Cytokine production and T cell receptor expression by salivary gland T cells and intraepithelial T lymphocytes for the regulation of the IgA response. The IgA effector sites such as the salivary glands and the intestinal tract contain several distinct T cell subsets which possess unique biologic characteristics. Freshly isolated CD3+ T cells from the salivary glands, the LP region of the small intestine and IELs all harbor T cells which spontaneously produce Th1 (IFN-gamma)- and Th2 (IL-5 and IL-6)-type cytokines. Interestingly, a high frequency of IL-5-producing Th2-type cells is always associated with the occurrence of increased numbers of IgA plasma cells (e.g., the salivary glands and the LP region of the small intestine). Further, the salivary gland CD3+ T cells can be divided into three distinct subsets including those of CD4+, CD8- (12-23%), CD4-, CD8+ (18-25%) and DN (6-16%) T cells. In terms of TCR expression, CD4+, CD8- and DN T cells exclusively expressed alpha/beta TCR and gamma/delta TCR, respectively. One of the unique features of the salivary gland T cells is that like IELs, relatively high numbers of gamma/delta TCR-bearing cells are seen in the CD4-, CD8+ T cell fraction. Since our study has provided important new evidence that these gamma/delta TCR-bearing T cells from IELs of mice orally immunized with TD antigen possess the capability of abrogating oral tolerance to antigen-specific immune responses including the IgA isotype, one can visualize that gamma/delta TCR+ T cells can be essential regulatory T cells which protect (or enhance) alpha/beta TCR+, CD4+ Th cells for maximum IgA responses at IgA effector tissues including the salivary glands, and the gastrointestinal tract in the presence of an active state of systemic unresponsiveness or oral tolerance.


Prospects for human mucosal vaccines.

Prospects for human mucosal vaccines. The selective induction of antibodies in external secretions and mucosal T cell-mediated immunity are desirable for the prevention of various systemic as well as predominantly mucosa-restricted infections. An enormous surface area of mucosal membranes is protected primarily by antibodies that belong, in many species, to the IgA isotype. Such antibodies are produced locally by large numbers of IgA-containing plasma cells distributed in subepithelial spaces of mucosal membranes and in the stroma of secretory glands. In humans and in some animal species, plasma-derived IgA antibodies do not enter external secretions in significant quantities and systemically administered preformed IgA antibodies would be of little use for passive immunization. Systemic administration of microbial antigens may boost an effective S-IgA immune response only in a situation whereby an immunized individual had previously encountered the same antigen by the mucosal route. Immunization routes that involve ingestion or possibly inhalation of antigens lead to the induction of not only local but also generalized immune responses, manifested by the parallel appearance of S-IgA antibodies to ingested or inhaled antigens in secretions of glands distant from the site of immunization. Convincing evidence is available that antigen-sensitized and IgA-committed precursors of plasma cells and T cells from IgA inductive sites (e.g., BALT, GALT, and tonsils) are disseminated to the gut, other mucosa-associated tissues, and exocrine glands. However, due to the limited absorption of desired antigens from the gut lumen of orally immunized individuals, repeated large doses of antigens are required for an effective S-IgA response. Novel antigen delivery systems for the stimulation of such responses has been briefly reviewed here. These, of course, include genetically engineered bacteria and viruses, CT/CFB, liposomes and microspheres. Live attenuated or genetically manipulated bacteria expressing other microbial antigens have been used for selective colonization of GALT. Unique antigen packaging and the use of adjuvants suitable for oral administration hold promise for an efficient antigen delivery to critical tissues in the intestine and deserve extensive exploration. The oral immunization route appears to have many advantages over systemic immunization; however, one must consider alternate IgA inductive sites and compartmentalization within the Common Mucosal Immune System. In addition to providing immunity on mucosal surfaces, which are the most common sites of entry of infectious agents, the mucosal routes of administration are more acceptable and do not require stringent criteria applicable for injectable vaccines, storage problems may be simplified, and large populations of individuals can be immunized simultaneously without the assistance of highly trained health personnel.


A mechanism of passive immunization with monoclonal antibodies to a 185,000 M(r) streptococcal antigen. The cell surface streptococcal antigen (SA) I/II of 185,000 M(r) is an immunodominant molecule that expresses one or more adhesion determinants. A series of 14 monoclonal antibodies (MAb) to defined parts of SA I/II were generated and some of these were used in passive immunization of macaques. Topical administration of selected MAb to the teeth of macaques prevented colonization of endogenous or implanted exogenous Streptococcus mutans for a period of 1 year. Significant reduction of both smooth surface and fissure caries was found in macaques who had MAb (Guy's 1) applied to their teeth, as compared with saline-treated animals. A series of in vivo passive immunization experiments was then carried out in 57 human subjects. Topical application of MAb to SA I/II prevented colonization of both artificially implanted exogenous strains of S. mutans, as well as natural recolonization by indigenous S. mutans. The properties of the protective MAb were then investigated and the epitope specificity within the SA I/II molecule was found to be essential but not the isotype specificity of the immunoglobulin (Ig). The requirement for complement activating and the phagocyte binding sites of the Fc fragment of MAb was not essential, as the F(ab')2 fragment of the MAb was as protective as the intact IgG, but the Fab fragment failed to prevent recolonization of S. mutans. Prevention of recolonization was specifically restricted to S. mutans, as the proportion of other organisms, such as S. sanguis, failed to show a significant change. The surprising feature of these experiments was that protection of re-colonization of S. mutans lasted up to 2 years, although MAb was applied for only 3 weeks and functional MAb was detected on the teeth only 3 days following application of the MAb. The long-term protection could therefore not be accounted for by a persistence of MAb on the teeth, but may be due to a shift in the microbial balance in which other bacteria occupy the ecological niche vacated by S. mutans, resulting in colonization resistance to S. mutans. Gene cloning and sequencing the SA from S. mutans, S. sobrinus and S. sanguis identified a conserved region (residues 955-1213) which on Southern hybridization and partial DNA sequence analysis was also found in 19 alpha-haemolytic oral streptococci. The results suggest that the SA molecule may constitute a family of adhesins in oral alpha haemolytic streptococci.(ABSTRACT TRUNCATED AT 400 WORDS)


Liposomes and conjugate vaccines for antigen delivery and induction of mucosal immune responses. In this brief review, emphasis was placed on the effectiveness of liposomes as carriers/vehicles of soluble antigens and as adjuvants for use in oral vaccine development. Evidence was provided that oral administration of antigen in liposomes resulted in a mucosal response which was higher than that obtained when the oral vaccine consisted of antigen alone. Specific mucosal responses were enhanced by incorporating lipophilic MDP into the antigen/liposome vaccines. Antigens shown to be effective in inducing a protective mucosal response when given in an oral liposome vaccine were anti-idiotypic antibodies, purified S. mutans GTF, CHO and Ag I/II. Evidence is also provided that CTB may be an effective oral adjuvant when coupled to proteins or peptides by either chemical or genetic methods. Further studies, however, will be required to characterize the effectiveness and safety of CTB in conjugate vaccines for inducing specific mucosal responses and to develop practical means to prepare oral liposome vaccines for use in humans.


Molecular, immunological and functional characterization of the major surface adhesin of Streptococcus mutans. In the 15 years since the last major NIH conference that dealt with anti-caries vaccines, we have learned much. Certainly, whole bacteria or bacterial fractions may not be proper immunogens due to the possibility of inducing tissue cross-reactivity. Our own experience (van de Rijn et al., 1976) illustrates that pitfall. But even in the era of genetically engineered vaccines, we first must understand the biological functions of our chosen immunogen before employing that pure protein in a vaccine. Our recent work (Brady et al., 1991c) indicates that antigen P1, a ubiquitous protein found on several oral streptococci, may possess different, but possibly overlapping, functional domains influencing reactions with fluid-phase salivary agglutinin (aggregation) versus fixed agglutinin (adherence). A proper vaccine would induce antibodies against the latter domain(s) thereby retarding colonization. An improper vaccine that induces antibodies against aggregation-related domains on P1 would lessen the host's ability to clear those bacteria from the oral cavity. After carefully identifying appropriate functional domains and obtaining sub-clones of the larger gene that yield truncated polypeptides typical of adherence-specific regions that are also immunogenic, we may be in a position to create the most effective vaccine. In studies employing the polymerase chain reaction (PCR) and standard cloning procedures, we have already begun to produce such polypeptides. Once a library of polypeptides is assembled, they may be tested for functional activity and for lack of induction of cross-reactivity with nonpathogenic streptococci (i.e., S. gordonii). Certain of these recombinant-specified polypeptides could serve as the basis for an anti-caries vaccine. Alternatively, peptides may be synthesized that resemble these sub-molecular regions for use in a vaccine or as competitive inhibitors of adherence but not aggregation. Clearly, a vaccine against dental caries remains a real possibility for the future.


Antigen-induced late-phase airway obstruction in the guinea pig? Hartley or Dunkin-Hartley guinea pigs (n = 136) were actively sensitized to ovalbumin or Ascaris suum protein by five different regimes. Specific airway resistance (sR(AW)) was measured in conscious animals by a plethysmographic procedure before, immediately following and at various intervals (up to 96 h) after aerosolized antigen or vehicle challenge. Each sensitization and challenge regime produced an immediate allergic response with positive responses (defined as a 2-fold increase in sR(AW)) in 78-100% of animals. Recovery from the immediate response followed by late-phase responses was observed in only two out of a group of four animals. The results failed to substantiate literature reports of a high incidence of late responses in the guinea pig at 4-8, 17-24 or 72 h after allergen challenge.


Antigen-induced hyperresponsiveness to methacholine in ventilated, anesthetized guinea pigs. Guinea pigs were actively sensitized to ovalbumin and exposed 2-3 weeks later to an aerosol of ovalbumin or saline. Changes in lung function were assessed 0.5, 1, 6, 24 and 72 h later by measuring the peak increase in pulmonary inflation pressure induced by i.v. methacholine during constant-volume ventilation. Responses to methacholine were significantly potentiated at 0.5, 1, 6 and 24 h but not at 72 h following exposure to antigen. Hyperresponsiveness to methacholine was maximal at 0.5-1 h and, in terms of magnitude, comparable to the early increase in airway reactivity found in mild asthmatics after allergen challenge. Whether the hyperresponsiveness to methacholine induced by antigen in the guinea pig can be attributed solely to an increase in airway reactivity or is due, at least in part, to decreased lung compliance requires further study.


Adenosine-induced bronchoconstriction in an allergic rabbit model: antagonism by theophylline aerosol. Recently, there has been an increasing interest in the involvement of adenosine as a mediator of allergic asthma. In the present study, we have employed an allergic rabbit model to study the airway responses to adenosine. The rabbit litter mates were injected i.p., within 24 h of birth, with ragweed pollen extract to preferentially produce ragweed-specific IgE. This allergic rabbit model demonstrates many clinical features of asthma similar to human allergic asthma. Aerosolized adenosine (0.156-10 mg/ml) caused a concentration-dependent bronchoconstriction. The normal rabbits (non-immunized) did not respond to adenosine within that concentration range. Theophylline significantly inhibited the adenosine-induced bronchoconstriction. These early studies suggest that adenosine differentiated asthmatic airways from normal airways and adenosine-induced bronchoconstriction in this model was mediated through specific cell surface receptor(s).


