A study of cytokeratin in human normal bladder epithelium and bladder carcinoma cell lines]

Normal epithelial and carcinoma cells of human bladder were investigated for the cytokeratin which is one of the intermediate filaments and comprises cytoskeleton using the immunofluorescence method. Carcinoma cell lines used were JTC-30, JTC-32, HUB-41 and T-24. In normal urothelium, keratin fibers were fine and straight with unchanged diameter and distributed regularly in the cytoplasm. By contrast, keratin fibers in bladder carcinoma cells were kinked and changed in diameter and were distributed irregularly in the cytoplasm. The above findings were most obvious in T-24 which formed undifferentiated carcinoma when transplanted to nude mice, and keratin fibers were dominantly located in the perinuclear area. The changes of keratin fibers appeared to be parallel to the grade of histological anaplasia of the tumor formed by implantation of bladder carcinoma cell line cells to nude mice. These observations suggest that the morphology of cytokeratin is a useful indicator for evaluating the grade of malignancy in transitional cell carcinoma.     

Evaluation of nicotine,cotinine, thiocyanate,carboxyhemoglobin, and expired carbon monoxide as biochemical tobacco smoke uptake parameters

Summary In a cross-sectional study on 236 individuals in Japan (174 males, 62 females; 149 smokers, 87 non-smokers) plasma nicotine (pnic), cotinine (pcot) and thiocyanate (pSCN), urinary creatinine ratios of nicotine (unic), cotinine (ucot) and thiocyanate (uSCN) as well as carboxyhemoglobin (COHb) and expired carbon monoxide (COex) were determined. All tobacco smoke uptake parameters (TSUP) were significantly elevated in smokers as compared to non-smokers. The discriminant power (smokers vs non-smokers) rank in the following order: ucot pcot unic > pSCN COHb pnic > COex uSCN. All parameters except for pnic are significantly correlated with the self-reported number of cigarettes smoked per day. The reason for the poor correlation of pnic with daily cigarette consumption is the short half-life of pnic coupled with the arbitrary time of blood drawing in relation to the last time of smoking.Dr. Muranaka, the chief author of this paper, was the director of our hospital. He died suddenly on 18 April 1986. This article is therefore the last monument to be planned and achieved under the late Dr. Muranaka's direction.     

Production of superoxide anion, prostaglandins, and hydroxyeicosatetraenoic acids by macrophages from hypersensitivity-type (Schistosoma mansoni egg) and foreign body-type granulomas.

Macrophages isolated from hypersensitivity (Schistosoma mansoni egg) and foreign body- (Sephadex bead) type granulomas were evaluated with regard to superoxide anion (O2-) production and arachidonic acid metabolism. Granuloma macrophages from schistosome-infected mice were examined during both the acute and modulated phases of the disease. In addition, the populations were characterized phenotypically by measurement of Ia antigen expression. Based on differences in the parameters studied at least three different macrophage populations could be identified in acute, modulated, and foreign body-type lesions, respectively. Macrophages from acute lesions (8-week granuloma macrophages) produced significant amounts of O2-, prostaglandins, and monohydroxyeicosatetraenoic acids without the addition of an exogenous stimulus. These cells also showed a high degree of Ia expression. In contrast, macrophages from modulated (20-week granuloma macrophages) and foreign body (foreign body granuloma macrophages) lesions required stimulation with phorbol ester to evoke significant O2- production and minimally metabolized arachidonic acid. However, 20-week and foreign body granuloma macrophages could be distinguished by their high and low degrees of Ia expression, respectively. The role of lymphokines and other intercellular signals in determining macrophage activation states within granulomas is discussed.     

Frequent rearrangement of chromosomal bands 1p22 and 11q13 in squamous cell carcinomas of the head and neck

We report the finding of clonal structural chromosome abnormalities in short-term cultures from 15 squamous cell carcinomas of the head and neck region. When the distribution of chromosomal breakpoints in these 15 tumors and in the 16 head and neck carcinomas previously described are assessed, a marked clustering is seen at bands 1p22 and 11q13, which are rearranged in eight and nine tumors, respectively. No other band was involved in aberrations in more than five tumors. Cytogenetic evidence of gene amplification was seen in four tumors, three times in the form of homogeneously staining regions (twice located in 11q13), and in one tumor as double minutes. Among the candidate genes for such amplification are BCLI, INT2, and HSTI, all of which map to 11q13, and NRAS, which maps to 1p22. All these oncogenes have previously been shown to be amplified in subsets of head and neck carcinomas. We conclude that bands 1p22 and 11q13 are nonrandomly involved in chromosomal rearrangements in head and neck carcinomas and suggest that activation of oncogenes located in these bands may proceed via cytogenetic mechanisms.     

Enzyme linked immunosorbent assay (ELISA) for human IgG (Gm) allotype determination

An inhibition enzyme-linked immunosorbent assay (inhibition-ELISA) was developed for the quantitative determination of human IgG (Gm) allotypes using rabbit anti-Gm antisera, alkaline-phosphatase-conjugated goat anti-rabbit IgG and, as the calibrant, purified human myeloma proteins possessing the relevant Gm allotype. The assay is reproducible and can detect as little as 10 ng/ml of G1m(a), G2m(n) or G3m(st), and 100 ng/ml of G1m(f) or G3m(g). Using this assay, the "gene dosage effect" and "allelic balance" in healthy Japanese were studied.     

Production of human interferon-gamma in serum-free medium.

Interferon (IFN)-gamma was produced with a high yield in cultures of human peripheral mononuclear cells by combined stimulation with OK-432 and staphylococcal enterotoxin B. Human mononuclear cells cultured in serum-free medium produced several times as much IFN as those in RPMI-1640 medium containing 10% fetal bovine serum. A synergistic effect of OK-432 and staphylococcal enterotoxin B on the production of IFN-gamma was demonstrated. Ultrogel AcA54 column chromatography of crude IFN showed a single peak with an apparent molecular weight of 43,000. Our production system for human IFN-gamma offers a feasible approach to preparation of large quantities of purified IFN-gamma for structure studies, antibody production, and clinical applications.     

In vitro adhesion of eosinophils to infective larvae of Wuchereria bancrofti

A reproducible in vitro test was developed to quantitatively study the adhesion of human eosinophils to Wuchereria bancrofti infective larvae. Eosinophils, regardless of the donor, selectively adhered to the larvae in the presence of immune serum. The reaction reached a maximum by 90 minutes at room temperature and remained unchanged up to 6 hours. The adherent eosinophils, however, did not induce any apparent morphologic change in the larvae.

The phenomenon appeared to require, primarily, IgG anti-larval antibodies. Heat-inactivation of the serum did not prevent the reaction from occurring, although addition of fresh normal serum enhanced the intensity of adhesion.

Maximal adhesion of eosinophils was obtained when the larvae were viable and in the presence of immune serum and fresh normal serum during incubation with the leucocytes.

Normal serum was found to induce this adhesion reaction. The responsible factor could be removed by absorption of normal serum with cotton. However, this procedure had no effect on the reactivity of sera from filariasis cases.

The reaction was almost totally inhibited by EDTA and citrate. The anti-inflammatory steroid, betamethasone, had a moderately inhibitory effect. An unexplained finding was an enhancing effect on the reaction when histamine was added to non-reactive normal serum.

     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.