Role of endotoxin in alterations of hepatic drug metabolism by diphtheria and tetanus toxoids and pertussis vaccine adsorbed. Administration of diphtheria and tetanus toxoids and pertussis vaccine adsorbed (DTP vaccine) or endotoxin (LPS) resulted in marked alterations in hepatic drug-metabolizing enzymes in endotoxin-responsive (R) and non-endotoxin-responsive (NR) mice. A single human dose (0.5 ml) of DTP vaccine increased hexobarbital-induced sleep times to 1.6- to 1.8-fold above those of controls in both strains of mice. This effect persisted for 7 days. In contrast, Bordetella pertussis LPS-treated mice showed an increase at 1 day (3.0-fold for R mice and 1.5-fold for NR mice), which returned to control levels by day 7. Furthermore, cytochrome P-450 levels were decreased 30 to 40% 24 h after DTP vaccine administration in both R and NR mice, while after LPS administration they were decreased 30% in R mice and less than 10% in NR mice. Both spleen and liver weights of R and NR mice were increased 7 to 14 days following DTP vaccine administration. However, LPS treatment had no apparent effect on liver weights, and spleen weights of R mice were elevated from days 3 to 7. Histopathologic tissue examination showed random, multifocal inflammation with hepatocyte necrosis after DTP vaccine administration to both R and NR mice and an absence of lesions in LPS-treated mice. Premixing LPS with polymyxin eliminated the increased sleep times, but premixing DTP vaccine with polymyxin did not affect the increased sleep times. Levels of tumor necrosis factor and interleukin-6 in plasma of R mice were markedly increased after DTP and LPS treatment, while NR mice had reduced increases. These results suggest that LPS contributes to the alterations in R and NR mice seen within the first 24 h of vaccine administration but that it is not likely to contribute to the effects observed at later time points.


Chelating agents inhibit activity and prevent expression of streptococcal glucan-binding lectins. Several of the cariogenic mutans streptococci produce cell wall-associated glucan-binding lectins (GBLs). The lectins bind alpha-1,6-linked glucans and have no affinity for other polysaccharides or anomeric linkages. When citrate or lactate was included in the growth medium, expression of the activities of the GBLs of Streptococcus cricetus and S. sobrinus was prevented. Furthermore, chelating agents, including citrate, lactate, EDTA, and acetylacetone, were able to reversibly inhibit glucan-induced aggregation of GBL+ streptococci. In addition, the chelating agents prevented sucrose-dependent streptococcal adhesion to glass surfaces and dispersed preformed adherent masses of the streptococci. Neither citrate nor other chelating agents modified the activities of glucosyltransferases. Expression of the lectin could only be achieved by the addition of manganous ion to the growth medium. Chloramphenicol and other metabolic inhibitors prevented synthesis of GBL in cells obtained from manganese-deficient medium and shifted to manganous ion-sufficient medium. The GBL may be a manganoprotein, the manganese of which may be perturbed, but not removed, by chelating agents. During synthesis of the GBL, manganous ion may be required in order for the protein to achieve an active conformation. Citrate or other chelating agents may have promise as anticaries agents.


Coxiella burnetii superoxide dismutase gene: cloning, sequencing, and expression in Escherichia coli. A superoxide dismutase (SOD) gene from the obligate intracellular bacterium Coxiella burnetii has been cloned, and its DNA sequence has been determined and expressed in Escherichia coli. The gene was identified on pSJR50, a pHC79-derived genomic clone, by using the polymerase chain reaction with degenerate oligonucleotide primers corresponding to conserved regions of known SODs. Sequences resembling conventional E. coli ribosomal and RNA polymerase-binding sites preceded the C. burnetii 579-bp SOD open reading frame. An E. coli SOD-deficient double mutant (sodA sodB) that carried pSJR50 had growth and survival responses similar to those of the wild type when the transformant was challenged with 0.05 mM paraquat and 5 mM hydrogen peroxide, respectively. These observations indicated that the C. burnetii gene was functionally expressed in E. coli. Staining of native polyacrylamide gels for SOD activity demonstrated that pSJR50 insert DNA codes for an SOD that comigrates with an SOD found in C. burnetii cell lysates. The enzyme was inactivated by 5 mM hydrogen peroxide, which is indicative of an iron-containing SOD. Additionally, the predicted amino acid sequence was significantly more homologous to known iron-containing SODs than to manganese-containing SODs. Isolation of the C. burnetii SOD gene may provide an opportunity to examine its role in the intracellular survival of this rickettsia.


Immune response to outer membrane antigens of Moraxella catarrhalis in children with otitis media. The systemic and local antibody responses to homologous strains of Moraxella catarrhalis were investigated in 14 children with otitis media. A total of 8 children (57%) demonstrated a rise in serum antibody of the immunoglobulin G (IgG) (5 of 14), IgM (5 of 14), or IgA (6 of 14) classes of immunoglobulin to outer membrane antigens. Local antibody consisted of IgG (100%), IgM (29%), and IgA (71%). The IgG and IgA specific antibody present in middle-ear effusions appeared to represent local production rather than passive diffusion from the systemic circulation. These data suggest that young children develop an antibody response to M. catarrhalis in the middle ear during otitis media but fail to develop systemic antibody in a uniform manner.


Expression of the eukaryotic Trypanosoma cruzi CRA gene in Yersinia enterocolitica and induction of an immune response against CRA in mice. The 70-kb plasmid pYV of Yersinia enterocolitica directs the secretion of a set of proteins, called Yops, that are produced during infection of humans and animals. Trypanosoma cruzi, the agent of American trypanosomiasis, synthesizes a cytoplasmic protein termed CRA that is considered to be T. cruzi specific. To produce CRA in Y. enterocolitica, we constructed a hybrid yopE-CRA gene that we integrated into plasmid pYV by homologous recombination. Recombinant Y. enterocolitica produced a chimeric Yop-CRA protein that was secreted in large amounts in the surrounding medium. This protein reacted with sera directed against either CRA or YopE. To test the ability of the recombinant strain to induce an immune response against CRA, we inoculated C57BL/6J mice by gastric intubation with live recombinant bacteria. A clear antibody response directed against CRA was detected in the mouse serum. The CRA-presenting Y. enterocolitica strain also carried a bioluminescence detection marker, which allowed us to monitor colonization of the intestinal lumen of infected mice. No significant differences were observed between the infectivity of the CRA antigen-producing and -nonproducing Y. enterocolitica strains, despite the fact that one of them no longer produced YopE.


Evidence that autologous idiotypic regulation of anti-arginine-glycine-aspartic acid autoantibodies may influence development and progression of syphilitic lesions in infected rabbits. The 83-kDa receptor protein of Treponema pallidum (TpN83) recognizes and binds fibronectin (Fn) at the amino acid sequence RGD site. By using experimentally infected animals, we have demonstrated that immunoglobulin G antibodies to this antigen and autoantibodies to the RGD site of Fn are putative components of immune complexes. This, and other findings, led us to initially hypothesize that anti-idiotypes (anti-Id) of an anti-TpN83 response are autoantibodies to RGD. Alternatively, we reasoned that if anti-Fn autoantibodies played a role in the pathogenesis of syphilis, then down-regulation of such a response by the Id network might directly affect progression of the disease. To test the hypothesis, rabbits were immunized with either affinity-purified TpN83 antigen or the synthetic Fn-7 peptide, KYGRGDS, and subsequently challenged with T. pallidum. Compared with results obtained with unimmunized, control rabbits, accelerated lesion development was noted in the rabbits immunized with TpN83. Pronounced, though unexpected, differences with respect to lesion development and progression were noted in the animals immunized with Fn-7 and then challenged intravenously; a minimal number of lesions appeared with a delayed onset. These lesions, like the localized chancres seen following intradermal challenge, were smaller and minimally ulcerated, and they healed rapidly. The Fn-7-immunized rabbits all differed from the controls in that anti-Id to anti-RGD F(ab')2 were demonstrable within 4 weeks following infection; decreases in anti-Fn autoantibody levels were associated with concomitant increases in anti-Id levels. Immunoglobulin Gs (anti-Id) from these animals following elution from anti-RGD F(ab')2 immunoaffinity columns also reacted with affinity-purified TpN83 antigen in immunoassays. These results suggest that down-regulation of autoreactive clones by manipulation of the idiotypic network in experimental syphilis warrants further investigation.


Iron acquisition and hemolysin production by Campylobacter jejuni. Campylobacter jejuni strains were tested for their ability to acquire iron from various iron sources present in humans. Hemin, hemoglobin, hemin-hemopexin, and hemoglobin-haptoglobin stimulated the growth of C. jejuni strains in low-iron medium. Transferrin, lactoferrin, and ferritin were unable to provide iron to the strains tested. Derivatives of the naturally transformable C. jejuni strain 81-176 were isolated on the basis of their inability to use hemin as an iron source. These mutants were also unable to use hemoglobin, hemin-hemopexin, or hemoglobin-haptoglobin as iron sources. Some mutants lacked a 71,000-Da iron-regulated outer membrane protein, while others appeared to retain all of their outer membrane proteins. Growth curves and a recombination experiment that exploited natural transformation were used to further characterize the mutants. A hemolytic activity was shown to be produced by several C. jejuni strains, but it did not appear to be iron regulated.


