae neuraminidase (VCN) increased the cytotoxicity to a level twice or more as high as that of untreated cells, but the same treatment of spleen cells from normal mice had no or little effect. On the other hand, neither in spleen cells from tumor-bearing mice nor in those from normal mice, the VCN treatment had no effect on their binding to M-HeLa cells. The suppression of NK activity by preincubation with serum from tumor-bearing mice or prostaglandin E2 was completely abolished by VCN treatment. The above results indicate that VCN treatment of lymphocytes might augment NK activity by an antagonistic effect against an immune suppressive factor.

The survival rate of 19 patients who underwent living-related kidney transplantation after donor-specific blood transfusions (DST) was compared with that of 32 historical controls receiving transplants without DST. The graft survival rate of the DST group was 82% after two and three years. The graft survival rate of the DST group was significantly better than the 53% rate after two years obtained with the 32 historical controls (p less than 0.05). We tested sera from 16 DST-treated recipients to study the beneficial effect of DST on kidney allograft survival using the mixed lymphocyte culture (MLC) serum inhibition test. The results demonstrated that MLC inhibitory factors were induced in the serum of the recipient after completion of DST. This inhibition of MLC was observed by treatment of responder lymphocytes with serum obtained three weeks after DST plus rabbit complement. The inhibitory effect was also specific for responder cells in anti-donor MLC. Regarding the correlation with rejection episodes, these MLC inhibitory factors were often observed in the non-rejection group (p less than 0.05). The data suggest that such factors may be anti-idiotypic antibodies and be associated with prolonged graft survival.

The anticancer drug sensitivity of human cancers was tested by the human tumor clonogenic assay (HTCA). Of 152 human cancer specimens tested, 63 (41%) formed more than 30 tumor cell colonies in control plates and could be used to evaluate the drug sensitivity of tumor cells. In 42 (93%) of 45 clinical trials in 24 patients, a parallel correlation was observed between the in vitro anticancer drug sensitivity measured by the HTCA and the clinical response of tumors to anticancer drugs. These results suggest that the HTCA is a good technique for the in vitro test of the anticancer drug sensitivity of human cancers.

Tissue reactions at the cement-bone and artificial implant-bone interface were examined light and electron microscopically in thirty-six patients who underwent revisory operation of hip or knee replacement. The reactions were classified into three types: inert tissue, active tissue with giant cell proliferation, and active tissue with predominant foamy cell proliferation. The third type of reaction was found only in total hip replacement with bone cement. No evidence of allergic reaction to implanted materials was found in any replacement, though active cellular infiltrations were observed around loosened prostheses especially in cemented arthroplasty. The tissue reactions always occurred around instable or loosened prostheses. Thus, the present study shows that mechanical instability is the primary cause of such undesired tissue reactions.

An adriamycin (ADM)-resistant subline was established by continuous exposure of the SBC-3 cells, a cell line of human small cell lung cancer, to increasing concentrations of ADM, followed by the cloning procedure. The resistant sublines (SBC-3/ADM) thus established were 30-fold more resistant to ADM than the parent SBC-3 cells, in terms of the 70% lethal dose determined by soft agar clonogenic assay. The doubling times of the SBC-3 and SBC-3/ADM cells were 36 h and 22 h, respectively. When transplanted into athymic nude mice, the parent as well as resistant cells formed tumors, and serial passage was successful. Although the transplanted tumors from the two cell lines were very similar in histology, the resistance of the SBC-3/ADM cells to ADM developed in vitro was maintained in serially transplanted tumors. The uptake studies with [3H]daunomycin revealed decreased influx and enhanced active efflux of the drug in the resistant cells, whereas cytogenetic analysis showed that the cell lines had an identical karyotype. These results indicate that ADM resistance may be attributed to alternations in membrane transport, resulting in reduced intracellular accumulation of the drug.