Modification of allergen-induced airway obstruction and bronchial hyperresponsiveness in the allergic rabbit by theophylline aerosol. The effects of theophylline on allergen-induced airway obstruction and bronchial hyperresponsiveness were investigated in allergic rabbits. This allergic rabbit model was developed in our laboratory and stimulates the human model of allergic asthma in several aspects. Four allergic rabbits with hyperreactive airways were challenged with ragweed to elicit early- and late-phase asthmatic responses and subsequent increased airway responsiveness. Two to three weeks later, the rabbits received theophylline (5 mg/ml, nebulized for 3 min) prior to a second allergen challenge. Theophylline significantly inhibited the allergen-induced early- and late-phase asthmatic responses by 38% (P < 0.05) and 49% (P < 0.05), respectively. Further theophylline inhibited the allergen-induced increase in lung resistance by 42% (P < 0.05) during the late-phase responses. Theophylline also inhibited the allergen-induced bronchial hyperresponsiveness by 39% (P < 0.05) at 24 h. These data suggest a potential anti-inflammatory effect of theophylline in preventing allergen-induced asthmatic responses and bronchial hyperresponsiveness.


Quantitation of eosinophil Major Basic Protein cytotoxicity to rodent respiratory epithelium. Eosinophil Major Basic Protein (MBP) may be a potent effector in damaging airway epithelium and inducing acute (2-3 h) hyperresponsiveness to agonists in primates. Accordingly, interactions between human eosinophil MBP and guinea-pig airway epithelium were quantitated biochemically. MBP was extracted from human eosinophils and purified by size-exclusion HPLC. This resulted in a single protein band on electrophoresis, which cross-reacted with antisera raised to peptides derived from the predicted sequence of human MBP. This human MBP caused modest, but statistically significant, damage to respiratory epithelium (16.4% increase in efflux of 51Cr from guinea-pig tracheal rings) after 3 h of incubation with 10(-4) M concentration, but not with lower concentrations. These data demonstrates that MBP cytotoxicity to intact epithelium can be rapidly measured in vitro, and suggests that rodent airway epithelium may be relatively resistant to the cytotoxic effects of MBP.


Multiple antigen challenge produces pulmonary eosinophilia but not pulmonary hyperresponsiveness in actively sensitized guinea pigs. Exposure of actively sensitized boosted guinea pigs to aerosolized antigen, 3 times on alternate days, produced pulmonary eosinophilia but not pulmonary hyperresponsiveness to methacholine Cl measured 3 days after the last antigen challenge. These data suggest that the presence of large numbers of eosinophils in the airways and tissues of the lungs is not sufficient to produce nonspecific pulmonary hyperresponsiveness. These data also suggest that actively sensitized and boosted guinea pigs respond differently to repeated antigen exposure than do asthmatics or wild caught allergic cynomolgus monkeys.


Chasing the elusive animal model of late-phase bronchoconstriction: studies in dogs, guinea pigs and rats. Antigen inhalation in sensitized dogs, guinea pigs and rats resulted in a marked, late-phase, eosinophil-rich, influx of inflammatory cells into the bronchial lumen. Attempts to demonstrate an associated late-phase bronchoconstriction were disappointing. We were unable to demonstrate a late-phase bronchoconstriction in either rats or dogs, even when dogs were pretreated with metyrapone to reduce blood cortisol levels. In ovalbumin-sensitized guinea pigs, challenged with low doses of ovalbumin, we observed an immediate bronchoconstriction, a late-phase bronchopulmonary eosinophilia but no late-phase bronchoconstriction. However, inhalation of very high doses of antigen in mepyramine-treated sensitized guinea pigs did induce a moderate late-phase bronchoconstriction.


Repeated aeroallergen challenge induces lung dysfunction but not bronchial hyperresponsiveness in conscious guinea pigs. Adult male Hartley-strain guinea pigs were sensitized by 10 min exposure to aerosolized 1% ovalbumin (OA; 10 mg/ml in normal saline containing 4% heat-killed B. pertussis vaccine and 0.02% antifoam B emulsion). One week after sensitization, animals were placed in an exposure chamber and challenged (nebulized OA 0.5%) until each animal showed labored breathing. Maximal exposure time was 10 min. Diphenhydramine (20 mg/kg, i.p.) was given 1 h before each OA challenge to protect the animals from bronchospasmic death. Antigen challenge was repeated twice a week for 2 weeks. The specific airway resistance (sR(aw)) changes in response to increasing concentrations of aerosolized acetylcholine (Ach) were determined. The data obtained in this study demonstrated that repeated antigen challenge produced a significant bronchial tone i.e. an increase in sR(aw) and a decline in specific airway conductance (sG(aw)) and failed to induce bronchial hyperreactivity to aerosolized acetylcholine (Ach) in conscious guinea pigs.


Characterization of aeroallergen-induced dyspnea in unrestrained guinea pigs by bias-flow-ventilated whole body plethysmography. Aeroallergen-induced dyspnea in guinea pigs was associated with an increase in amplitude in the box pressure fluctuations (212%) and pseudo-flow signal (604%) and an 80% decline (from 0.19 to 0.04 s) in relaxation time (the time it takes the box pressure signal to drop from its peak to 1/3 of its peak value). All of these lung dysfunction changes were highly significant (P < 0.001). Pyrilamine (1 mg/kg, p.o., -2 h) inhibited dyspnea (delta P and delta F) by 50-53%. This technique allows quantitative analysis of allergic dyspnea in conscious, unrestrained guinea pigs.


Pulmonary responses of the guinea pig to inhaled A23187: a brief review. The pulmonary effects of inhaled A23187 are reviewed. Guinea pigs challenged with this divalent cationic ionophore rapidly develop airway obstruction, which is maintained for at least 4 h. Pulmonary inflammation and increased airway responsiveness are also observed. Pharmacologic manipulations suggest that these actions are due to the release of multiple mediators. We have found A23187 challenge to be valuable as an approach for testing potential asthma drugs.


Lack of late-phase airway responses in conscious guinea pigs after a variety of antigen challenges. Guinea pigs were actively sensitized by parenteral injections of ovalbumin (OA), house dust extract (HD) or Ascaris suum extract (As) in a variety of multidose regimens. At least 3 weeks after the initial sensitization injection, aerosols of the appropriate antigen were administered to conscious guinea pigs in a double-chamber body plethysmograph. OA elicited the most consistent and intense bronchoconstriction (BC) as measured by decreases in specific airway conductance (sGAW). The airway responses to As were clearly separable into responders and nonresponders. HD produced essentially no BC. However, intense lacrimation and rhinorrhea occurred in all HD-sensitized, but not unsensitized, animals. No late-phase changes in sGAW or increased reactivity to other spasmogens were seen up to 8 h after any antigen challenge. Eosinophil influx of magnitude similar to that measured by 24 h post-antigen bronchoalveolar lavage (BAL) occurred with all the three antigens. Animals which did not bronchoconstrict to As experienced an equal or greater pulmonary eosinophilia as airway responders. The present data with HD and As suggest that acute BC in response to antigen provocation is not a prerequisite for the eventual pulmonary eosinophilia. The lack of late-phase airway reactions in these models raises a doubt in the direct extrapolation to airway responses in allergic human asthma. The acute lacrimation and rhinorrhea to HD may suggest utility as a model of allergic rhinitis.


Comparison of the airway hyperreactivity produced by single and multiple antigen exposures in sensitized guinea pigs. Chronic airway hyperreactivity is a hallmark feature of asthma, but animal models of airway hyperreactivity often utilize a single antigen challenge. Therefore, we compared the airway hyperreactivity produced by single and multiple antigen challenges in ovalbumin-sensitized guinea pigs. Significant (2-fold) leftward shifts in dose-response curves for i.v. methacholine- or LTD4-induced bronchoconstriction in anesthetized and ventilated animals occurred 24 h following a single ovalbumin challenge. This nonspecific airway hyperreactivity was prevented by pretreatment with ketotifen or dexamethasone. However, airway hyperreactivity was no greater 24 h following the last of 3 daily antigen challenges than after 1 challenge and was absent 72 h following one antigen challenge. These results raise concern over the similarity of antigen-induced airway hyperreactivity in guinea pigs to the chronic airway hyperreactivity in asthmatics.


Acute hypersensitivity to aerosolized histamine induced by aerosolized ovalbumin in guinea pigs. Acute airway hyperresponsiveness can be induced after exposure to aerosolized ovalbumin in sensitized guinea pigs. The purpose of the present studies was to determine if "pro-inflammatory agents" would potentiate and prolong antigen-induced pulmonary hyperresponsiveness to histamine in guinea pigs. Guinea pigs were sensitized to aerosolized ovalbumin by exposing them to a 3 min aerosol, generated ultrasonically from a 10% ovalbumin solution on day 0 and day 7. On day 13 the guinea pigs were exposed to a 3 min aerosol of deionized water or a pro-inflammatory agent (1 microgram/ml PAF, 1 mg/ml LPS, or 4% B. pertussis vaccine). Twenty-four hours later, on day 14, the conscious guinea pigs were challenged with a 3 min aerosolized ovalbumin exposure (under isoproterenol cover) and the individual guinea pig responsiveness to aerosolized histamine was determined 2 and 24 h later in an anesthetized modified Konzett-Rossler preparation. Under these experimental conditions, ovalbumin challenge to sensitized guinea pigs produced only an acute hyperresponsiveness (about a 3-10-fold shift) to aerosolized histamine, which lasted less than 24 h. The pro-inflammatory agents neither potentiated nor prolonged the duration of the hyperresponsiveness.


Specific temperature dependence of diamine oxidase activity and its thermal stability in the presence of polyvinylalcohol. An inflexion point on the dependence of the swine kidney diamine oxidase activity upon the temperature was found at 40-43 degrees C, suggesting a conformational transition. The activation energies with putrescine as substrate calculated from the Arrhenius plots were 38.23 kcal/mol for the temperature interval 25-40 degrees C and only 15.14 kcal/mol for the range 45-60 degrees C. These values suggest two different conformations, one corresponding to the interval below 40 degrees C and another one between 43-60 degrees C, with an intermediate transitory form corresponding to the inflexion point at 40-43 degrees C. For various temperature decades within 10-60 degrees C, peculiar Q10 values in the range 1.37-3.00 (differing from the usual value Q10 = 2), were obtained. The non-strictly Arrhenius curves, the activation energies and the inflexion point were quite similar with and without 0.05% polyvinylalcohol. This particular temperature effect found for swine kidney diamine oxidase is similar to the one reported for bovine serum amine oxidase. An increased enzyme thermal stability was obtained in the presence of high molecular weight polyvinylalcohol.