Influence of saliva on aggregation and adherence of Streptococcus gordonii HG 222. The influence of saliva on the aggregation and adherence of Streptococcus gordonii HG 222 was studied. The aggregation was measured spectrophotometrically, and the adherence of S. gordonii to microtiter plate wells was measured in an enzyme-linked immunosorbent assay system. The aggregation of HG 222 was induced primarily by mucous saliva, whereas the adherence of HG 222 to microtiter plates was mediated by both mucous and serous saliva. Fractions of submandibular saliva, obtained by gel filtration and containing low-molecular-weight mucins (MG-2), induced both bacterial aggregation and adherence. Purified MG-2 induced aggregation and promoted adherence, whereas high-molecular-weight mucins (MG-1) did not. After incubating clarified human whole saliva with HG 222, only MG-2, and not MG-1, was bound by the bacteria. Proline-rich proteins (PRPs) and proline-rich glycoprotein (PRG) promoted the adherence of HG 222. These proteins in solution bound to HG 222 but did not induce aggregation of the bacterial cells. PRPs and PRG in solution were not able to inhibit adherence to microtiter plate wells coated with the same components. Purified alpha-amylase hardly promoted adherence to microtiter plates but, in the soluble state, readily bound to HG 222. In conclusion, these results indicate that the aggregation of S. gordonii HG 222 is mediated primarily by MG-2. These mucins also promote adherence. Several other salivary components, such as PRPs and PRG, are also involved in the adherence of HG 222.


Toxicity and immunogenicity of Neisseria meningitidis lipopolysaccharide incorporated into liposomes. To obtain nontoxic and highly immunogenic lipopolysaccharide (LPS) for immunization, we incorporated Neisseria meningitidis LPS into liposomes. Native LPS and its salts were incorporated by the method of dehydration-rehydration of vesicles or prolonged cosonication. The most complete incorporation of LPS into liposomes and a decrease in toxicity were achieved by the method of dehydration-rehydration of vesicles. Three forms of LPS (H+ form, Mg2+ salt, and triethanolamine salt) showed different solubilities in water, the acidic form of LPS, with the most pronounced hydrophobic properties, being capable of practically complete association with liposomal membranes. An evaluation of the activity of liposomal LPS in vitro (by the Limulus amoebocyte test) and in vivo (by monitoring the pyrogenic reaction in rabbits) revealed a decrease in endotoxin activity of up to 1,000-fold. In addition, the pyrogenic activity of liposomal LPS was comparable to that of a meningococcal polysaccharide vaccine. Liposomes had a pronounced adjuvant effect on the immune response to LPS. Thus, the level of anti-LPS plaque-forming cells in the spleens of mice immunized with liposomal LPS was 1 order of magnitude higher and could be observed for a longer time (until day 21, i.e., the term of observation) than in mice immunized with free LPS. The same regularity was revealed in a study done with an enzyme-linked immunosorbent assay. This study also established that antibodies induced by immunization belonged to the immunoglobulin M and G classes, which are capable of prolonged circulation. Moreover, liposomal LPS induced a pronounced immune response in CBA/N mice (defective in B lymphocytes of the LyB-5+ subpopulation). The latter results indicate that the immunogenic action of liposomal LPS occurs at an early age.


Interleukin-2 receptors in experimental Chagas' disease. Mammals infected with the protozoan parasite Trypanosoma cruzi develop suppressed cellular and humoral immune responses. This immunosuppression has been correlated with reduced T-cell responses involving deficient interleukin-2 (IL-2) production and is apparently mediated primarily by suppressor macrophages. Various forms of immunosuppression in other systems have been associated with increased levels of soluble IL-2 receptors (sIL-2R), and in the present study levels of sIL-2R in the sera of T. cruzi-infected mice during the course of infection were examined in enzyme-linked immunosorbent assays. It was found that serum levels of sIL-2R were elevated only during the third week of acute infection, a time of intense immunosuppression. In addition, IL-2R on the surface of T cells were examined by flow cytometric analyses to determine whether there is an alteration in the number of IL-2R-positive cells and whether there is a change in expression of these receptors as infection progresses. The results revealed no significant change in the percentage of cells expressing IL-2R, nor did T cells become suppressed in their ability to express IL-2R in response to concanavalin A during the course of infection.


The integrin-binding domain of invasin is sufficient to allow bacterial entry into mammalian cells. Yersinia pseudotuberculosis is able to enter normally nonphagocytic host cells by multiple pathways, the most efficient of which is mediated by invasin, a 986-amino-acid bacterial outer membrane protein. It has previously been shown that the C-terminal 192 amino acids of invasin are sufficient to bind mammalian cells. To determine if additional regions of the invasin protein are necessary to promote entry, we developed a novel assay that tests the ability of various invasin derivatives to confer on Staphylococcus aureus the ability to enter animal cells. We determined that the 192-amino-acid cell-binding region of invasin, when used to coat the bacterial cell surface, was also sufficient to promote cellular penetration. These results suggest that the simple binding of invasin to its receptors is sufficient to mediate entry and that the bacterium plays a largely passive role in the entry process.


Effect of pyocyanine, a pigment of Pseudomonas aeruginosa, on production of reactive nitrogen intermediates by murine alveolar macrophages. In this study we investigated the effect of pyocyanine, a pigment produced by Pseudomonas aeruginosa, on production of reactive nitrogen intermediates by macrophages. We found that addition of pyocyanine to cultures of murine alveolar macrophages inhibited the capacity of these cells to produce reactive nitrogen intermediates (measured as nitrite) in a dose-dependent manner without altering cell viability, cytokine-induced Ia expression, or production of tumor necrosis factor.


Safety and immunogenicity of a booster dose of Vibrio cholerae CVD 103-HgR live oral cholera vaccine in Swiss adults. Adult volunteers received a booster dose (4 x 10(8) CFU) of attenuated Vibrio cholerae CVD 103-HgR oral vaccine 15 or 24 months after primary immunization. The immune response was modest, presumably due to rapid clearance of the vaccine strain by a primed immune system.


Identification of an acute-phase reactant in murine infections with Trypanosoma brucei. A 42-kDa protein appeared at a much higher concentration in plasma from Trypanosoma brucei-resistant (C57BL/6) mice after infection than in plasma from trypanosome-susceptible (C3H/He) mice. This protein was purified by sequential steps of gel filtration, protein A-Sepharose affinity chromatography, isoelectric focusing, and ammonium sulfate precipitation. The purified protein was identified as a subunit of the acute-phase reactant haptoglobin. Causes of elevated plasma haptoglobin and its implications for resistance to trypanosomiasis are discussed.


Results of the third immunology of leprosy/immunology of tuberculosis antimycobacterial monoclonal antibody workshop. An international workshop was sponsored by the World Health organization to screen new antimycobacterial monoclonal antibodies and to identify antibodies which could be recommended as standard reagents giving consistent results under differing assay conditions. Fifty-eight antibodies were submitted to the workshop by eight independent laboratories. Nineteen of the antibodies recognized antigens distinct from those identified in earlier workshops, defining at least 10 new protein antigens. Monoclonal antibodies characterized in the workshop provide a set of convenient reagents for further characterization of mycobacterial antigens.


Induction of circulating group II phospholipase A2 expression in adults with malaria. High levels of interleukin 1 and tumor necrosis factor are found in both cases of malaria and cases of septic shock. Since both interleukin 1 and tumor necrosis factor induce expression of the proinflammatory enzyme phospholipase A2 (PLA2), we examined serum PLA2 levels in 14 adults with malaria. Mean serum PLA2 activity was elevated 40-fold above normal (P less than 0.001). Serum PLA2 activity correlated with PLA2 immunoreactivity (r = 0.987; P less than 0.001) by an enzyme-linked immunosorbent assay specific for human group II PLA2, showing that serum PLA2 in cases of malaria is host derived. This article describes the novel finding of elevated PLA2 levels in cases of malaria, further strengthening the notion that mediators of the host response in cases of malaria are similar to those in cases of septic shock.


In vivo complement activation and binding of C3 to encapsulated Cryptococcus neoformans. Tissues from mice infected with Cryptococcus neoformans were examined by immunofluorescence to determine the extent of deposition of complement component C3 on encapsulated cryptococci. The relative percentages of cryptococci in each tissue having readily visible C3 were greatest for liver and lung tissues, with the kidney tissue having the next highest percentage and the spleen having the lowest percentage. Binding of C3 fragments to cryptococci in brain tissue was essentially absent.


Inefficiency of in vivo candidacidal mechanisms in experimental subcutaneous infections with Candida albicans in mice. A murine model of subcutaneous Candida albicans infections was used to evaluate host defenses against inocula of from 10(1) to 10(8) yeast cells. In these experiments, small inocula did not produce abscesses that drained to the skin surface, whereas larger ones did. Also, small numbers of organisms often remained at the infected sites for up to 21 days after inoculation with either small or large numbers of organisms. The data from these studies suggest that the in vivo candidacidal mechanisms in these infections are relatively inefficient and that they therefore may require some additional mechanism to control proliferation of the remaining organisms.