Using a cell line (SBC-3/ADM) of human small cell lung cancer, which is 30-fold more resistant to adriamycin than the parent cell line (SBC-3), the activity of a variety of anticancer agents was analyzed by soft agar clonogenic assay to search for a means of circumventing drug resistance. The SBC-3/ADM cells were markedly resistant to some anthracycline antibiotics in comparison with the SBC-3 cells: 28-fold for daunomycin, 26-fold for 4'-epiadriamycin, 18-fold for THP-adriamycin, and 8.4-fold for aclarubicin. However, the cells were as sensitive to mitoxantrone, one of the anthraquinone derivatives, as the parent cells. The cells were resistant to structurally or pharmacodynamically unrelated compounds such as vincristine, mitomycin C, and an active form of ifosfamide, whereas they were susceptible to cisplatin to some extent. The in vitro radiosensitivity of both cell lines was also evaluated, and they were found to be equally sensitive to X-ray. These results suggest that mitoxantrone and cisplatin may exert sufficient activity for small cell lung cancer which has acquired resistance to adriamycin, and that consolidative chest irradiation may be clinically useful after combination chemotherapy including adriamycin.

The influence of surgical stress on the natural killer (NK) activity of peripheral blood lymphocytes in patients with carcinoma of the lung or gastrointestinal system was studied. The peripheral blood lymphocytes of the patients showed a marked decrease in NK activity against K-562 cells as target cells 1-2 days after surgery. The activity remained lowered for 2 weeks after thoractomy and for 1 week after laparotomy. No appreciable suppression of NK activity was observed with normal human peripheral blood lymphocytes preincubated with postoperative patient sera. Peripheral blood mononuclear cells obtained postoperatively from patients lost NK activity after ultraviolet irradiation, without any detectable loss of viability. Such irradiated mononuclear cells showed inhibition of NK activity after a 24-hour preincubation with peripheral blood lymphocytes from normal subjects. Similar suppressive activity was demonstrable in a fraction of mononuclear cells with adhesiveness to plastic petri dishes, while non-adherent cells had no such activity. When added immediately to the cytotoxicity assay system without the 24-hour preincubation, patient mononuclear cells caused no inhibition of NK activity, whereas adherent cells from normal subjects enhanced NK activity. The findings seems to indicate that, following surgical stress, plastic dish-adherent peripheral blood mononuclear cells become deprived of NK helper activity and exert suppression, thus causing postoperative depression of NK activity.

The elimination of fibroblast-like cells from primary cultures of fetal human livers was studied. A fibroblast-like cell line (HuF), which was obtained by subculturing fetal human liver cells 4 or more times, was briefly treated with hydrocortisone (HC) or putrescine (PUT). The growth of HuF cells was inhibited by HC at a concentration of 10(-2) M and by PUT at a concentration higher than 10(-3) M. Long-term treatment of HuF cells with 10(-3) M HC inhibited the growth of the cells. Primary cultures of fetal human livers were made in medium containing HC or PUT, and morphological and functional examinations were made. The cultures were predominantly composed of epithelial-like cells, with few fibroblast-like cells, when the HC concentration was 10(-5)M to 10(-3) M. A high amount of albumin was secreted at these concentrations of HC. On the other hand, at 10(-3) M PUT, many epithelial-like cells were seen, but albumin was undetectable. The present results indicate that albumin-producing epithelial-like cells can be selectively maintained in medium containing HC, in primary cultures of fetal human livers.

The presence of antibodies against adult T cell leukemia antigen (ATLA) was studied in 59 patients with chronic respiratory diseases. Of 13 patients with diffuse panbronchiolitis, 3, who developed adult T cell leukemia, had the anti-ATLA antibody and 8 had the related, anti-ATLA-like antibody. Of 13 cases of idiopathic interstitial pneumonia, 8 had the anti-ATLA-like antibody. Except for only one case, these antibodies were not detected in 33 patients with bronchial asthma or sarcoidosis and 20 healthy adults examined. These results suggested that the test of these antibodies would be useful for the diagnosis of diffuse panbronchiolitis and idiopathic interstitial pneumonia which frequently develop lung cancers.

To investigate the involvement of actin filaments in concanavalin A (Con A)-induced cap formation and cytochalasin B (CB)-induced zeiotic knob migration, the distribution of F-actin was studied in Con A-treated and CB-treated Ehrlich ascites tumor cells (EATC) by fluorescence microscopy using heavy meromyosin conjugated with a fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide, (DACM-HMM). In non-treated cells, the diffuse fluorescence of DACM-HMM was observed in the cytoplasm, particularly intensely under the plasma membrane and around the nucleus. In Con A- and CB-treated cells, the fluorescence was seen at Con A-induced-capped and CB-induced-knob-accumulated regions. This fluorescence was more intense in CB-treated cells. To study the actin filaments in these fluorescent regions more clearly, the soluble components of the cells were eliminated by treatment with Triton X-100 or saponin solution containing a low concentration of glutaraldehyde, and the detergent-treated and saponin-treated cells were observed under a transmission electron microscope. Concentrated actin filaments were observed directly beneath the Con A-induced capping area and CB-induced zeiotic knob-accumulation area. The area of concentrated actin filaments appeared to correspond to the electron dense area observed in the identical region in the cells fixed without detergent treatment. More actin filaments were observed in CB-treated cells than in Con A-treated ones.