Decreased histamine content and metabolism in mammary cancer tissue from C3H mice. Histamine (HA) level and its metabolism in adenocarcinoma mammae, spontaneously growing cancer in C3H mice, were examined in relation to the type of tumor, intensity of tumor vascularization and the presence or absence of a secretory function. Histamine concentration being in mammary gland one of the highest among mammalian organs (418 nmol/g) was decreased by 90% in tumor (34 nmol/g). Similarly, histidine decarboxylase (HDC) activity dropped from approximately 7.6 pmol/min/g in normal gland to an undetectable level in adenocarcinoma mammae. Of the two main HA degrading enzymes, namely, diamine oxidase and histamine N-methyl-transferase (HMT), only HMT could be detected in mammary gland, either healthy or neoplastic, and its activity was about 5-fold lower in tumor than in the control tissue (1 nmol/g/min vs. 5.2 nmol/g/min). The pattern of changes in histaminergic parameters evoked by the tumor was similar irrespective of the morphological type it represented, characteristics of its vascular bed or whether or not it showed secretory activity.


Effects of ketotifen on airway responses to allergen challenge in the actively sensitized brown Norway rat. The purpose of the study was to examine the effect of ketotifen on the airway responses and the recruitment of the inflammatory cells into the airways of sensitized rats after antigen challenge. Twenty-five Brown Norway rats, 7-9 weeks old, were actively sensitized to ovalbumin (OA) (1 mg s.c.) and Bordetella pertussis vaccine (10(9) bacilli i.p.). At 14 days after sensitization rats were anesthetized with urethane (1.1 g/kg i.p.) and intubated endotracheally. Aerosols of OA (5% W/V in saline for 5 min) were administered to control rats (Group A; n = 9), to a low-dose ketotifen group (Group B; 1 mg/kg PO; n = 8) and a high-dose ketotifen group (Group C; 10 mg/kg; PO for 10 days; n = 9). Pulmonary resistance (RL) was measured at baseline, and every 15 min for up to 8 h after OA. The magnitude of the early response was 241 +/- 51% in A (% baseline RL; mean +/- SE), and significantly less in B(119 +/- 7%) and C(131 +/- 16%) (p < 0.01). The late response was significantly lower in C than A but not B. The total cell number in bronchoalveolar lavage at 8 h after OA challenge was significantly higher in A than B and C (p < 0.01). The lymphocyte and eosinophil counts were reduced in B and C compared to A (p < 0.05). A positive correlation was found between the late response and total number of cells recovered in the BAL (r = 0.78) (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Neutralization of the oedematogenic activity of Bothrops jararaca venom on the mouse paw by an antibothropic fraction isolated from opossum (Didelphis marsupialis) serum. The pharmacological modulation of mice paw oedema produced by Bothrops jararaca venom (BJV) has been studied. Intraplantar injection of BJV (1-30 micrograms/paw) produced a dose- and time-related oedema, which was maximal 30 min after injection, reduced gradually thereafter and disappeared over 48 h. BJV heated at 100 degrees C for 5 or 15 min blocked local hemorrhage and caused partial inhibition of its oedematogenic activity. The BJV oedema was not inhibited by the anti-histamine meclizine, the inhibitor of histamine and serotonin, cyproheptadine, PAF-acether antagonist WEB 2170 or by the anti-leukotrienes C4/D4, LY 171883. Dexamethasone, aspirin, indomethacin, and the dual cyclooxygenase and lipoxygenase inhibitor BW 755C inhibited BJV-induced oedema indicating that arachidonic acid metabolism products via the cyclooxygenase pathway participate in its genesis and/or maintenance. The antibothropic fraction (ABF) (25-200 micrograms/paw) isolated from Didelphis marsupialis serum neutralized the oedema induced by the venom with and without heating, the hemorrhage induced by BJV and partially blocked the oedema induced by bradykinin and by cellulose sulphate. The oedema produced by histamine, serotonin, PAF-acether or leukotriene C4 was not inhibited.


Effect of clonidine on experimental brain edema in the rat. Several experimental brain edema models are currently available for drug evaluation. Brain edemas are essentially vasogenic and/or cytotoxic, and eicosanoids are involved in the development of these edemas. Thus, a new model developed in our laboratory, which was obtained by phospholipase A2 intracerebral injection was used to study the antiinflammatory effect of clonidine. The copper wire edema model was chosen as reference. Edemas were evaluated by determining the swelling and Na+ and K+ tissue concentrations of each hemisphere. Drugs were administered intraperitoneally. Dexamethasone was the only drug to inhibit copper wire-induced edema, whereas indomethacin and clonidine as well as dexamethasone exhibited marked antiedematous activity in our model. The effect of clonidine, which could be inhibited by prior administration of yohimbine, suggests that central alpha 2-adrenergic stimulation is involved in reducing experimental brain edema.


Anti-inflammatory properties of zinc protoporphyrin disodium (Zn-PP-2Na). Anti-inflammatory properties of zinc protoporphyrin disodium (Zn-PP-2Na) were studied. Zn-PP-2Na exhibits anti-allergic action against type III and IV reactions (passive Arthus reaction in rats and tuberculin-induced footpad reaction in mice), but does not affect type I and II reactions (homologous passive cutaneous anaphylaxis in mice and reversed cutaneous anaphylaxis in rats). Zn-PP-2Na also clearly inhibits type II collagen-induced arthritis in mice. The agent inhibits general arthritis symptoms, anti-type-II collagen antibody production and type II collagen-induced delayed type hypersensitivity (DTH) in arthritic mice. Zn-PP-2Na, however, did not affect carrageenin-induced paw edema and histamine- and serotonin-induced skin reactions in rats. Zn-PP-2Na inhibits IL-1-induced mouse lymphocyte proliferation, but does not affect PMA-induced O2- generation from guinea-pig neutrophil. These results indicate that Zn-PP-2Na inhibits type II collagen-induced arthritis in mice due to the antagonism of IL-1 activity and the inhibition of DTH against type II collagen.


Comparative study of effects of cyclosporins A and G on collagen arthritis in mice. The effects of the immunosuppressive agents cyclosporin G (CsG) and cyclosporin A (CsA) on collagen arthritis were compared in mice. When administered subcutaneously daily on days 0-13 after immunization with type II collagen, CsG and CsA were both capable of suppressing the development of collagen arthritis in mice as well as the immunological response to native type II collagen in a dose-dependent manner. Histopathologically, no marked inflammatory lesions were observed in diarthroidal joints from mice treated with 100 mg/kg per day of CsA or 800 mg/kg per day of CsG. However, an analysis of dose response showed CsG to be 8 times less potent than CsA in inhibiting the development of arthritis.


Effect of adjuvant arthritis on the disposition of acebutolol enantiomers in rats. Disease states such as arthritis may interact with the kinetics of beta-blockers. Acebutolol (AC) is a chiral beta-blocker which is available as a racemate. The beneficial properties of AC, however, is attributed mainly to the S-(+)-enantiomer. The disposition of AC enantiomers and their active, chiral metabolites, diacetolol (DC) were examined after oral administration to healthy and adjuvant-induced arthritic (AA) female Sprague-Dawley rats. Arthritis was induced by tail base injection of Mycobacterium butyricum. Swelling of hind and forepaws were apparent in 10-16 days in AA but not controls. Control and AA rats were sacrificed at 0.5, 1, 2, 4, 6 and 8 h after a 25 mg/kg oral AC dose and blood was collected (n = 6). Significant three to tenfold increases in the initial plasma concentrations (0.5-2 h) of AC were observed in AA. Enantiomers were equally affected, thus AC S:R ratio was not changed. Higher plasma concentrations of the metabolite were only significant at 2 h. The ratio of DC:AC, however, was unaffected by AA. The DC S:R ratio was significantly decreased at 0.5 and 1 h in AA. The limited protein binding of AC (10%) was neither stereoselective nor affected by AA. Reduced intrinsic clearance in AA may be responsible for these observations.


Immunodepressive activity of FCE 23762 on humoral and cell-mediated immune responses in normal mice: comparison with doxorubicin. FCE 23762 (3' desamino-3'[2(s)methoxyl-4-morpholinyl]doxorubicin) is a new doxorubicin (Dx) derivative that has been selected for clinical testing for its favourable antitumor characteristics, which include efficacy on Dx-resistant tumors. Immunosuppression is an undesirable side-effect of anti-cancer chemotherapy and the therapeutic efficacy of Dx is probably also related to its low immunotoxicity. It was, thus, of interest to compare the effects of FCE 23762 and its parental drug on the immune responses. Both compounds were injected i.v. into healthy mice at equitoxic doses and according to different treatment schedules. Single doses of FCE 23762 and Dx, given concomitant or after the antigen, suppressed at the same degree and dose-dependently the primary anti-SRBC antibody response. Following a multiple treatment schedule after the antigen, FCE 23762 was less suppressive than Dx on both primary and secondary antibody production. Differently from Dx, that was completely inactive, FCE 23762 moderately inhibited DTH reaction to SRBC, only at the highest single dose tested or for repeated administrations given simultaneously or after priming. Both drugs were totally ineffective in delaying skin allograft rejection. Since spleen cellularity and ex vivo lymphocyte proliferation to Con A and LPS were similarly impaired by the two drugs, the differentiated immunodepressive activity of FCE 23762 and Dx cannot be merely associated to their cytotoxic and antiproliferative action. The hypothesis of a selective effect on different regulatory cell subsets and/or immune mechanisms is discussed.


The retinal distribution of ganglion cells with crossed and uncrossed projections and the visual field representation in the opossum. The retinal distribution of ganglion cells with crossed and uncrossed projections in the South American opossum, Didelphis marsupialis, was revealed by delivering HRP to one optic tract or to retinal targets of one hemisphere. The cells with uncrossed projections are restricted to the temporal retina, comprising 1/3 of the total retinal area, with a sharp transition at the naso-temporal boundary. Besides being distributed over the nasal 2/3 of the retina, cells with crossed projections are intermingled with those with uncrossed projections over the entire temporal retina. Quantitative analysis about the representation of the horizontal meridian on four specimens revealed that the maximum density of cells with uncrossed projections is on the average located at 3.2 mm (SD = 0.21), i.e. 34.8 deg, temporal to the optic disk, falling to 10% at 2.1 mm (SD = 0.14) or 22.8 deg. On the other hand, the peak for cells with crossed projections is more nasally placed at 1.8 mm (SD = 0.18), i.e. 19.6 deg. Between these two maxima, the site wherein the densities of cells with crossed and uncrossed projections are about equal is on the average about 2.7 mm (SD = 0.25) form the optic disk, i.e. 29.3 deg. This estimate supports the hypothesis that the retinal intersection of the vertical meridian lies within the region of split representation of crossed and uncrossed ganglion cells. In addition, it was observed that the opossum's retina has a large contingent of cells with uncrossed projections temporal to an eccentricity of 2.7 mm from the optic disk, where it represents roughly 2/3 of the ganglion cells. These data corroborate the relevance of the opossum as a non-primate model for visual work.