Phosphatidylserine found in intestinal mucus serves as a sole source of carbon and nitrogen for salmonellae and Escherichia coli. Salmonella choleraesuis (a pig pathogen), Salmonella typhimurium (a virulent strain in mice), and three strains of Escherichia coli (including a human enterohemorrhagic strain, a human urinary tract isolate, and a human fecal isolate) grew as well in vitro utilizing the lipids derived from mouse cecal mucus as the sole source of carbon and nitrogen as they did in mouse crude cecal mucus. Further analysis of the total lipid extracts of mucus dialysates showed that the acidic lipid fraction supported growth nearly as well as the total lipid fraction. Interestingly, among the many purified acidic lipids from mucus which were tested and analyzed, including several phospholipids, only phosphatidylserine was found to support the growth of all of these enteric bacteria, including Salmonella milwaukee, a human pathogen. The possible role of growth on pure phosphatidylserine in the pathogenesis of salmonellae is discussed.


A conjugated synthetic peptide corresponding to the C-terminal region of Clostridium perfringens type A enterotoxin elicits an enterotoxin-neutralizing antibody response in mice. A synthetic peptide homolog corresponding to the C-terminal 30 amino acids of Clostridium perfringens type A enterotoxin (CPE) was conjugated to a thyroglobulin carrier and used to immunize mice. Conjugate-immunized mice produced antibodies which neutralized native CPE cytotoxicity, at least in part, by blocking enterotoxin binding. This peptide may be useful for the development of a vaccine to protect against CPE-mediated disease.


Secondary caries.

Secondary caries. This paper reviews secondary caries which is the major reason for failure of restorations and therefore worthy of attention if operative dentistry is to be cost effective. The basic premise of the review is that the secondary caries process is difficult to diagnose and cannot be permanently treated away by operative management. Preventive therapy, dependent on the patients' self-care, is essential. However, the quality of the restorative material and the skill with which it is handled are also relevant to the prevention of further failure.


Types of resin-based inlay materials and their properties. The composition of 'chairside' resin-based inlay materials is similar to that of direct resin-based filling materials. The handling and curing procedures vary for the different inlay materials. Some systems involve precuring inside the oral cavity and a final cure at elevated temperature outside the oral cavity. Others are only cured extra-orally. The advantage of the inlay technique is that polymerisation shrinkage of the composite occurs before bonding to tooth structure. After cementation, therefore, the inlay gives good marginal adaptation in occlusal restorations. Some of the curing procedures for inlays give good conversion. The systems with good conversion also show better mechanical properties than the equivalent resin-based material cured according to the direct filling technique.


Luting agents for ceramic and polymeric inlays and onlays. The requirements for luting agents for ceramic and polymeric inlays and onlays are reviewed and discussed. The current overriding consideration seems to be the chemical adhesion of the restoration to tooth substance and this is challenged as being an overambitious goal. The systems in use are principally the dual cure resin based cements and these have some disadvantages such as restricted and slow cure, chemical degradation and, most importantly, shrinkage on setting. This shrinkage, when the material is used in conjunction with a dentine bonding agent, is almost certain to guarantee the rupture of any bond formed. It is suggested that the objectives of the luting agent should be rethought with reference back to the well established principles of cast metal restorations.


The WHO Oral Health goals for the year 2000 in South Africa. This paper outlines a policy to attain the WHO Oral Health goals for the year 2000 by the implementation of primary and secondary prevention. The Republic of South Africa (RSA) with its complex political and social formation and an interesting mix of 1st and 3rd world populations provides the space for challenging and innovative approaches.


Dental development in the midwest of America. During the early nineteenth century, dentistry was just beginning its existence as a profession and emerging from a trade into a profession in the United States. Dental schools were being established to replace preceptorships as a means of training and members of the profession became aware of the need to draw up laws for the regulation of the professions. The Midwest of the United States participated in these changes and many of the dental practitioners of the Midwest started the reforms that ultimately led to the establishment of dentistry as a profession. Some of these originators and their contributions are reviewed.


Protective gloves.

Protective gloves. The worldwide emergence of acquired immune deficiency syndrome (AIDS) has led to the wide use and acceptance of gloves as a measure of protection against infection in dental practice. Problems caused by the frequent use of gloves are already known from experiences in the surgical sector and have taken on a new urgency: these include impermeability, the difficulties of disinfection, and adverse skin reactions. In addition, there are specific dental questions such as the interference of gloves with electrical sensitivity tests and also with the taking of impressions. This paper discusses topical aspects of glove use and possible solutions to current problems.


Secretion of autocrine growth-promoting activity by renal-carcinoma cells treated with 5-fluorouracil. A study was made of the auto-proliferative activity of human renal-carcinoma cells in the supernatant from a carcinoma-cell culture in serum-free medium to which an anticancer agent had been added (5-FU). The human renal-cancer cells used in this study were of 3 strains: ACHN, VMRC-RCW and NT. When each line was cultured in medium containing no 5-FU, the supernatant showed almost no activity for stimulating DNA synthesis. However, when the line was cultured in the presence of 5-FU, the supernatant showed autocrine growth-promoting activity which strongly stimulated DNA synthesis of 3 renal-cancer cell lines in a dose-dependent manner. Activity could be detected in a 4- to 6-kDa fraction by gel filtration. This fraction increased the DNA-synthesis-promoting activity of epidermal growth factor, transforming growth factor-beta, basic fibroblast growth factor and insulin, and was acid- and heat-stable. It was also stable against pepsin and dithiothreitol. DNA synthesis in BALB/c 3T3, adult rat hepatocytes and rabbit renal tubular cells was not affected by this fraction which was thus considered not to affect non-cancerous cells. Renal-cell carcinoma responds poorly to anticancer agents, and the autocrine activity of the fraction may possibly be a factor accounting for this resistance.


Human carcinoembryonic antigen cDNA expressed in rat carcinoma cells can function as target antigen for tumor localization of antibodies in nude rats and as rejection antigen in syngeneic rats. We have tried to develop a new model consisting of rats transplanted with syngeneic colon carcinoma PROb cells transfected with cDNA coding for the carcinoembryonic antigen (CEA), the human tumor marker most commonly used as target for MAbs. The antigenic density of the 4 CEA-expressing clones selected for a precise characterization ranged from 5 x 10(4) to 1 x 10(6) CEA molecules per cell. In all clones the CEA was shown to be attached to the membrane by a phosphatidylinositol (PI) anchor. Using a panel of radiolabeled MAbs directed against the 5 major epitopes described on the CEA molecule, we showed that all these CEA epitopes were expressed by the 4 transfectants. Southern-blot analysis showed that the entire CEA cDNA was present in the transfectants. Western-blot analysis, however, showed that the size of the CEA expressed by the 4 transfectants was slightly smaller than that of CEA produced by 2 reference human colon-carcinoma cell lines. Two clones, expressing 1 x 10(5) and 1 x 10(6) CEA molecules per cell, respectively, were grafted s.c. in nude mice and rats. Injection of radiolabeled anti-CEA F(ab')2 fragments into these animals showed specific tumor localization with the highest percentages of injected doses for the transfectants expressing the highest CEA level. When grafted into immunocompetent syngeneic BDIX rats, the CEA-expressing clones induced a strong antibody response against CEA and tumor rejections in a majority of the animals. Although the analysis of the immune response against the CEA-cDNA-transfected carcinoma cells is under investigation, the present results demonstrate that human CEA could function as a rejection antigen when transfected into rat carcinoma cells.


Establishment and characterization of human uterine leiomyosarcoma heterotransplanted into nude mice. Human uterine leiomyosarcoma is a rare gynecological malignancy with a generally poor prognosis. We have established a human uterine leiomyosarcoma tumor line in nude mice, designated UTS-1, and describe the characteristics of this tumor. The UTS-1 tumor doubled in 12.1 days and retained the histological characteristics of leiomyosarcoma, even after 14 serial generations. Ultrastructurally, the tumor is characterized by nuclear pleomorphism typical of smooth muscle, intracytoplasmic filaments with dense bodies, a relative paucity of micropinocytotic vesicles, and an incomplete external lamina. Immunohistochemically, the UTS-1 cells reacted with antibodies against vimentin, desmin, smooth-muscle actin and myosin, but not with antibodies against keratin, CEA and S-100 protein. Serum levels of AFP, CA125, CEA and SCC ranged within normal limits in tumor-bearing mice. The serum level of immunosuppressive acidic protein correlated well with an activity of the tumor. Estrogen and progesterone receptors were not detected in the tumor. Chromosomal analysis showed a human karyotype with some marker chromosomes and a modal number of 85 chromosomes. The UTS-1 tumor should prove a useful model to explore the biological characteristics and treatment of human uterine leiomyosarcoma.