The effects of ethanol on rat Kupffer cells were studied functionally and morphologically. Eight g ethanol per kg body weight per day was intragastrically administered to rats for 7 days. An isocaloric glucose solution was administered to control rats. The phagocytic activity of the reticuloendothelial system was measured by the carbon clearance method (57 mg carbon particles per kg body weight) on the 7th day. Kupffer cells having phagocytized carbon particles were counted under the light microscope. Kupffer cells were also observed by scanning electron microscopy. Both the carbon clearance and Kupffer cell number were lower in ethanol-administered rats (32 +/- 8 X 10(-4) mg/ml; 0.6 +/- 0.3/0.01 mm2 liver lobule) as compared to control rats (63 +/- 15; 3.1 +/- 1.0). Microvilli and filopodia of Kupffer cells were fewer in ethanol-administered rats than in control rats. Carbon clearance correlated with Kupffer cell number per 0.01 mm2 liver lobule and liver weight. These results suggest that the decrease in carbon clearance induced by ethanol is due mainly to the decrease in Kupffer cell number and partly to the decrease in Kupffer cell activity as demonstrated by the disappearance of microvilli and filopodia.

Primary cultures of surgically obtained noncancerous portions of human liver tissues were made. Liver tissues were poorly dissociated with collagenase, but well dissociated with dispase. The yield and viability of cells were improved somewhat when dissociated with collagenase followed by dispase. The mean cell yield was 1.1 X 10(6) cells/g liver. The epithelial-like morphology of the dissociated liver cells was maintained for about one week, but thereafter degenerative alteration of cells was observed. In liver explant culture, an active outgrowth of cells was observed for more than one month. Albumin production in culture fluids from dissociated livers was detectable for about 2 weeks, but later became undetectable, while that from explant culture was detectable for at least one month. These data demonstrate that adult human hepatocytes can be isolated from noncancerous portions of livers with relatively high yield, and that albumin production of the dissociated cells is detectable for several days.

To study chromatin structure at the sites of DNA replicated in permeable cells, deoxyribonuclease I (DNase I) sensitivity of newly replicated DNA in permeable mouse sarcoma cells was compared with that of newly replicated DNA in intact cells. About 35% of the DNA replicated in permeable cells was hypersensitive to DNase I, and the remaining DNA showed the same DNase I sensitivity as that of parental chromatin DNA. The sensitivity of DNA replicated in permeable cells was higher than that of DNA newly replicated in intact cells, and was close to that of DNA replicated in the presence of cycloheximide. The sensitivity of DNA pulse-labeled with [3H]deoxythymidine triphosphate by replication in permeable cells was reduced significantly by chasing with cold deoxythymidine triphosphate. The present results suggest that chromatin structure at the sites of DNA replicated in permeable cells is similar to that at the sites of DNA replicated in living cells in the absence of protein synthesis, and that some structural change (possibly toward the maturation) of newly replicated chromatin occurs after the DNA replication in permeable cells.

Development of the ear, especially the middle ear, was studied histologically in ddN and CF mice. Primordia of the 3 ossicles and the otic capsule appeared on day 12 of pregnancy. The stapedial primordium was observed as a mass of mesenchymal cells lateral to the primordium of the otic capsule, attaching to the medial part of the facial nerve. On day 13, the stapedial primordium continued to develop with the Reichert's cartilage. On day 14, the malleus and incus were differentiated. On day 15, the 3 ossicles were mostly completed in shape, and the stapedial footplate had a bilaminar structure at this stage. This structure appeared to correspond to the lamina stapedialis in the developing human stapes.

With the purpose of revealing the biological effects of the X-ray irradiation the authors extracted phospholipids from the liver of irradiated animals and proved that this substance has the action to inhibit the growth of the bone marrow cells, the motility of pseudo-eosinophilis and the erythropoiesis in tissue culture, suggesting that the injury will mainly be induced by the toxic substances produced by irradiation.