[Acute toxicity of ammonia in the production process of postlarvae of Penaeus paulensis Pérez-Farfante, 1967]. Larval shrimp Penaeus paulensis showed a tendency to decrease in ammonia tolerance as the larva metamorphosed from nauplius to postlarvae stage. The 24-h LC50 were 4.04, 1.70, 2.72, and 1.42 mg/l NH3-N on nauplii, zoea, mysis and postlarvae, respectively. The zoea stage and the initial postlarvae substages were very susceptible to ammonia. The 96-h LC50 values on zoea and postlarvae were 0.69 mg/l and 0.32 mg/l, against 0.80 mg/l NH3-N on mysis. The eggs of Penaeus paulensis were very affected by ammonia, which provoked a progressive decrease in the hatching rate and induced morphological deformities in hatched nauplii. A "safe level" of ammonia was estimated at 0.032 mg/l NH3-N on the basis of 24-h LC50 for eggs.


[Acute toxicity of ammonia to Artemia sp]. The acute toxicity of total ammonia-N, (NH3 + NH4+), and un-ionized ammonia-N, NH3-N, on newly hatched Artemia nauplii and Artemia adults was measured in 24, 48, 72, and 96-h semi-static bioassays system. There was a significant difference (P < 0.05) in medial lethal concentrations (LC50) obtained during the tests. The LC50 values on nauplii ranged from 650 mg/l, in 24-h, to 399.1 mg/l total ammonia-N, in 96-h, while the LC50 values on adults ranged from 1290.4 mg/l to 600.5 mg/l total ammonia-N, in the same period. Two methods for calculations of un-ionized ammonia toxicity are analyzed and discussed.


Maturation of the filaria Litomosoides sigmodontis in BALB/c mice; comparative susceptibility of nine other inbred strains. When inoculated subcutaneously, the infective larvae of L. sigmodontis undergo complete development and produce a patent microfilaraemia in mice of the BALB background (BALB/c, BALB/K and BALB/B, with respectively the H-2d, H-2k et H-2b haplotypes). The most susceptible strain is BALB/c with all mice harbouring adult filariae and 47% of mice presenting with a patent microfilaraemia. Mice with the B10 background (B10, B10Br and B10D2, with respectively the H-2b, H-2k et H-2d haplotypes) are almost completely resistant to infection. Adult filariae were recovered from all mice of the CBA/Ca, CBA/HN, C3H/HeN, DBA/2N strains. However, the site and structural development of the parasite varied in each strain. Absence of microfilaraemia is associated with absent or abnormal spicules, reduced number of female filariae and small size of female filariae. These results show that the Major Histocompatibility Complex only modulates the developmental pattern of filariae within the limits imposed by background genes. Male CBA/HN and C3H/HeN were more susceptible to infection than female mice. Inverse phenomenon was observed with strains BALB/c; and, no host sex effect was seen in DBA/D2N.


A survey of sexual behaviour of Singaporeans. In order to plan education programmes against sexually transmitted diseases (STDs) and HIV infection, a population-based survey on partner relations was conducted on 2,115 Singaporean men and women aged between 15 and 49 years. Of the 605 (60.1%) sexually experienced men and 647 (58.3%) sexually experienced women, 510 (84.3%) men and 644 (99.5%) women were married. Of the marrieds, 465 (76.9%) men and 638 (98.6%) women had their first sexual encounter with the person who was or later became their spouse. Of the sexually experienced men, 97 (16%) engaged in casual sex in the previous year, of which 76 (78.4%) were encounters with commercial sex workers. Of these encounters 55 (72.4%) were unprotected or partially protected by condoms. A history of casual sex in the previous four weeks was given by 18 men, of whom 14 (77.8%) were married. Nine (64.3%) of these married men had sex concurrently with both their wives and commercial workers. Persons at high risk were those who started sexual activity early, unmarried, sexually active men with no or low education, and persons who travelled. Of the sexually experienced respondents, 1,242 (99.2%) had heard about condoms, and 431 (34.4%) were currently using them as contraceptive devices. Attitudes towards condoms and willingness to use them to prevent STDs and HIV infection were favourable. Men were more permissive in their attitudes towards sex. The level of permissiveness in men and women was not corroborated by their reported behaviour, an indication of either under-reporting and/or a disparity between attitude and practice.


Clinical aspects of a successful embryo cryopreservation programme from IVF cycles in Singapore General Hospital. An analysis in November 1991 showed a successful cryopreservation programme between May 1989 and October 1991 in Singapore General Hospital. This programme has resulted in 9.1% more pregnancies in addition to those from conventional in-vitro fertilisation (IVF) methods, increasing pregnancy rates during the same period, from 27.8% per IVF cycle to 36.9% per cycle. The current policy is to transfer 3 fresh embryos in the IVF cycle and freeze the remainder using a modified Testart's method. Clinical factors that influence the success rate include the number of embryos transferred, stage of development of embryos and genital tract infections. Age of patient and grade of embryos did not seem to have affected pregnancy rates. Patients with anovulatory cycles enjoy similar pregnancy rates using a regulated cycle regime for frozen-thawed embryo transfer.


Rheumatoid arthritis in Singapore: carpal predominance and lower incidence of erosive changes. Radiographs of the hand and wrists in 33 patients with a diagnosis of classic or definite rheumatoid arthritis were reviewed. The mean duration of disease was about five years. A scoring system for quantitative analysis was used to assess the presence and severity of erosions, joint space narrowing and deviation of joint alignment. No radiological abnormality was seen in 30% of patients while in another 30%, only minor radiological changes were seen. Moderate to severe radiological abnormality was seen in only 40% and in this group of patients all showed more severe disease in the wrists than in the finger joints. Erosions in the fingers were only seen in 21% of patients and erosions in the wrists were present in 57%. Previous reports have indicated that disease in the wrists can be more severe than the hands in up to 60% while the incidence of erosions can be expected to range from 40% at 3 months to 95% at 10 years. Although the sample size in this study is small, the findings suggest that the radiological pattern of rheumatoid in the hands in Singapore is quite different from that classically described in temperate countries as the incidence of erosions appears lower while disease in the wrists can be expected to be more severe than in the fingers in almost all cases. The usual radiological criteria used to aid diagnosis of the disease may not be applicable.


A cadaveric study of the potential damage in arthroscopic examination of the temporomandibular joints. An arthroscopic study on 22 temporomandibular joints in 13 cadavers was undertaken. Dissections were carried out to correlate the arthroscopic with macroscopic findings in the joints. Iatrogenic damage to the temporomandibular joints and their surrounding tissues was also examined. Correlation between arthroscopy and macro findings was 91.0%. There was no damage to the facial nerve and superficial temporal vessels. Iatrogenic damage to the temporomandibular joint included scuffing of the articular surfaces of the glenoid fossa and articular eminence (31.8%), scuffing of the condylar articular surface (9%), one disc perforation (4.5%) and one perforation of the medial capsule (4.5%). Prevention of iatrogenic injuries is discussed.


Benign lower jaw tumours and their management: a ten year experience in Nigeria. Lower jaw tumours in Nigeria often grow to enormous size. A high cure rate can be achieved by wide excision of the tumours and bone grafting. Of 23 patients who underwent mandibular reconstruction in Enugu, Nigeria, over a ten year period, loss of bone graft occurred in three patients (14.28%), but no recurrence of tumour was noted over a follow-up period ranging from 2-12 years. An acceptable aesthetic and functional result followed bone grafting in most cases.


Renal bone disease in patients on haemodialysis: biochemical and radiological assessment. Renal bone disease is an important cause of morbidity in patients on dialysis. The prevalence of renal bone disease, especially aluminium related bone disease, has not been studied in the Singapore dialysis population. As such, we studied 45 haemodialysis patients for renal bone disease using biochemical and radiological parameters. Selected patients underwent a renal biopsy. There were 29 males and 16 females, mean (+/- SEM) age, 44.6 +/- 13.4 years. The duration of haemodialysis ranged from two months to ten years, mean 18.5 months. 75.4% of patients had hyperphosphatasemia, 24.4% had hypocalcemia and two patients had hypercalcemia. There was a wide range in the serum parathyroid hormone levels and 55.4% of patients had serum parathyroid hormone levels > 1000 pmol/L. Patients with symptoms and radiological abnormalities had significantly higher serum parathyroid hormone and alkaline phosphatase levels than those without (P < 0.005). The desferrioxamine infusion test was positive, with an increment in serum aluminium (DL) > 100 mg/L in five patients. Skeletal survey was positive for renal bone disease in 24.4% of patients. There was a significant correlation between the serum parathyroid hormone level, DA1 and the duration of dialysis (r = 0.752, p < 0.001 and r = 0.837, p < 0.001 respectively). There was no correlation between serum parathyroid hormone, calcium, phosphate levels and DA1. The serum haemoglobin concentration and ferritin levels did not show a correlation with DA1. Bone biopsy revealed hyperparathyroid bone disease in two patients, aluminium-related bone disease in one patient and mixed uraemic osteodystrophy in one patient.(ABSTRACT TRUNCATED AT 250 WORDS)


A double-blind, randomised, cross-over study comparing the 50g OGTT and the 75g OGTT for pregnant women in the third trimester. There is little consensus in the conduct and interpretation of the oral glucose tolerance test (OGTT) in pregnancy. In Singapore much data has been accumulated about the 50g OGTT while the converse is true of the 75g OGTT. A double-blind, randomised, crossover study of 56 subjects in the third trimester was conducted to compare both glucose challenges to determine if there was a difference in glucose handling in the context of a two hour OGTT. A significant difference (p < 0.001, t = 5.76) was found at two hours where the mean glucose concentrations were 5.2 +/- 0.8 mmol/L and 6.2 +/- 1.4 mmol/L for the 50g and 75g OGTT respectively. These findings suggest that the 75g OGTT is potentially more effective in unmasking pregnant subjects with impaired glucose tolerance.


An assessment of serum lipid and lipoprotein levels in patients with ischaemic heart disease. A total of 166 patients who had one or more attacks of myocardial infarction and those with angina pectoris, forty-five relative of 18 hyperlipidemic survivors of ischaemic heart disease, and 330 healthy persons (controls) were investigated for serum lipid profiles. Fifty-six of the 166 patients were hyperlipidemic. The commonest abnormalities in lipoproteins were Types IIa, IIb and IV. 75.5% of the 45 relatives investigated were hyperlipidemic. The familial studies showed that hyperlipidemias occurred in the family members of persons with ischaemic heart disease suggesting that hyperlipidemia could play an important role in predisposing familial clustering of coronary heart disease. A family history of heart disease may be a useful marker for identifying persons who are more likely to have high levels of blood lipids for possible treatment.