Expression of p53 protein in cutaneous melanoma. The purpose of the present study was to examine the expression of p53 protein in malignant melanomas of the nodular sub-type, with special reference to the role of p53 alterations in the development of metastases. Increased expression of p53 protein was found in 29 of 30 cases using PAb1801 monoclonal antibody (MAb) and flow-cytometric detection on archival material. A large proportion of the cells were positive in a majority of the cases. The level of expression was not correlated to DNA ploidy or to subsequent development of local metastases. However, a significant decrease in p53 protein expression was observed in metastatic lesions, as compared with the corresponding primary tumors. Our results indicate that p53 alterations may be an early event in melanoma development, since almost all cases were affected, and that metastatic spread appears to be parallelled by decreased p53 protein expression.


Effect of MHC class-I transfection on local tumor growth and metastasis in an H-2-negative clone derived from a chemically induced fibrosarcoma. GR9 is a chemically induced fibrosarcoma composed of clones with different H-2 class-I expression. These clones differ with respect to local growth and spontaneous metastasis. The B9 clone (H-2 negative) is highly tumorigenic (local growth) but of low metastatic potential (spontaneous metastasis assay). We have analyzed the effect that transfection of H-2Dd and H-2Kd genes on this clone have upon local growth, lung colonization after i.v. injection and ability to form spontaneous metastases. The results showed that the effect on local growth of transfection of the Kd-gene was stronger than that of the Dd gene. In addition, B9 co-transfected with H-2Kd and Dd genes showed the highest immunogenic properties in syngeneic BALB/c mice. Interestingly, the pSV2-neo transfected clone gave almost the same result as that obtained with Dd transfection. Lung colonization after i.v. injection of the different clones (experimental metastasis), paralleled the results obtained for local growth: the number of lung nodules followed the cadence KdDd less than Kd less than Dd less than pSV2. Survival of mice was always inversely correlated with local growth, e.g., all mice injected with 5 x 10(5) B9 H-2KdDd transfected cells survived. In contrast, no mice injected with the B9 control did. These differences were abrogated in irradiated and nude BALB/c mice. Finally, all transfected clones remained non-metastatic in a spontaneous metastasis assay, behaving as the control, non-transfected B9 cells.


Aspects of medical history and exocrine carcinoma of the pancreas: a population-based case-control study in The Netherlands. During 1984-88 a population-based case-control study was carried out in The Netherlands, in collaboration with the International Agency for Research on Cancer, to examine the possible relationship between aspects of medical history and exocrine pancreatic carcinoma in 176 cases and 487 controls. About 58% of patients were interviewed directly. We observed an inverse relationship between medical treatment for allergy-related conditions and the development of pancreatic cancer (30 cases vs. 130 controls, OR 0.57, 95% CI 0.36 to 0.90). A history of gallbladder problems, gallstones, cholecystectomy, stomach or duodenal ulcer, pancreatitis, appendicitis, diabetes or tonsillectomy was not related to risk. In direct responses, compared with once daily, a positive relationship was seen for stool frequency, 10 years ago, of less than once daily (18 cases vs. 40 controls, OR 2.10, 95% CI 1.09 to 4.04). In men, diabetes treated with insulin and diagnosed more than 1 year previously was significantly and positively related to risk (5 cases vs. 1 control, OR 11.66, 95% 1.28 to 105.95). In brief, the results of the present study suggest that a history of allergy-related conditions may protect, whereas a past stool frequency of less than once daily may enhance the risk of cancer of the pancreas. Other elements of the medical history were not consistently related to risk.


Anthropometric and reproductive variables and exocrine carcinoma of the pancreas: a population-based case-control study in The Netherlands. During 1984-88 a population-based case-control study was carried out in The Netherlands, in collaboration with the International Agency for Research on Cancer, to examine the possible relationship between aspects of medical history and exocrine pancreatic carcinoma in 176 cases and 487 controls. About 58% of patients were interviewed directly. In women, a significant, positive dose-response effect of height was seen (p-value trend less than 0.005). Compared with ages 14 or more, women with an early age at menarche, i.e., 11 years or less, had a 3-fold increase in risk (15 cases vs. 23 controls, OR 3.07, 95% CI 1.35 to 7.00). Other apsects of the reproductive history were not related to risk. In brief, the results of the present study support the hypothesis that, in women, early menarche and greater adult stature may be early predictors of the development of cancer of the pancreas later in life.


K-ras point mutations in human colorectal carcinomas: relation to aneuploidy and metastasis. Material from paraffin sections of 109 human colorectal carcinomas, mostly obtained at autopsy, was analyzed for the presence of K-ras point mutations at codon 12, position 2. Mutations at this position were found in 23 cases (21.1%). Aneuploid colorectal carcinomas showed a significantly higher prevalence of K-ras point mutations than diploid tumors, suggesting an involvement of ras mutations in the development of aneuploidy. No differences in the prevalence of K-ras mutations were observed with respect to the patients' age, sex and tumor type. In metastases, the type of ras gene mutation was always identical to that of the respective primary tumor. Mutations were not found in metastases from primary tumors devoid of ras mutations. This renders a clonal selection of K-ras mutated cells from a wild-type primary tumor during the metastatic process unlikely. However, nearly twice as many ras gene mutations were seen in metastatic than in non-metastatic primary tumors.


Immunoreactivity, pharmacokinetics and bone marrow dosimetry of intrathecal radioimmunoconjugates. Ten patients with neoplastic meningitis were treated with a variety of 131I-monoclonal antibody (MAb) conjugates, chosen to bind to their particular malignancy. Pharmacokinetic studies revealed that MAbs leave the ventricular compartment, enter the sub-arachnoid space and then pass into the blood. Once the MAbs enter the blood compartment, their clearance is determined by factors such as circulating anti-mouse Ig and circulating antigens. These lead to complex formations and the clearance of the conjugate by the reticuloendothelial system. In one individual, the anti-mouse Ig response observed systemically was not mirrored within the CSF, which has implications for planning future therapy of this type. In other patients, formation of immune complexes was due to binding to circulating antigen within the blood. The major toxicity associated with the intrathecal administration of 131I-MAbs was bone-marrow suppression. The doses to the bone marrow, resulting from the form of therapy, were calculated but showed no direct correlation with WHO grade 3/4 toxicity. Doses to the ventricular lining were also calculated, but due to the complex geometry of the compartment, calculation of potential tumour doses was not practicable.


Micronuclei in bronchial biopsy specimens from heavy smokers: characterization of an intermediate marker of lung carcinogenesis. Bronchial micronuclei, small fragments of extra-nuclear DNA formed during cell division, provide a non-specific but quantifiable marker of DNA damage. Micronuclei have been used to assess carcinogen exposure and as an intermediate endpoint in chemoprevention trials. As part of an ongoing chemoprevention trial, heavy smokers underwent screening bronchoscopy, with biopsies taken at 6 standardized sites. Micronuclei counts were obtained for each site in each of the 40 volunteers found to have squamous metaplasia. Unlike squamous metaplasia, the average micronuclei counts among these heavy smokers were not associated with smoking history. Micronuclei counts were also not associated with the presence or extent of metaplasia. A striking degree of intra-individual variability was observed by comparing the micronuclei counts from different biopsy sites within individuals. The findings suggest that use of micronuclei from single sites may be misleading as a marker of carcinogen exposure or as an estimate of cancer risk. Serial measurements in individuals may provide the most useful information concerning carcinogenic exposure and the impact of chemopreventive agents.


Breast cancer in a low-risk population. The influence of age and menstrual status on disease pattern and survival in Saudi Arabia. Data on 130 women with invasive breast cancer, seen at our institution between April 1981 and November 1990, were retrospectively reviewed to assess the influence of age and menstrual status on the pattern and prognosis of their disease. Patients were mostly young (median age 40 years) and in 21 patients (16%) the diagnosis was established at the age of 30 years or younger. Eighty-six patients (66%) were under 50 and 82 patients (63%) were pre-menopausal. Pre-menopausal patients were more numerous than expected in stage III and less numerous in stage II. On the other hand, the differences between observed and expected values for both stage I and stage IV in the 2 menopausal groups were not significant. Compared with post-menopausal patients, pre-menopausal patients with 1-3 or greater than 3 positive lymph nodes were more numerous than expected. Differences between pre-menopausal and post-menopausal patients have persisted after categorizing patients into 2 age-groups with a cut-off point at 50 years. Comparable initial assessment and therapeutic modalities were offered to the 2 menopausal groups. At the time of analysis (January, 1991) all patients had a complete follow-up. Over a median follow-up of 46 months, the overall median survival (+/- SE) was 85.7 (+/- 4.4) months with a survival probability (+/- SE) at 5 years of 62% (+/- 5%). The proportional hazard model of Cox has identified advanced stage (stages III and IV) and involvement of lymph nodes as the only independent adverse predictors of survival with estimated hazard rates of 2.9 and 2.8, respectively. Unadjusted analysis, adjusted analysis and stratified survival functions failed to reveal any survival difference based on age or menstrual status. We conclude that, in a low-risk population and despite apparent baseline differences in demographic and disease characteristics between pre-menopausal and post-menopausal breast cancer patients, neither age nor menopausal status had a significant influence on survival. Our results should guide future cancer-care programs in Saudi Arabia.


Distribution of autologous tumor-specific cytotoxic T lymphocytes in human metastatic melanoma. The study of specific immunity in human cancers has been hampered by the elusive distribution and heterogeneity of effector cells. In this study, we have investigated the distribution of autologous melanoma-specific cytotoxic T lymphocytes (CTLs) in 18 different distant metastases from melanomas (9 non-visceral and 9 visceral metastases). Uncultured cells from tumors were provided directly for the establishment of T-cell clones using limiting dilution analysis to avoid any possible effects of in vitro sensitization of T cells to coexisting tumor cells. Autologous tumor specific CTL clones were detected in 6 of 18 tumors (33%, 4 non-visceral and 2 visceral metastases). The majority of CTL clones (35 of 46 and 17 of 19) in 2 patients with HLA class-I A2 haplotype failed to lyse either A2+ or A2- allogeneic melanoma cells, although anti-class-I (monomorphic) MAb inhibited their cytotoxicity. The remaining 11 of 46 and 2 of 19 CTL clones showed A2-restricted cytotoxicity. Autologous tumor-specific cytotoxicity was also detected after polyclonal culture of these tumor-infiltrating lymphocytes (TILs) in 8 of 16 tumors (50%, 5 non-visceral and 3 visceral metastases). These results suggest that tumor-specific T cells exist at tumor sites in at least one-third of distant metastases of melanomas and could be induced by the addition of IL-2 in at least half of the tumors. Tumor-specific T cells were detectable more often in non-visceral than in visceral metastases.


Expression of histo-blood-group-A/B-gene-defined glycosyltransferases in normal and malignant epithelia: correlation with A/B-carbohydrate expression. Malignant transformation of oral and bladder epithelia is often associated with loss of histo-blood-group-A- and -B-carbohydrate antigens, whereas these antigens, which are absent in normal adult distal colon (but present in fetal colon) reappear in malignant distal colon. In order to gain insight into the genetic basis of the biosynthetic regulation for these changes, we have correlated the expression of the A- and B-carbohydrate antigens with that of the A/B-gene-defined glycosyltransferases in colon, bladder and oral carcinomas by immunohistology. A newly developed anti-A/B-transferase monoclonal antibody (MAb) was used to demonstrate the in situ localization of transferase expression at the individual cell level with correlation to carbohydrate antigen expression, and gave the essential information that the transferase is derived from the ABO gene complex. The reappearance of A- and B-carbohydrate antigens in carcinomas of the distal colon was found to be unrelated to the expression of the A/B-transferase proteins, which were expressed throughout normal adult colon in accordance with previous enzymatic studies. In contrast, the loss of A- and B-carbohydrate antigens in malignant bladder and oral epithelia was accompanied by concordant loss of enzymes.


Morphological, immunohistochemical and biochemical characterization of 6 newly established human ovarian carcinoma cell lines. Six permanent human tumor cell lines (OV-MZ-1 to 6) were established from 6 patients with serous adenocarcinomas of the ovary. These cell lines were derived from both solid tumors and ascites, from pre-treated and untreated patients, and are available over a range of in vitro passage numbers. The tumor cells grow as monolayers and develop foci of "piled-up' cells in confluent cultures. Flow cytophotometry showed that all the lines exhibited DNA hyperdiploidy with DNA tetraploidy in one cell line and DNA aneuploidy in the other cell lines. The mean population doubling time ranged from 24 to 52 hr. Transmission electron microscopy demonstrated that the tumor cells of all cell lines exhibited features of epithelial differentiation such as desmosomes and intracellular gland-like lumina. Immunocytochemical analysis showed that the co-expression of cytokeratins and vimentin, which is a feature of ovarian serous cystadenocarcinomas in situ, was fully preserved in the majority of cell lines. The main cytokeratin polypeptides expressed were numbers 7, 8, 17, 18 and 19. The tumor-associated antigen CA-125, but not CEA, was shed in the culture supernatant. This was in accordance with FACScan analysis of the cell lines and the level of CA-125 and CEA in the patients' serum. The estrogen and progesterone receptors were negative both in the cell lines and in the original tumors. These new ovarian carcinoma cell lines will be valuable models for further investigations into a variety of biological properties.


Orthotopic and ectopic organ environments differentially influence the sensitivity of murine colon carcinoma cells to doxorubicin and 5-fluorouracil. We determined the effects of organ environment on the response of murine CT-26 colon carcinoma cells to 2 structurally and pharmacologically distinct chemotherapeutic agents. CT-26 cells were injected i.v. (to produce lung lesions), s.c., into the cecal wall, and into the spleen (to produce spleen and liver lesions). Doxorubicin (DXR) at 10 mg/kg, 5-fluorouracil (5-FU) at 20 mg/kg, or saline (control) was injected intravenously on different schedules after tumor-cell implantation. The in vivo responses of the tumors growing in the cecum, spleen, liver, lung and subcutis were compared. Colon carcinomas growing in the subcutis were most sensitive to DXR. Tumors growing in the spleen and cecum were most sensitive to 5-FU and less so to DXR. Tumors in the liver were highly resistant to both drugs, whereas experimental lung metastases were sensitive to 5-FU but resistant to DXR. The differential responses of the tumors to the drugs were not due to drug distribution. The level of protein-kinase-C activity was elevated in the spleen, liver and cecum tumors as compared with s.c. tumors and correlated with the in vivo DXR resistance of the tumor cells. This correlation suggested that organ environment may modulate the chemosensitivity of tumor cells, at least in part, by perturbing signal transduction pathways. Collectively, the data indicate that the organ environment has profound effects on the response of tumor cells to chemotherapy. A molecular understanding of this phenomenon should facilitate the design of more effective systemic chemotherapy for cancer metastases.


Porphyria cutanea tarda and human immunodeficiency virus infection. Porphyria cutanea tarda (PCT), a relatively uncommon disease, has recently been reported in patients infected with the human immunodeficiency virus (HIV). Although PCT and HIV infection may co-exist by chance, the increasing number of reported cases suggest that HIV or an associated factor triggers the development of PCT in predisposed individuals. We report four additional cases of PCT in HIV seropositive patients and review the previously reported cases. The possible links between PCT and HIV are discussed. We believe the diagnosis of PCT should prompt investigation for HIV infection in all patients.


Confluent and reticulated papillomatosis: clinical, light and electron microscopic studies. A case of a 17-year-old Japanese-Canadian boy with a 2-year history of skin lesions relevant to confluent and reticulated papillomatosis (CRP) is reported with electron microscopic findings. He had scattered skin lesions of brownish-colored, rough-surfaced, fine-scaled patches and plaques distributed on the nape, shoulders, and both flanks, extending to the chest and back. Importantly, they showed reticular or confluent arrangement. There was no family history of the same skin lesions. The histology showed epidermal papillomatosis and mild acanthosis with a marked hyperkeratosis. Electron microscopically, the skin lesions consisted of the following abnormal findings: (1) a marked alteration of cornified cell structures showing snake coil-like, or triangle-like stacks, (2) a marked increase in the number of lamellar granules in the granular layer, (3) an increased number of melanosomes in the horny layers, and (4) no significant fine structural changes of epidermal melanocytes. This is the first electron microscopic report on CRP suggesting that the pathophysiology of this disease is related to abnormal keratinocyte differentiation.


Joint complaints in psoriasis patients.

Joint complaints in psoriasis patients. Four hundred and fifty-nine psoriasis patients seen at the Department of Dermatology, Bowman Gray School of Medicine of Wake Forest University, responded to a questionnaire concerning current or past joint complaints. Of these, 17% reported a previous diagnosis of psoriatic arthritis, and over 53% had a current or past history of arthralgias. Additional information on those patients reporting arthralgias focused on the cutaneous sites of involvement with psoriasis and which joints were most likely to be involved in patients with various types of arthritis. Previous treatments for skin involvement with psoriasis were more aggressive in those patients with a diagnosis of psoriatic arthritis. A significant proportion of patients presenting to dermatologists for treatment of psoriasis have joint complaints, and the percentage of patients with psoriatic arthritis is greater than generally appreciated by non-dermatologists.


Squamous cell carcinoma in southern and northern Australia. Squamous cell carcinoma (scc) of the skin was studied in two similar populations, one living in the temperate zone of Australia, the other living in the tropics. In the tropics, the patients were significantly younger, the man to woman ratio approached unity, and women had significantly more sccs on the legs. In the temperate zone, men had significantly more on the head and neck, but women had significantly more on the upper and lower limbs.


Dermatology in a war zone: a Persian Gulf experience. The clinical experience of two US Army dermatologists during the recent Gulf War (Operation Desert Shield/Storm) are presented with comparison with dermatologic experience in previous wars and in civilian practice.


Nonbullous bullous pemphigoid.

Nonbullous bullous pemphigoid. Three patients had a rare form of bullous pemphigoid, clinically similar to the erythematous type of bullous pemphigoid without vesiculobullous lesions. All the patients displayed immunofluorescence features of bullous pemphigoid. This form of the disease presents a substantial diagnostic problem because it lacks the principal morphologic feature of bullous pemphigoid, namely the vesicles and bullae.