Pattern of dermatophyte infection in Singapore. Dermatophyte infection is the fourth commonest skin disorder seen in the National Skin Centre in Singapore. A prospective study was carried out from June 1990 to March 1991 to determine the epidemiology and pattern of dermatophyte infections seen in this centre. Two hundred patients who had a clinical diagnosis of dermatophyte infection and confirmed by direct microscopy were included into the study. Cultures for dermatophyte were done on Sabouraud's dextrose agar with chloramphenicol and cycloheximide. There were 148 male and 52 female patients, of whom 146 (73%) had a positive culture from one or more sites. One hundred and thirty-nine (95.2%) patients had dermatophyte infection while the remaining seven (4.8%) patients had non-dermatophyte infection. The sites involved were the groin (92 patients), feet (52 patients), trunk (26 patients), hands (18 patients), face (9 patients), legs (9 patients), scalp (8 patients) and axilla (7 patients). The commonest dermatophyte isolated was Trichophyton rubrum (81/139). It was also the commonest organism isolated in all the sites except the scalp. The other dermatophytes grown were, in order of frequency, Trichophyton interdigitale (21/139), Epidermophyton floccosum (19/139), Trichophyton mentagrophytes (9/139), Microsporum ferrigineum (4/139), Microsporum canis (4/139) and Microsporum gypseum (1/139). Scalp infections occurred in children and were due either to Microsporum canis or Microsporum ferrigineum. The organisms isolated in our study were either ubiquitous or common in the Asia-Pacific region indicating that certain dermatophytes are more prevalent in certain geographical regions.


Myopia and educational attainment in 421,116 young Singaporean males. Data of 421,116 Singaporean males aged 15 to 25 (mean 17.75) years who underwent compulsory medical examination in 1974-84 and 1987-91 were used to estimate the prevalence of myopia and to study the correlation between the prevalence and severity of myopia and educational attainment. The estimated myopia prevalence rate was 26.3% in 1974-84 and 43.3% in 1987-91. This rise in the rate was accompanied by an increase in the proportion of males who achieved higher levels of education over the same period. The overall myopia prevalence rate was 30.4%. Both the prevalence and severity of myopia were higher as the level of education attained increased. The myopia prevalence rate was 15.4% in males with no formal education and increased steadily through groups with intermediate education to 65.2% among those with GCE 'A' level education, 57.5% among diploma holders and 65.1% among university graduates in 1987-91. Seventy out of 173 (40%) myopes with no formal education compared to 1035 out of 1612 (64%) myopes with university degrees had unaided visual acuity worse than 6/60 in 1987-91. Our findings confirm indications from other sources that the association between the prevalence and severity of myopia and education attainment is real. A combination of genetic and environmental factors may be the cause of this association.


A descriptive study of emergencies admitted to a surgical department. A study was undertaken to evaluate the pattern of emergency admissions and their management by the surgical firm in a medium size (700 beds) general hospital. Over a three month period, 258 patients were admitted to one surgical team that was on take every third day. All patients were entered into a protocol that was updated daily by a registrar. In four (13.3%) of the 30 days on take, patients had to be referred to a nearby hospital due to shortage of emergency beds. The accuracy of diagnosis by the Accident and Emergency (A&E) resident, surgical resident and surgical team were 75.7, 77.2 and 88.4% respectively. The mean delay before attendance by an intern after admission was 47 (SD +/- 29) mins. Forty-four (17%) of these admissions were probably unnecessary. A further 22 (8.5%) patients had to be referred to other disciplines, indicating an initial wrong diagnosis. A substantial number of investigations were unnecessarily done on an emergency basis. Ninety-three (36.0%) patients required surgery. The median duration of hospital stay was three days. The overall morbidity was 4.2% and mortality 1.2%. The study was valuable in revealing the deficiencies in the existing emergency service; leading to new proposals to achieve our ultimate aim of providing high quality patient care.


An investigation of solvent-induced neuro-psychiatric disorders in spray painters. An index case of chronic toxic encephalopathy is described in a 28 year old male spray painter with eleven years of exposure to solvents at work. The patient had progressive affective symptoms following repeated past episodes suggestive of acute solvent intoxication. He also showed impaired performance on tests of auditory memory, perceptual speed, visual scanning, and manual dexterity, as well as electromyographic evidence of prolongation of distal terminal latency and slowing of conduction velocities. The case investigation was followed up with an epidemiological study of the factory where he worked, to determine if other spray painters might have similar excessive complaints of neuropsychiatric symptoms and/or objective neurobehavioural deficits. This involved 15 other male spray painters employed for at least five years, and a sex- and age-matched group of 15 control workers not exposed to solvents. The spray painters reported significantly more numbers of neurobehavioural symptoms than the controls. There were little differences in neurobehavioural test results, except for aiming test (psychomotor co-ordination) and Benton visual retention test (visual memory). Spray painters with four or more neurobehavioural symptoms also performed more poorly on Digit Symbol (perceptual speed) compared with those with less number of symptoms. The epidemiological results provide strong circumstantial basis for the diagnosis of chronic toxic encephalopathy in the patient, and also indicates a need for vigilance of the hazards of central nervous system disorders in workers with chronic exposure to solvents in Singapore.


Obstetric admissions to the intensive care unit--a retrospective review. A five-year retrospective study of obstetric admissions to the Surgical Intensive Care Unit (SICU) in the National University Hospital, Singapore was carried out with the aim of determining the incidence, causes and outcome of these admissions. Most of the patients were admitted following emergency caesarean sections. Obstetric complications was the reason for admission in 56.8% with hypertensive disease of pregnancy being the major cause and haemorrhage accounting for the rest. Anaesthetic complications accounted for 21.6% of admissions and these included difficult intubation, aspiration pneumonitis, cardiac arrhythmias and respiratory depression. Medical complications due to cardiovascular disease, autoimmune disease and malignancy also accounted for 21.6% of admissions. Only 37 out of 16264 deliveries (0.22%) required intensive care support. The median of duration of stay was one day.


The management of spasticity--a review of options available in rehabilitation. The author gives a literature review of the management of spasticity carried out. Physical agents used such as ice, heat and vibration are described. Methods used by physical therapists are discussed. Drugs most commonly used in the treatment of spasticity are Diazepam, Baclofen and Dantrolene. Phenol has been used by injection into motor points, peripheral nerves and intrathecally. Other drugs that have been used are also discussed. Neurosurgical procedures include neurectomies, neurotomies, rhizotomies and more radical procedures such as myelotomies and cordectomies. Orthopaedic procedures involve muscle and tendon lengthening, release of contractures and tendon transfers. Surgery is discussed in terms of function. In considering the options, available care must be taken to achieve and maintain optimal functional level.


Urinary symptoms and urodynamic diagnosis of patients in one geriatric department. Fifty five patients between 65 and 89 years old who had their urinary problems assessed by urodynamics study were reviewed. The most common urinary symptom among males was urge incontinence, while retention and urge incontinence occurred with equal frequency among females. The commonest cause of retention in males was bladder outlet obstruction, while atonic neurogenic bladder was the most common cause in females. Urge incontinence was strongly associated with an unstable bladder, small bladder volume and Parkinson's disease. Retention of urine, and an atonic neurogenic bladder strongly correlated with diabetes mellitus. Three patients (out of 31) with unstable bladders also had detrusor external sphincter dyssynergia. Of these, two had Parkinson's disease. Although three patients were thought to have stress incontinence after history and physical examination, only two had stress incontinence with detrusor instability on urodynamic studies. The last patient had atonic bladder with overflow.


Massive bleeding per rectum from a caecal pregnancy. An unusual case of massive bleeding per rectum caused by erosion into the caecum of placental tissue from an ectopic pregnancy is presented. Despite its rarity, abdominal pregnancies may have to be considered in sexually active fertile women presenting with difficult torrential bleeding from the lower gastrointestinal tract.


Polyneuropathy following intra-muscular sodium aurothiomalate for rheumatoid arthritis--a case report. A patient with rheumatoid arthritis developed rapid onset of peripheral neuropathy whilst on treatment with intra-muscular (IM) gold (Sodium Aurothiomalate). At the time of admission, the arthritis was relatively quiescent. Electromyogram showed evidence of sensorimotor polyneuropathy. Investigations excluded other causes of polyneuropathy. Sural nerve biopsy did not reveal inflammation or vasculitis. The patient's condition improved after cessation of gold therapy. Gold induced neuropathy should be considered in a patient with rheumatoid arthritis who presents with polyneuropathy while on gold therapy.


Tracheal rupture from incorrect positioning of endotracheal tube. Significant ischaemic tracheal damage from endotracheal intubation is uncommon when the lateral wall pressure exerted by the cuff does not exceed the mean capillary perfusion pressure of the mucosa. This is facilitated by the modern endotracheal tubes with high-volume-low-pressure cuffs. We report a case of tracheal rupture due to an incorrectly positioned softcuffed tube. The need to review tube position radiologically and to make immediate adjustment cannot be overemphasised.


Clear cell carcinoma of the lung--a case report. Clear cell carcinoma of the lung is a rare entity. We report the first case seen locally. The patient is symptomatic, presenting with a large pulmonary tumour. Histologically, there were clusters of large tumour cells with ample clear cytoplasm and absence of mucin, glandular and squamous differentiation. Urine analysis, blood carcinoembryonic antigen level and abdominal CT scan were normal.


Acute surgical abdomen in systemic lupus erythematosus--an analysis of 10 cases. Gastrointestinal manifestations in a lupus patient may be due to different aetiologies such as vasculitis or a surgical condition. Problems of diagnosis are frequently encountered because the clinical presentations may mimic each other. We analysed ten lupus patients with acute surgical abdomen to identify the clinical, laboratory and radiological features that may aid in early diagnosis and management. Three patients with surgical abdomen had concomitant active lupus. Intra-abdominal sepsis and bleeding peptic ulcer disease constituted two major causes of laparotomies. Overt signs of peritonitis might not be present due to steroid masking effect. There were no specific clinical features, laboratory or radiological tests that could distinguish gastrointestinal vasculitis from acute surgical abdomen. Blood cultures, C-reactive protein and CT abdomen were useful adjuncts in the management of abdominal sepsis. No correlation was found between the timing of surgery, mortality, steroid dosage and wound complication.


Hepatotoxicity of amiodarone.

Hepatotoxicity of amiodarone. Amiodarone, a commonly used antiarrhythmic agent, has numerous adverse effects. The purpose of this case report is to highlight its hepatotoxicity, an unusual complication of long term amiodarone therapy. Our patient is a 76-year-old man with underlying ischaemic heart disease and recurrent ventricular tachycardia. Eleven months after commencing amiodarone, he developed asymptomatic raised aminotransferases which resolved following drug withdrawal. Amiodarone was then reintroduced and four years later, the patient developed hepatomegaly, worsening liver biochemistry and histopathological changes consistent with early cirrhosis. His symptoms improved following discontinuation of amiodarone. However, hepatomegaly and a low serum albumin still persist four years later.


Echocardiographic diagnosis of quadricuspid aortic valve. Isolated quadricuspid aortic valve is a rare congenital anomaly. We present a case diagnosed on cross-sectional echocardiography. To the best of our knowledge, this is the first case report on echocardiographically diagnosed quadricuspid aortic valve in Singapore.