Systemic ketoconazole treatment for Fusarium leg ulcers. Fusarium oxysporum was isolated from a large foot ulcer in an otherwise healthy 69-year-old man. Although tissue invasion could not be proven histologically, systemic antifungal treatment was administered with satisfactory response. Fusarium species are common soil-inhabiting organisms and plant pathogens. In humans, Fusarium is considered an opportunistic agent in skin ulcers, interdigital spaces, and burned skin, but can also cause mycotic keratitis, onychomycosis, and rarely deep-seated or disseminated infections, especially in an immunocompromised host. The distinction between skin infection and saprophytic growth, as well as optimal treatment regimens for the two types of infection, have not been clearly defined. We describe a case of leg ulcers caused by Fusarium oxysporum in a 69-year-old man treated successfully with oral ketoconazole. "Silent" immunologic disturbances were found in this apparently healthy patient. The case illustrates a relatively benign infection caused by Fusarium that responded to systemic antifungal drug treatment.


Alopecia universalis treated with oral cyclosporine A and prednisolone: immunologic studies. Alopecia universalis is a refractory condition. Although the cause of this disease is unknown, immunologic abnormalities have recently been suspected. Thus, we treated six cases of refractory alopecia universalis with immunotherapy. Oral administration of cyclosporine A (2.5 mg/kg) and prednisolone (5 mg/day) resulted in marked symptomatic improvement. Cyclosporine A did not produce any side effects because the administered dosage was relatively low. At present, more than 6 months after the cessation of treatment, recurrence of alopecia has not been seen. Oral administration of low-dose cyclosporine A and prednisolone is considered to be an effective treatment for this disease. Immunologic examination of peripheral blood demonstrated improvement of immunologic function. In particular, CD8-positive T cells, NK cells, and C3, which had been reduced, were increased. A reduction in active CD4 cells, eosinophils, and circulating immune complexes was observed. Histology with fluorescent antibodies showed T-cell infiltration around the hair matrixes. This phenomenon was no longer observed after treatment. These improvements in immunologic function were seen in parallel with the resolution of the clinical symptoms, indicating that immunologic abnormalities are related to this disease.


Severe acute form of adult dermatomyositis treated with cyclosporine. The classical treatment of severe forms of dermatomyositis includes high doses of steroids and/or cytotoxic agents. Acute forms are frequently life threatening. Because cyclosporine is a fast-acting immunosuppressive drug, it appears to be a good candidate for the treatment of refractory forms of acute dermatomyositis. We report a dramatic improvement of a severe, acute, steroid-resistant adult form after cyclosporine administration. A rapid clinical and biochemical improvement is reported, and the reversibility of immunologic abnormalities is emphasized.


A phase II study of crisnatol mesylate in patients with ovarian carcinoma. Fourteen patients with advanced ovarian cancer received a 72 hour infusion of a new DNA intercalator, crisnatol mesylate, administered intravenously. There was no evidence of antitumor efficacy. A syndrome of nausea and vomiting associated with vertigo, dizziness and ataxia was observed in nearly all patients. Two of the patients developed severe CNS toxicity manifested in one by a grand-mal seizure and in the other by peripheral neuropathy. Further explorations into the potential efficacy of crisnatol mesylate administered intraperitoneally are underway.


Phase II clinical and pharmacological study of didemnin B in patients with metastatic breast cancer. Sixteen evaluable patients with metastatic breast cancer were entered into a phase II trial of didemnin B. They received the drug at an initial dose of 5.6 mg/m2 every 21 to 28 days. Major toxicities noted were myalgia and nausea and vomiting while myelosuppression was mild. There were no complete responses; however, two minor responses were observed. The pharmacokinetics of didemnin B were studied in 10 patients who received the drug as 30 to 60 min i.v. infusions. A sensitive competitive inhibition enzyme immunoassay was used to quantitate didemnin B levels. Drug was observed to be rapidly cleared from plasma in a biphasic manner (t1/2 alpha = 0.12 hr, t1/2 beta = 4.8 hr). Although the assay could not identify the presence of specific metabolites, the increase of apparent didemnin B levels in plasma at later time points suggested the formation of unidentified metabolites which cross reacted with the antibody in the analytical procedure. In vitro experiments indicated that didemnin B was not bound to bovine serum albumin and only a minor portion (24%) of drug was found associated with red blood cells.


Phase II clinical study of pirarubicin in hormone resistant prostate cancer. Twenty one patients with hormone resistant prostate cancer were entered in a phase II study of pirarubicin 70 mg/m2, as a single intravenous injection given at 21 day intervals. All patients had leukopenia (9 severe or life threatening) and 2 died of septicemia. Thrombocytopenia occurred in 5 patients (one life threatening) and anemia in 12 patients. One partial response of 3 months duration was documented. Pirarubicin 70 mg/m2 given intravenously at 21 day intervals causes severe hematological toxicity and has minimal therapeutic activity in patients with hormone resistant prostate cancer.


Phase II evaluation of piroxantrone in renal cell carcinoma. A Southwest Oncology Group Study. The Southwest Oncology Group (SWOG) studied the response rate and toxicity of piroxantrone (150 mg/m2 q 21 days) in patients with advanced metastatic renal cell carcinoma. Among 32 eligible patients, there were no partial nor complete responses. There were two mixed responses. Significant white cell toxicity, anemia, nausea, and vomiting were observed. Mild or moderate degrees of fever, malaise, and stomatitis occurred. No significant cardiac toxicity was noted. Piroxantrone does not have significant activity as a single agent in advanced renal cell carcinoma.


Evaluation of in vitro drug screening leads using experimental models of human ovarian cancer. Five compounds which were identified as potential new anticancer drugs in in vitro screening with the human tumor colony forming assay were selected for further evaluation using in vitro and in vivo models of human ovarian cancer. Three of five compounds were found to inhibit in vitro colony formation of ovarian cancer cell lines derived from both untreated and combination chemotherapy refractory patients. One compound was also found to prolong survival in a human ovarian carcinoma xenograft model system. This compound, chloroquinoxaline sulfonamide, was selected for development and has shown preliminary indication of activity in phase I clinical testing.


Bis-daunomycin hydrazones: interactions with DNA. A series of bis-daunomycin hydrazones were synthesised from diester diamide linking groups derived from alpha,omega-dicarboxylic acids. All members of the series bis-intercalated into DNA, as evidenced by doubling of the lengthening of rod-like DNA compared to daunomycin, and by a 1000-5000 fold slower dissociation from DNA than daunomycin under detergent sequestration conditions. The bis-hydrazones exhibited neighbour exclusion, and occupied 6 bp under saturating conditions of drug. A unique DNA sequence specificity was apparent from transcriptional footprinting of 100 bp of DNA, with the greatest preference for 5'-CACA sites.


Phase II trial of 5 day continuous intravenous infusion of 6-thioguanine in patients with recurrent and metastatic squamous cell carcinoma of the head and neck. Fifteen patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck received a 5 day continuous I.V. infusion of 6-thioguanine repeated every five weeks. Dose limiting toxicity was primarily hematological with grade III/IV leucopenia and thrombocytopenia seen in seven patients. Nausea and vomiting was moderate and well controlled with antiemetics. No complete or partial responses were observed, with a median time to progression of 58 days and a median survival of 227+ days for the entire group. Based on these results we do not recommend I.V. 6-thioguanine for the treatment of this disease.


Phase II study of amonafide in advanced and recurrent sarcoma patients. The activity of amonafide, a benzisoquinoline-1,3-dione, was assessed in 15 patients with advanced or recurrent sarcoma (11 previously treated). Eligible patients had ECOG performance status 0-2, and acceptable renal, hepatic and bone marrow function. Amonafide 300 mg/m2 was given intravenously over one hour daily on five consecutive days, every 3 weeks. Leukopenia and granulocytopenia were the most common and severe toxicities (grade 3 or 4 toxicity in 20% and 27% of patients, respectively). Local irritation and nausea/vomiting, the most common nonhematologic toxicities, were generally mild. No objective responses were seen, though 2 patients had brief stabilization of disease. Amonafide at this dose and schedule has no activity against advanced, recurrent sarcoma.


Coat protein properties suggest that azuki bean mosaic virus, blackeye cowpea mosaic virus, peanut stripe virus, and three isolates from soybean are all strains of the same potyvirus. The interrelationship of a number of potyviruses infecting legumes has been investigated by comparing molecular properties of their coat proteins. Comparison of the coat proteins by the techniques of amino acid analysis and PAGE was inadequate to distinguish strains from distinct potyviruses. However, high-performance liquid chromatographic peptide profiles of tryptic digests of coat proteins of these legume-infecting potyviruses enabled such assignments to be made. These data indicate that amino acid sequences of coat proteins of azuki bean mosaic virus, the Type and W strains of blackeye cowpea mosaic virus, three isolates (74, PM, PN) of a potyvirus obtained from soybean in Taiwan, and the Blotch and Mild Mottle strains of peanut stripe virus (PStV) may be very similar to the known sequence of PStV Stripe coat protein. In contrast, peptide profiles of coat proteins from soybean mosaic virus, clover yellow vein virus, bean yellow mosaic virus, potato virus Y, and tobacco etch virus were dissimilar to each other and to the profile of PStV Stripe, suggesting that their coat protein sequences were also quite different. Based on observations of the coat protein structure of many potyviruses, the results suggest that the potyvirus isolates with similar coat proteins are strains of the same potyvirus.


Mapping and sequence analysis of the capsid protein gene of cowpea mottle virus. Twelve cDNA clones were generated, covering approximately 95% of the cowpea mottle virus (CMeV) genome from the 3' end to near the 5' end. The entire capsid protein sequence of 1,104 nucleotides was contained in two clones located near the 3' terminus. The codons represented 367 amino acids (M(r) 39,611). The postulated amino acid sequence of CMeV capsid protein had 36% homology to turnip crinkle virus and 26% homology to carnation mottle virus in the arm and S domains, but western blots showed no serological relationship to either. On the basis of the organization and expression of its genome and its physicochemical properties, CMeV is assigned to the carmovirus group. Like other carmoviruses, CMeV generates three dsRNAs which are co-terminal at the 3' end in infected tissues, but CMeV differs from other carmoviruses in the absence of encapsidated subgenomic RNAs.


A topological model for hepatitis B surface antigen. A model of hepatitis B surface antigen has been derived, based on extensive sequence analysis and biochemical data. The surface antigen sequences of the human, woodchuck, ground squirrel and duck hepadnaviruses were examined using hydrophobicity, hydrophobic moments, flexibility and secondary structure prediction. The helix phase diagram, which is a modified version of Eisenberg's hydrophobic moment plots and which specifically addresses the problem of transmembrane helices, was used to examine the predicted helices. In this model four transmembrane helices are predicted. The N and C termini and the second hydrophilic region, which bears the major B-cell antigenic determinants, are external. It is suggested that the transmembrane helices may pack to form a channel through the membrane and may also be involved in the mechanisms of cell entry. A significant difference between the duck hepadnavirus and the mammalian HBsAg sequences was found, hence care must be taken when extrapolating data between the duck and the human surface antigen.


Cytoplasmic localization of the DNA virus frog erythrocytic virus. In situ hybridization, using a biotinylated clone of frog erythrocytic virus (FEV), was conducted to determine the location of viral sequences in bullfrog erythrocytes. FEV-specific hybridization signals were found to correspond to mature cytoplasmic viral particles and assembly sites. These data are consistent with electron microscopic observations of viral assembly in the erythrocyte cytoplasm. Although FEV has morphological and biochemical properties similar to frog virus 3, our data suggest that the site of DNA replication and assembly of FEV is more similar to that of the poxviruses.


Transfusion therapy in elective total hip arthroplasty. A retrospective audit of transfusion practice in 150 consecutive elective primary total hip arthroplasties was undertaken, to examine blood usage and to determine the potential for reduction in its use. Predetermined criteria were used to measure unnecessary transfusions. Transfusion was considered unnecessary if the discharge haematocrit exceeded 36%, or if patients who lost less than 30% of their estimated blood volume, were transfused. Using these criteria, overtransfusion occurred in 42-45% patients. The intra-operative blood transfused correlated well with intra-operative blood lost. All other variables showed no significant correlation with both intra-operative and post-operative blood transfused. Females lost less and were transfused significantly more than males. The study corroborates previous reports of blood overuse. These results suggest that adopting standards of practice to measure and to monitor transfusion practice (quality assurance programmes) would be a worthwhile objective.


Intravenous nutritional support and the surgeon: where next? Over the past twenty five years the development of total parenteral nutrition has in many ways revolutionised the practice of surgery. It has enhanced survival in otherwise high mortality operations such as oesophageal surgery, especially with anastomotic complications. It has changed significantly the management of fistulae, either post operative or associated with diseases such as Crohn's enteritis. Here a basic general principle is applied--that a fistula will close if there is no distal obstruction and the throughput can be diminished. This can be achieved by withholding oral feeding and using the parenteral route. It has allowed survival in the short gut syndrome from whatever cause and it is interesting to see the degree of "intestinal adaptation" that occurs once the first critical year is survived with the help of intravenous nutrition. The assessment of nutritional status is difficult and while the level of serum albumin may be taken as a clinical standard, it is obvious that many patients survive extensive surgery with low albumin levels and also that there appears to be a lag period to the restoration of albumin levels, even with otherwise successful nutritional support and with other parameters being satisfactory. Even complex formulae using a combination of laboratory and antropometric parameters is not fully satisfactory as an absolute assessment of nutritional status. It is now interesting to see that nutrition can affect both immune competence and even carcinogenesis. The lipid element in intravenous nutrition may cause dysfunction of immunity and vitamin status, gastric and platelet function with impaired oxygen diffusion leading to increased wedge pressures.(ABSTRACT TRUNCATED AT 250 WORDS)


Factors affecting glycosaminoglycan concentration in normal human plasma. Acid Glycosaminoglycans (GAGs) were measured, in terms of hexuronic acid, following alkaline treatment and Ecteola chromatography, in plasma obtained from healthy volunteers, blood-donors, amateur soccer players and university students, and from hospitalized subjects at the end of their convalescence. Diurnal variations of plasma GAG concentration, with significant decrease during the morning, were obtained in students and patients, suggesting hormonal influences. Furthermore, moderate modifications of plasma GAG concentration were observed in students following cyclo ergometer exercise which were consistent in each subject with cortisol mediated changes. However, the absolute value of plasma GAG concentration appears to be depending on the physical training of the subject, being significantly higher in the soccer players and in the blood-donors than in the other groups of subjects, chiefly composed of sedentary individuals. The intramuscular connective tissue is then suggested to represent a main site of origin of plasma GAGs.


Fructose 1,6-bisphosphate-activated pyruvate kinase from E. coli: ligand promoted conformational changes. The ligand-dependent susceptibility to heat inactivation and to tryptic digestion and the intrinsic fluorescence of the fructose 1,6-bisphosphate-activated pyruvate kinase from Escherichia coli were investigated in the absence and in the presence of physiological ligands. With respect to the enzyme alone, binding of the allosteric activator fructose 1,6-bisphosphate makes the protein sensitive to tryptic attack and thermolabile, while binding of phosphoenolpyruvate and Mg2+, but not of either ligand separately, induces in the enzyme a highly thermostable conformation, the attainment of which does not require an ordered binding sequence of the two ligands. The apparent loosening of the enzyme structure induced by fructose bisphosphate suggests that the activation it exerts at low phosphoenolpyruvate concentration might be due to an increased accessibility of substrate to the active site.


Testing the null hypothesis in small area analysis. The goal of small area analysis is often to demonstrate that hospital admission rates or procedure rates vary greatly among regions, suggesting the occurrence of unnecessary admissions or procedures in some regions. Recent articles have shown that such variation may be largely due to chance, even if no underlying differences exist among the small areas; thus, it is important to test if the observed variation is larger than expected by chance. In this article we discuss how the appropriate method for testing the null hypothesis depends on the distribution of the number of admissions at the person level. If it is not possible for an individual to have more than one admission for a given procedure, the appropriate test is a simple chi-square test. If multiple admissions are possible, a modified chi-square test can be used to account for the excess variability due to multiple admissions. Failure to make the correct modification to the chi-square test in this latter case can result in spurious results. This underscores the importance of collecting data on multiple admissions in order to estimate the distribution of the number of admissions at the individual-patient level.


Inhibitory effect of bestatin on the growth of human lymphocytes. Bestatin at mid to high concentration had inhibitory effect to [3H]thymidine incorporation of human lymphocytes. It also decreased the PHA-P, Con A and PWM-induced mitogenicity of human lymphocytes. On the contrary, bestatin had growth stimulatory effect to murine lymphocytes, and enhanced the Con A, LPS-induced mitogenicity of murine lymphocytes. These growth inhibitory (human) and stimulatory (mouse) effect of bestatin was found to be independent of adherent cells (macrophage and dendritic cell), indicatory directly working to T or B lymphocytes.