[Contribution of intra-operative measurement of intact parathormone in surgery for primary hyperparathyroidism]. Using a sensitive two-site immunoradiometric assay which detects intact parathormone (iPTH), we studied the decrease in peripheric and jugular plasmatic iPTH during surgical removal of abnormal parathyroid (s). In the next future, results of intact parathormone (iPTH) assay will be given in 45 minutes. In a prospective study of 33 patients operated on for hyperparathyroidism or for cold thyroid nodule, the serum levels of intact PTH was measured intraoperatively in peripheric and in jugular blood. The preoperative mean serum iPTH concentration was 119.23 +/- 172.48 pg/ml and fell to 34.5 +/- 32.21 pg/ml after surgery in 14 cases of primary hyperparathyroidism (p < 0.001). Thirteen out of 14 patients had serum iPTH values less than 65 pg/ml within 15 minutes after parathyroidectomy. The preoperative mean serum iPTH concentration in the 5 secondary hyperparathyroidism was 781.2 +/- 403.19 pg/ml. This value fell to 124 +/- 66.91 pg/ml after parathyroidectomy (p < 0.04). No significant decrease was observed in the mean serum concentration of the 14 patients operated on for cold thyroid nodule. Patients suffering from single parathyroid adenoma presented a significant gradient in jugular plasmatic PTH concentration between the adenoma side and the contralateral one. This gradient decreased during effective parathyroid adenomectomy (309.7 +/- 313.3 pg/ml to 3.7 +/- 35.1 pg/ml). Intraoperative serum iPTH concentration will provide a valuable tool to appreciate the effectiveness of surgical removal of parathyroid glands and to detect the location of parathyroid adenoma when the surgical research is negative.


[Epidemiology of goiter in Auvergne].

[Epidemiology of goiter in Auvergne]. An epidemiological study about goiter, related to the French national survey concerning school children aged 10-16, was carried out in region Auvergne. The prevalence of goiter was 12.52%, including 2.8% patients with WHO stages I and II, thus being slightly below national average (16.7%). Iodine urinary excretion was 62.9 +/- 34.3 micrograms per gram creatinine (French mean value 85.0), although sometimes major variation was noted in the same area. Later, two studies were performed in adults, in two places of Haute-Loire and Puy-de-Dôme, respectively Arsac-en-Velay and Besse-en-Chandesse. Both places were selected because of high frequency of goiter in children. Prevalence in adults was 16.1% in Arsac-en-Velay including 12.5% stages I and II, and 8.9% in Besse-en-Chandesse including 5.9% stages I and II. Mean iodine urinary excretion was respectively 59.78 micrograms/gram creatinine and 43.45 micrograms/liter. Thus, these surveys confirm a significant prevalence of goiter in Region Auvergne, with endemic places, and a generally low iodine urinary excretion.


[Partial 3-beta-hydroxysteroid dehydrogenase deficiencies]. An overview of the partial 3 beta-hydroxysteroid dehydrogenase deficiency is presented. The classical, congenital or early postnatal form is characterized by a salt-losing syndrome and/or ambiguous genitalia. The late-onset forms, only recognized for the last ten years and diagnosed with an increasing frequency, are to be systematically suspected in the presence of clinical hyperandrogenism with or without oligomenorrhea. This deficit, involved in both adrenal and gonadal tissues, seems to be transmitted by an autosomal recessive gene. An ovarian 3 beta-hydroxysteroid dehydrogenase deficit can be a primary cause of some cases of polycystic ovary syndrome and the relations with this affection are disputed. The increased ratios of delta 5 steroids/delta 4 steroids ensure the diagnostic conviction while the elevated ratio of 17-hydroxy-pregnenolone/17-hydroxyprogesterone and the normal ratio of 11-desoxycortisol/cortisol allow to eliminate the possibility of a 21-hydroxylase or 11 beta-hydroxylase deficiency, respectively. The treatment is based above all on the glucocorticoid utilization, which can lead to the return of menses and the ovulatory function, but the cutaneous symptoms of hyperandrogenism will be better controlled by cyproterone acetate out of situations of stress.


[Genetic susceptibility of insulin-dependent diabetes]. Type I Insulin-Dependent Diabetes Mellitus (IDDM) is an autoimmune disease characterised by the destruction of pancreatic Beta cells. There is an evidence for a contribution of genetic factors to the development of the disease and it is widely documented that HLA antigens contribute to the disease susceptibility. HLA-DR3 and HLA-DR4 associations have been firstly described in Caucasian. Recent studies at the gene level have elucidated this HLA association more precisely, pointing out the prominent role of HLA-DQ locus genes. The hypothesis has been proposed that some critical amino-acid at position 57 of DQ Beta chain and at position 52 of DQ Alpha chain both contribute to disease susceptibility. According to the functional role of HLA class II molecules, these particular residues may affect the antigen binding and T cell recognition and therefore contribute to the triggering of the pathological auto-immune response.


[Immunologic mechanisms implicated in autoimmune destruction of beta cells in type I diabetes]. Lymphocytic infiltration of the islets of Langerhans results from activation of immunity effector cells during the period preceding clinical onset of diabetes. After escape from thymic selection, the autoreactive cell clones proliferate in the peripheral hemopoietic organs prior to invading the islets of Langerhans. The selection, expansion and activation of autoreactive cells are under genetic control. The development of quantitative tests of cellular immunity should allow determination of the importance of the anti-islet immunologic reaction, suspected at present from the presence of specific autoantibodies in genetically predisposed subjects at high risk of insulin dependence.


[Risk markers of insulin-dependent diabetes in the normal population]. Risk factors for IDD have been studied in relatives of patients, in whom the risk is far higher than in the children from the background population. Several factors have been identified as the genetic background at the HLA level (DR and DQ regions) and autoantibodies like islet cells antibodies (ICA) and insulin autoantibodies (IAA). 8,363 sera from schoolchildren (age 6-17 yr) have been tested for the presence of ICA. 150 sera (1.8%) were found positive; the prevalence rate of high ICA titres (> or = 20 uJDF) was very low: 0.2%. ICA titres remained stable over a 8 mth follow-up period. IAA have been measured on the first 2,000 sera and the prevalence rate was 1.2%. Only 3 sera were found positive for the two antibodies, one coming from a diabetic girl. The distribution of the susceptibility alleles at the DQ-HLA region was similar among the ICA-positive and ICA-negative children.


[Metabolic markers of type I prediabetes]. Studies of the early metabolic alterations of type 1 diabetes aim at the quantification of the residual beta cell mass and its rate of destruction, as well as identifying predictive markers of insulin dependency. The first phase of insulin secretion during the intravenous glucose tolerance test has been mostly investigated. The test is satisfactorily reproducible provided it is standardized. The age and the insulin sensitivity of the subject should be taken into account when interpreting the results. Moreover, functional phenomenon that may be reversible may take part in the observed alterations of insulin secretion. The abolition of the first phase of insulin secretion always precedes insulin dependency, and has a good positive predictive value in subjects with anti-islet cell antibodies. However, some pre-diabetic subjects already have a low first phase insulin response from the first examination, which does not favour the hypothesis of a linear beta cell destruction and points to the heterogeneity of this stage of the disease. Conversely, profound metabolic alterations have been described with no progression towards insulin dependency, suggesting remission of the autoimmune process. The predictive value of the alterations of insulin secretion using other stimulus will be assessed by ongoing studies.


[Screening of type I diabetes in patients' families]. Insulin dependent or type 1 diabetes is an autoimmune disease with a strong genetic susceptibility linked to MHC and non MHC genes. Risk of the disease is 20 fold higher in first degree relatives of patients than in the general population. beta-cell destruction is progressive and marked by the appearance of antibodies to several islet constituents including insulin and glutamate decarboxylase. These markers allow disease prediction specially in children where a population with a 5 years risk approaching 100% can be defined. The intravenous glucose tolerance test can detect a progressive decline of the first phase of insulin secretion, preceding glucose intolerance and hyperglycemia. These screening programs will allow clinical trials currently limited to non specific immuno-suppressive agents such as cyclosporine in patients with preclinical diabetes. In the future, identification of targets and effector mechanisms of auto-immune destruction of the beta-cells will allow the evaluation of more specific approaches at earlier stages of the disease.


The effect of anti-CD3 on the induction of non-MHC restricted cytolytic activity. The effects of an anti-CD3 mAb on induction of non-MHC restricted cytolysis was investigated. Peripheral blood mononuclear cells (PBMC) from normal donors (29) and cancer patients (18) were cultured in 100 U/ml of interleukin-2 (rIL-2) with and without anti-CD3 mAb (OKT3, 10 ng/ml) for the first 48 hours of incubation. Thereafter, both PBMC cultures were maintained on rIL-2 up to 20 days. PBMC proliferation was enhanced 17-fold in number by day 20 when anti-CD3 mAb and rIL-2 was present during the first 48 hours but only 3-fold by day 20 when rIL-2 alone was present. Concomitantly anti-CD3 mAb but not Lym-1, an isotype matched control, inhibited the induction of lytic activity against both NK sensitive (K562) and NK resistant (Raji) target cell lines. Thus the inhibitory effect is dependent on anti-CD3 mAb stimulating the CD3/TCR T-cell receptor complex. While lytic activity was dependent on the concentration of rIL-2, inhibition of the induction phase of non-MHC restricted lytic activity was independent of the concentration of rIL-2. Flow cytometry analysis indicated that treatment with the anti-CD3 mAb increased the percentage of CD3 positive cells, CD4 positive cells and especially CD25 positive cells, but decreased th percentage of CD56 positive cells. Supernatants from anti-CD3 mAb stimulated cultures also inhibited the induction of non-MHC restricted lytic activity. Lymphokine analysis showed that supernatants of anti-CD3 mAb stimulated cultures had higher levels of TNF-alpha and IFN-gamma. However, TNF-alpha and IFN-gamma alone or in combination could not mediate the inhibitory effect. The inhibitory factor(s) was partially purified by sequential chromatography on matrices of controlled pore glass and Sepharose CL-6B. The molecular weight of the inhibitory factor(s) was less than 67K. These studies have identified a novel regulatory pathway controlling non-MHC restricted cytolysis. Perturbation of the T-cell CD3/TCR complex with the anti-CD3 mAb results in the secretion of a soluble mediator that down-regulates the induction of rIL-2 dependent non-MHC restricted cytolysis.


Monoclonal antibody SP-21 defines a sialosyl-Tn antigen expressed on carcinomas and K562 erythroleukemia cells. Monoclonal antibody (mab) SP-21 resembles the well established mab B72.3 by high affinity binding to peptide linked sialosyl-Tn disaccharide, NeuAc alpha 2-6 GalNAc alpha, and non-reactivity to the related trisaccharide NeuAc alpha 2-6 (Gal beta 1-3)GalNAc alpha. Although mab SP-21 may be classified, accordingly, into the family of mabs defining the sialosyl-Tn antigen, its fine specificity is distinct from the reference antibody B72.3 by binding of mab SP-21 to fetal mucins from human meconium or amniotic fluid. The distinct immunoreactivity of this mab is also documented by its specific cell staining of the erythroleukemia cell line K562, where it binds to a 105 KDa glycoprotein. Moreover a subpopulation of normal lymphocytes stains to this mab SP-21.


Anticancer activities of orally administered menogaril against human stomach and breast cancers implanted in nude mice. The therapeutic effects of orally administered menogaril, a semisynthetic analog of the anthracycline antibiotic nogalamycin, were studied on a panel of human stomach and breast cancer xenografts. The maximum tolerated dose (200 mg/kg) of menogaril was administered 3 times every 4 days and its growth-inhibitory effects on subcutaneously implanted tumors in nude mice were evaluated. Menogaril significantly retarded the growth of 3 out of 7 stomach cancers, SC-2, SC-9 and 4-1ST, and 3 out of 4 breast cancers, H-31, MC-2 and MX-1, with overall response rates of 43 and 75% for stomach and breast cancers, respectively. Some of these relatively responsive cancers were also treated by daily oral administration for 5 consecutive days, but the anticancer effects of the intermittent administration seemed to be better. These results suggest that menogaril may be effective against stomach and breast cancers when orally administered.


p53 mutation in human nasopharyngeal carcinomas. Nasopharyngeal carcinoma (NPC) is the third most common cancer in the southern provinces of China, but a rare cancer in other parts of the world. Epidemiological studies suggested a multifactorial etiology of NPC involving infection of Epstein Barr virus (EBV), genetic predisposition, environmental factors, such as consumption of salted fish, and other unknown factors. p53 mutation is a common event in many forms of human cancers but its possible involvement in the pathogenesis of NPC has not been examined. The presence of p53 mutation in NPC is studied by the sensitive PCR-SSCP analysis and direct DNA sequencing method. The frequent sites of p53 mutation (exons 4 to 8) reported in other human tumors were studied. Thirty-eight biopsied tumors of NPC and 4 NPC cell lines were examined for the presence of p53 mutation. No mutation of p53 resulting in change in amino acid sequence of the encoded p53 protein was identified in any of the biopsies tumors. RFLP studies of the biopsied materials of NPC also revealed no loss of heterozygosity at chromosome region 17p13 in 15 out of 15 informative cases, which further supports the conclusion that p53 mutation is an infrequent event in NPC. Apparently, p53 mutation has no significant role in the pathogenesis of this special group of human cancers. However, p53 mutation is frequently observed in cell lines derived from the primary NPC tumors. All the three NPC cell lines examined carry a missense p53 mutation, suggesting that mutation of the p53 gene may confer growth advantage to the tumor cells to become established in culture.


A randomized, comparative study of combination chemotherapies in advanced gastric cancer: 5-fluorouracil and cisplatin (FP) versus 5-fluorouracil, cisplatin, and 4'-epirubicin (FPEPIR). Kyoto Research Group for Chemotherapy of Gastric Cancer (KRGCGC). Sixty patients with inoperable or recurrent gastric cancers were randomly assigned into 2 treatment regimens: the FP regimen, consisting of cisplatin (CDDP, 50 mg/body, d1) + 5-fluorouracil (5-FU, 250 mg/body, d2-d5), or the FPEPIR regimen, CDDP+5-FU (FP) + 4'-epirubicin (EPIR, 30 mg/m2, d2), administered intravenously every 2 weeks. The objective responses were evaluated in 43 patients (FP: 21; FPEPIR: 22). The response rates were 24% (5PR/21) for FP and 27% (6PR/22) for FPEPIR. Objective responses were seen in 1 primary lesion and 4 metastatic lesions in patients on the FP regimen, and in 3 primary lesions and 4 metastatic lesions in patients on the FPEPIR regimen. The 6 month and 1 year survival rates were 34% and 13% for FP, and 55% and 27% for FPEPIR, respectively (not significant). Significantly higher survival rates were demonstrated in those patients with recurrent cancers (p < 0.01) and those who responded to the FPEPIR regimen (p < 0.05). Drug toxicities were assessed in 56 patients, and were in general mild and well tolerated. However, the FP regimen induced significantly less neutropenia (p < 0.01) and less complete alopecia (p < 0.01) than the did FPEPIR regimen. These results suggest that FPEPIR may be a more beneficial regimen for the treatment of gastric cancer than FP, and may be indicated for further clinical trials.


Laminin and 67 kD laminin binding protein in mouse B16 melanoma cells and 3T3 fibroblast spheroids. Multicellular spheroids which promote cell-cell and cell-matrix interactions were prepared in culture with mouse B16 melanoma cells (pigmented or non pigmented) alone or mixed with mouse 3T3 fibroblasts. Their volume and proliferation or necrosis rate were evaluated. As measured by dot blot immunoassay, laminin was mainly produced by fibroblasts rather than by melanoma cells. High levels of laminin B1 chain mRNA were detected only in spheroids composed of 3T3 fibroblasts. The levels of 67 kD laminin binding protein mRNA were high in all cell populations studied here.


A prospective study of tissue polypeptide specific antigen (TPS) in breast cancer diagnosis. The aim of the present study was to evaluate the clinical value of a new tumour marker TPS in sera from patients with breast cancer and patients with benign breast disease. The results for TPS were compared with those for TAG 12, CA 15-3 and MCA. The cutoff levels (90% specificity) determined for each test were 108.0 U/1 for TPS, 57.5 KU/1 for TAG 12, 24.8 KU/1 for CA 15-3 and 9.7 KU/1 for MCA. The diagnostic sensitivity of the TPS test was 0.21, for the TAG 12 test it was 0.2, 0.13 for the CA 15-3 test and 0.12 for the MCA test in detecting breast cancer. TPS, TAG 12, CA 15-3 and MCA tests were tested in a multivariate analysis to find the best combination of independent predictors of breast cancer. The most important predictor of breast cancer was TAG 12 followed by TPS. In order to calculate the contributions of tumour marker tests, a diagnostic score (DS) was developed. The diagnostic score (DS) was: DS = TPS x 0.0085 x TAG 12 x 0.0596 - CA 15-3 x 0.0866 - MCA x 0.2938 + 0.1738. The sensitivity of the DS in detecting breast cancer was 55% with a specificity of 90% and an efficiency of 64%. In conclusion, new tumour markers TPS and TAG 12 may have some value in breast cancer diagnosis. In particular, the results indicate the usefulness of computer assistance and a simple diagnostic score when tumour markers are used in breast cancer diagnosis.


The cytoskeleton as a subcellular target of the antineoplastic drug lonidamine. Lonidamine (LND), a dichlorinated derivative of indazole-3-carboxylic acid, has proved to exert a powerful antiproliferative effect and to impair the energy metabolism of normal and neoplastic cells. A target effect of the drug on the cell membrane structure was hypothesized. Thus, in order to elucidate better the mechanism of action of LND, the drug effects on the cell surface as well as on main cytoskeletal elements, i.e. actin microfilaments, microtubules and intermediate filaments, were investigated. In particular, an immunocytochemical and ultrastructural study was performed using two different cell lines: epithelial squamous carcinoma (A431) and melanoma (M14) cells. Treatment with 0.8 mM LND for 8 hr induced a remarkable rearrangement of the F-actin molecules with the disappearance of the stress fibers. As far as microtubules are concerned, formation of perinuclear patches of tubulin were detected after LND treatment. Intermediate filaments appeared to be differently affected by LND in the two cell types. Such changes were detected as an early phenomenon and the extent of the effects observed was positively related to the cell surface alterations and to the loss of cell viability, suggesting that the cytoskeletal elements might represent an additional target in the mechanisms of cytotoxic action of LND.


Cell transforming and oncogenic activity of 2,3,7,8--tetrachloro--and 2,3,7,8 tetrabromodibenzo-p-dioxin. We have developed a host-mediated assay system for detection of the transforming activity of chemical carcinogens on peritoneal macrophages, directly, as well as indirectly acting carcinogenic substances administered intraperitoneally to NMRI mice could be examined in this way. Resident macrophages were recovered by peritoneal lavage from treated and untreated mice and cultured in soft agar. After 5-6 days normal and transformed cells could be distinguished. Statistical analysis comparing cells from 2, 3, 7, 8-tetrachlorodibenzo-dioxin (TCDD)-treated animals with those from control mice proved that the test is positive at least on a significance level of 5%, using the t-test. TCDD revealed a cell-transforming potential that showed a dose-dependent response in this host-mediated assay. The co-carcinogenic activity of TCDD was established in experiments with diphenylhydantoin. Low doses of diphenylhydantoin which did not exhibit any transforming potential in our system gained a high oncogenic potential by the simultaneous administration of low doses of TCDD, which also had no transforming activity. We have compared the cell transforming potential of TCDD with its bromo analog TBrDD. The cell transforming potential of TCDD is 7 times that of TBrDD. We have succeeded in establishing a permanent cell lined from mice treated with TBrDD. The oncogenicity of this cell line was tested in athymic nu/nu mice. Animals treated subcutaneously with these cells (1 x 10(6) cells) developed tumors at the injection site. Using monospecific antibodies to tumor necrosis factor alpha (TNF-alpha), we have found that TCDD stimulates the secretion of TNF-alpha. The experimental data reported here lead to the conclusion that TCDD has a carcinogenic as well as a co-carcinogenic activity and has the property to induce TNF-alpha.


Effect of natural beta-interferon on estrogen receptor mRNA of breast cancer cells. We have demonstrated that natural beta-interferon (beta-IFN) enhances estrogen receptor (ER) mRNA of a human breast cancer cell line, CG-5. Cells were sensitive to the effect of beta-IFN at concentrations ranging from 10 to 100 IU/ml. The increase of ER mRNA was seen after 48 hr of treatment in at least three separate experiments. Our results are in agreement with the previously observed enhancement of receptor protein. In addition, they suggest that the IFN-induced promotion of the antiproliferative activity of drugs which act via ER may be due, in part, to increased receptor synthesis.


Transcriptional activation of the human immunodeficiency virus long terminal repeat sequences by tumor necrosis factor. The recombinant plasmid pBHIV-1 carrying the long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1), linked to the reported chloramphenicol acetyl transferase (CAT) gene, was introduced into human and rat fibroblasts. Stable transfectants were obtained which were resistant to genetecin and expressed CAT-activity from the HIV-1 LTR. The response to TNF alpha was studied. It was found that, at the optimum concentration of 100 IU/ml in human and 1000 IU/ml in rat fibroblasts, the expression of CAT was stimulated by 2.1 and 2.5-fold respectively. Our findings suggest that TNF-alpha in physiological concentrations can transcriptionally activate the HIV-1 LTR sequences and this may play an important role in the pathogenesis of HIV infection.


Growth inhibition of anaplastic glioma cells by nerve growth factor. Nerve growth factor (NGF) inhibited cellular DNA synthesis of rat T9 anaplastic glioma cells in a dose-dependent manner in the range of 0.5-5 micrograms/ml. Oxidation of 2 to 3 tryptophan residues of NGF, which had been known to destroy biological and immunological activity, greatly diminished its inhibitory effect on DNA synthesis. The inhibition was also abolished by anti-NGF IgG. Flow cytometric analyses and immunocytochemical assays of DNA synthesis using bromodeoxyuridine incorporation at various times during cell exposure to NGF revealed that the growth inhibition was attributable to gradual accumulation of growth-arrested cells at the G1 phase. Synthesis of nuclear regulatory proteins JUN and p53 was inhibited preferentially and progressively by NGF as inhibition of DNA synthesis increased.


Mode of EGF action on cell cycle kinetics in human breast cancer cell line MCF-7: some evidence that EGF acts as a "progression factor". EGF is known to play a very important role in the growth regulation of tumor cells. We have determined the effect of EGF in the absence and in the presence of serum on the cell cycle of MCF-7 cells synchronized in the G1 phase by serum deprivation. In the presence of 1% serum, EGF was found to increase DNA synthesis to 120% of control (P < 0.02), but did not modify the transition time from G1 into S phases, nor the cell doubling time during the first generation following the cell synchronization. The autoradiography analysis of 3H-thymidine labeled cells indicated that, following 24 h of EGF treatment, a constant additional number of cells (11 +/- 1.5%, P < 0.002) were recruited into the S phase in the presence as well as in the absence of serum. These data indicate that EGF exerts its mitogenic effect on MCF-7 cells by increasing the percent of S phase cells without modulating the cell doubling time. However, in the absence of serum a significant increase of thymidine incorporation in whole cells required 12 h of EGF treatment, whereas a 6 h-incubation with EGF was sufficient to stimulate DNA synthesis when synchronized cells were pretreated with serum for 6 h, suggesting that EGF sensitivity is dependent on the cell advance into the G1 phase at the moment of EGF addition. Topographical analysis of 3H-thymidine-labeled cells aimed at determining the spatial distribution of cells in culture revealed that EGF-stimulated cells were disposed near proliferative cells, indicating the local influence on cell proliferation. Taken together, our results suggest that in the MCF-7 cell line, EGF acts in the G1 phase by increasing the proportion of S cells without affecting the duration of the cell cycle. In our model, EGF seems to act as a "progression factor", in that it stimulates only cells already traversing a certain stage in the G1 phase under the action of serum factors, cell secreted diffusible products and cell-cell contact.


PSK (krestin) potentiates chemotherapeutic effects of tamoxifen on rat mammary carcinomas. A total of 20 mg of 7,12-dimethylbenz[a]anthracene (DMBA) was administered orally to 41 female Sprague-Dawley (SD) rats (control group), and 60 mg/kg of Krestin (PSK) were orally administered daily to 38 rats (PSK group) after DMBA administration. The average development period (9.6 weeks) of DMBA-induced tumors in the PSK group was significantly longer (P < 0.02) than that (7.9 weeks) in the control group. Average estrogen receptor (ER) levels of established tumors were almost the same between these two groups. However, the chemotherapeutic effect of tamoxifen (TAM) was significantly enhanced by PSK pretreatment.


Relationship between resonance energy and carcinogenic activity of benz[c]acridines. In order to investigate the correlation between the carcinogenic activity of benz[c]acridines and the electron density, the Huckel orbital method was used. Resonance energies, circuit resonance energies and bond currents of benz[c]acridines were calculated by Aihara's TRE theory. It was shown that these compounds had very stable aromatic characters with positive resonance energies and that the resonance energies per pi-electron values for the compounds with lack of the double bond corresponding to the K-region from the parent skeleton were more stable than those of their parent compounds. 7-Methyl-benz[c]acridine derivatives with the methyl groups at 7-, 7, 9-, 7, 10-, 7, 11-, 7, 9, 10-, or 7, 9, 11-positions except 5, 7-dimethylbenz[c]acridine had potent carcinogenic activity and significantly lower resonance energy per pi-electron.


Cytostatic effects of various alkyl phospholipid analogues on different cells in vitro. Phospholipid analogues were studied with regard to their cytostatic activity on different tumour cell lines and on murine bone marrow cells. Compounds compared for their activity were alkylglycero- and alkyl-phosphocholines with the corresponding serines and the alkylphosphocholines and -serines with the corresponding phosphono derivatives. Moreover, compounds containing cytidine 5'-diphosphate instead of the phospho (or phosphono-) choline or serine moiety were studied. rac-2-Chloro-2-deoxy-2-deoxy-1-0-hexadecyl-glycero-3-phosphocholine (cpd. Id), hexadecylphosphocholine (cpd. Ia) as well as hexadecylphosphonocholine (cpd. Ib) inhibited growth of tumour cells in suspension and monolayer culture and their colony and cluster formation in agar culture but not that of bone marrow cells. The exchange of choline for serine in these compounds results in the loss of this type of antitumour specificity. However, dodecylphospho-L-serine (cpd. IIc) is as specific as the choline derivatives Ia, b, d mentioned. Thus, for serine compounds the specificity for tumour cells might depend in a critical way on the length of the alkyl chain. The phosphono compounds Ib, IIb show almost the same activity as the corresponding compounds hexadecylphosphocholine (cpd. Ia) or hexadecylphosphoserine (cpd. IIa). The CDP-derivatives (IIIa, d, e, f) inhibited growth of tumour cells in suspension or monolayer cultures but not the colony and cluster formation in agar (i.e. they do not decrease the plating efficiency) from either tumour or bone marrow cells.


Receptors for estrogen and progesterone in breast carcinoma in situ. In contrast to the situation in invasive breast carcinoma, there are only few reports on the levels of receptors for estrogen (ER) and progesterone (PgR) in pre-invasive (in situ) breast carcinoma. In the present study ER and PgR levels were analyzed in 57 in situ human mammary carcinomas using a quantitative enzyme immunoassay method. Intraductal carcinoma was the dominating histopathological subgroup (75%). Within this subgroup the non-comedo and comedo variants comprised 66 and 34 percent respectively. Intralobular and papillary in situ subtypes were the second most frequent subgroups, representing 11% each. Using a cut-off level of 0.05 fmole/microgram DNA to define receptor poor and rich tumors we found that thirty-one out of the 57 cases (54%) were ER-rich. In the ER-rich tumors the receptor levels varied between 0.05 and 6.50 fmole/microgram with a mean concentration of 0.80 fmole/microgram DNA. Fifteen of 51 (30%) tumors were classified as PgR-rich tumors in which the levels ranged between 0.05 and 2.90 fmoles/microgram DNA, with a mean concentration of 0.45 fmoles/microgram DNA. Two thirds of the non-comedo, lobular and papillary variant were ER rich. A majority (80%) of comedo tumors were ER poor. Our findings show that 54% of in situ breast carcinomas have the molecular prerequisites for response to antiestrogen therapy which is of interest when planning adjuvant treatment protocols.


Transforming growth factor-beta is not the major soluble immunosuppressor in the microenvironments of human breast tumours. The immunosuppressive nature of the human breast cancer microenvironment was investigated. The soluble fraction of individual tumours was tested for its ability to influence the activation of peripheral blood lymphocytes (PBL) by interleukin-2 (IL-2) and to effect the continued proliferation of cells which had been pre-activated with IL-2. In all cases, both assays were profoundly inhibited (90-100%). None of this inhibition was due to cell killing by the tumour-derived soluble material (TDS). Sixty-two percent of TDS tested contained measurable transforming-growth factor-beta (TGF-beta) activity; following acidification TGF-beta was present in all TDS. However, in neither case was this material present in sufficient amounts to account for the degree of inhibition observed. In addition, neutralisation experiments failed to demonstrate consistent relief of inhibition in the presence of excess anti-TGF-beta antisera. These results demonstrate that TGF-beta s are not the major soluble immunosuppressive materials within human breast tumours.


Tumour levels of O6-alkylguanine-DNA-alkyltransferase and sensitivity to BCNU of human xenografts. Tumour levels of O6-alkylguanine-DNA-alkyltransferase (O6 AT) and glutathione content (GSH) were correlated with 1, 3-Bis (2-chloroethyl)-1-nitrosourea (BCNU) sensitivity in two human ovarian cancer xenografts (HOC8 and HOC18) and in two human glioblastoma xenografts (HG12 and HG15). HOC8 and HOC18, which were not responsive to BCNU treatment, showed O6 AT levels 14 and 23-fold higher than HG12 that was moderately sensitive to the same BCNU treatment. HG15, which was more sensitive to BCNU than HG12, showed significantly lower O6 AT levels. GSH levels were similar in all tumor xenografts. These data further stress the importance of O6 AT level as a relevant parameter for nitrosourea response in human tumours.


Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay. One of the important factors affecting the action of monoclonal antibodies (Mabs) or immunoconjugates on tumour sites depends on whether the Mab is internalized by the cancer cells in question. The underexplored subject of internalization is discussed in this paper, and a number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anti-carcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabeling assays, evidence for internalization of the anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min). Electronmicroscopy employing horseradish-peroxidase labeled anti-CEA Mab and control antibody permitted direct visualization of anti-CEA Mab-related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). Finally Western blots of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines provided evidence for internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320).


A simple fluorescent technique for screening cervical cells prior to nuclear analysis. Monolayer spreads of cervical cells were prepared and stained with haematoxylin and rhodamine-alpha-N-agmatine, a fluorescent marker for a cell surface protease. Mature epithelial cells from normal cervices lacked this cell surface enzyme and did not fluoresce. The abnormal cells possessed the cell surface enzyme, bound the probe and were quickly detected by fluorescence microscopy. The degree of abnormality of these fluorescent cells was determined by examination of their nuclear details, with the result that mild, moderate and severe dyskaryotic cells could be defined.


Differential stimulatory and inhibitory responses of human MCF-7 breast cancer cells to linoleic acid and conjugated linoleic acid in culture. Consumption of dietary fat has been linked to the high incidence of certain cancers. However, recent research has stimulated interest in conjugated linoleic acid (CLA), a newly recognized anticarcinogenic fatty acid. Human MCF-7 breast cancer cells were incubated for 12 d in culture medium supplemented with various concentrations (1.78-7.14 x 10(-5) M) of linoleic acid (LA) or CLA. Linoleic acid initially stimulated MCF-7 cell growth with an optimal effect at concentrations of 3.57-7.14 x 10(-5) M, but was inhibitory at similar concentrations after 8 and 12 d of incubation. In contrast, CLA was inhibitory to cancer cell growth at all concentrations and times tested. Cell growth inhibition by CLA was dose- and time-dependent. Growth retardation at the prescribed LA and CLA concentrations ranged, respectively, from 4 to 33% and 54 to 100% following 8 to 12 d of treatment. At similar LA and CLA concentrations, cytostatic and cytotoxic effects of CLA were more pronounced (8-81%) than LA. These in vitro results suggest that CLA is cytotoxic to MCF-7 cells.