A potential vaccine for cocaine abuse prophylaxis. Presently, there are estimated to be 1.5 to 2.0 million individuals infected with HIV-1 in the U.S. and about 12 million worldwide. In the U.S. over 90% of reported cases of AIDS occurred among two subgroups, homosexual males and intravenous substance abusers (IVSAs). Currently, there is no anti-cocaine addiction medication available. In order to explore vaccination as an alternative means for protection against cocaine addition, we immunized inbred male Fisher rats with either cocaine emulsification in complete Freund adjuvant (CFA) or with cocaine conjugated with keyhole limpet hemocyanin (KLH), as carrier plus CFA. Animals were initially immunized with 0.1 mg/animal of cocaine or cocaine-KLH. Animals were given a booster with the corresponding agents after 4 weeks. Ten animals/group were used. Controls received normal saline at the time of immunizations. These animals were injected, intraperitoneally, with 25 mg/kg of cocaine, and were examined for the analgesic effect of cocaine by the hot plate method. The average analgesic effect of cocaine was significantly reduced (p greater than 0.03) in animals immunized with cocaine-KLH (13.77 s) as compared to saline controls (21.6 s). Fifty percent of the animals (5/10) in the cocaine-KLH group and 33% of the animals (3/9) in the cocaine immunized group appeared completely resistant to the effect of cocaine on the central nervous system (CNS). We also have determined the levels of cocaine-specific antibodies produced by each animal by an ELISA method. Degree of protection from cocaine seems to correlate well with the amount of anti-cocaine antibodies produced by each animal (-0.61).(ABSTRACT TRUNCATED AT 250 WORDS)


Factors affecting interstate use of inpatient care by Medicare beneficiaries. This article examines the extent to which interstate inflow and outflow of patients affects their observed use of Medicare Part A inpatient care. Interstate patient flow can bias utilization rates and may be due to seasonal migration, interstate inpatient care market areas, or purposive seeking of specialized/high-quality care. Examination of state level patient flow data drawn from 1987 Medicare discharge indicate that most interstate patient flow occurs between adjacent states probably as an outgrowth of interstate markets. Regression analyses of patient flow data suggest that while seasonal migration is an important determinant of patient flow, its importance is secondary to that of indicators of the availability of specialized services. These findings suggest research questions that may be best answered in detailed analyses of inpatient utilization in interstate market areas and seasonal migration.


Augmentation of the radiation-induced antiproliferative effect in combined use of a derivative of nitrosourea, ACNU, with recombinant human tumor necrosis factor. The effect of combined use of a derivative of nitrosourea, ACNU, with recombinant human tumor necrosis factor (rhTNF) on the radiation-induced antiproliferative effect was examined using Meth A tumor cells. The radiation-induced antiproliferative effect was slightly augmented by the simultaneous addition of ACNU at 10 micrograms/ml in 5 Gy and 15 Gy irradiated groups. The antiproliferative effect was augmented in parallel with the radiation dose by the addition of rhTNF. Further augmentation of the proliferation inhibition was observed when both of ACNU (10 micrograms/ml) and 10 U/ml of rhTNF were added in combination with irradiation. Similar augmentation was observed when the target cells were treated with ACNU prior to irradiation and the addition of rhTNF.


Evaluation of the HCFA model for the analysis of mortality following hospitalization. From 1987 through 1990, the Health Care Financing Administration (HCFA) evaluated variations in the mortality rates experienced by patients admitted to hospitals participating in the Medicare program. This study was conducted to evaluate the adequacy of the model used for that purpose. Detailed clinical data were gathered on 42,773 patients admitted to 84 statistically selected hospitals. The effect of risk adjustment using the HCFA model, which is based on claims data, was compared to a risk-adjustment model based on physiologic and clinical data. Models that include claims data were markedly superior to those containing only demographic characteristics in predicting the probability of patient death, and the addition of clinical data resulted in further improvement. The correlation of ranks of hospitals based on a model that uses only the claims data and on one that uses, in addition, clinical data, was .91. As a screen for the identification of "high (mortality) outlier" hospitals, the claims model had moderate sensitivity (81 percent) and specificity (79 percent), a high negative predictive value (90 percent), and a low positive predictive value (64 percent) when compared to the clinical model. The two mortality models gave similar results when used to determine which structural characteristics of hospitals were related to mortality rates: hospitals with a higher proportion of registered nurses or board-certified physician specialists, or with a greater level of access to high-technology equipment had lower risk-adjusted mortality rates. These data suggest that the current claims-based risk-adjustment procedure may satisfactorily be used to characterize variations in mortality rates associated with hospitalization. The procedure could also be used as a basis for further epidemiological analyses of factors that affect the probability of patient death. However, it does not positively identify outlier hospitals as providers of problematic care.


Inhibition of DNA synthesis in peripheral blood mononuclear cells treated with phosphatidylserines containing unsaturated acyl chains. The immunosuppressive action of phosphatidylserine has been studied in mitogen-activated human peripheral blood mononuclear cells. The addition of phospholipid (10-60 nmol/10(6) cells) causes a dose-dependent inhibition of DNA synthesis induced by PHA, anti-CD3 mAb, allogeneic lymphocytes and tetradecanoylphorbol acetate plus ionomycin. In contrast, the interleukin-2-dependent DNA synthesis is less affected. Flow cytometric analysis and binding of radioiodinated interleukin-2 show that the phospholipid prevents the expression of interleukin-2 and transferrin receptors. Removal of monocytes by adherence does not change the action of phosphatidylserine. Furthermore, the phospholipid is equally effective in preparations depleted of CD4+ or CD8+ lymphocytes. Phosphatidylinositol partly reproduces the action of phosphatidylserine. Phosphatidic acid, phosphatidylglycerol and phosphatidylcholine are inactive. Also unsaturated phosphatidylserine analogues inhibit DNA synthesis whereas saturated phosphatidylserines do not. The data suggest that phosphatidylserine mainly affect the steps of T cell activation preceding the production of interleukin-2 and the expression of its receptor. The phosphorylserine headgroup and the unsaturated acyl chains contribute to this effect.


Determinants of need and unmet need among cancer patients residing at home. De-hospitalization of cancer treatment, particularly for those with advanced disease, can complicate adjustment and strain the capacity of caregiver networks to meet patients' daily needs. Outpatient staff should be able to recognize patients who need help to meet their daily needs as well as those who are not getting enough help. This study describes the physiological and social determinants of need and unmet need for assistance among 629 cancer patients with advanced disease initiating a course of outpatient chemotherapy and/or radiation therapy. Areas of needs examined through telephone interviews with participating patients were: personal care, instrumental tasks (housework, shopping, and cooking), and transportation. Physiological factors (metastases, disease stage, and functional status) were associated with need for assistance in all three areas. Also, older age (over 65) and low income predicted need for help with personal care, and women were more likely than men to report illness-related need for assistance with instrumental tasks and transportation. Unmet need was primarily associated with patients' social support system (e.g., children living nearby and perceived resiliency of network helpers). These findings highlight the need for outpatient staff to evaluate patients' informal care resources as well as patients' symptoms and impairments in deciding who should be referred for home care services.


Hospitalization style of physicians in Manitoba: the disturbing lack of logic in medical practice. Variations in hospital admission rates across small areas are ubiquitous, and it is increasingly assumed that high rates result from physicians' discretionary decisions. Data for elderly patients from the health insurance system of Manitoba were used to construct an index that divided physicians into four groups based on their propensity to admit patients to the hospital. I then determined whether physicians who are more prone to admit patients use hospitals for more discretionary purposes and admit patients who are less ill. Although the differences between physicians with different practice styles were in the expected direction, the most compelling finding was the similarity in characteristics of patients admitted by physicians with markedly different practice styles. Such findings suggest a very wide latitude in physicians' decisions to admit patients; this latitude is not well captured by a model that posits a logical relationship between physician treatment patterns and patient need.


Change in the Medicare case-mix index in the 1980s and the effect of the prospective payment system. Persistent increases in the Medicare case-mix index over the 1980s have been ascribed to changes both in medical treatment ("real changes") and in the way medical information is recorded ("coding changes") in hospitals. These changes have been attributed, in the absence of appropriate data and analyses, to the incentives of the Medicare prospective payment system (PPS). Using data for 1980-1986 from 235 hospitals, we estimate the effect on the Medicare case-mix index of a series of variables that reflect medical treatments and coding practices. Each of these underlying real or coding variables was changing prior to PPS and would likely have continued to change even in the absence of PPS. Furthermore, PPS may have had a distinct effect on these variables. These underlying trends and the PPS effects must each be estimated. Thus, the analysis begins by developing separate estimates for each of these real and coding variables (1) in the absence of PPS (autonomous effects) and (2) as a result of PPS (induced effects). Then, changes in the case-mix index are regressed against all of these variables to determine the degree to which specific autonomous real or coding variables or induced real or coding variables actually influenced measured case mix. Results show that real and coding changes each accounted for about half of the change in the Medicare case-mix index between 1980 and 1986, with the influence of coding starting to wane by 1986. PPS-induced factors explain about 80 percent of the change in measured case mix over time, autonomous factors about 20 percent. Especially powerful determinants of case-mix change included PPS-induced substitution of surgical for medical care and PPS-induced improvements in the accuracy of coding that led to assignment of patients to higher-weighted DRGs. Also, stringent Medicare peer review organizations appeared to restrain rises in case-mix indexes for their hospitals. Outpatient substitution for inpatient treatment, which others attributed to PPS, was well underway before PPS was announced.


Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction. The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)


Influence of the decrease of intracellular antigenic content on morphometric analysis: effect of the type and dilution of the first antibody. The aim of the study is to determine the effect degranulation of B cells on their immunohistochemical detection and to evaluate whether this effect depends on the technique of immunological detection. The biological model is the pancreatic insulin-containing B cell. To decrease the insulin content of the pancreas, insulin release was stimulated by five intraperitoneal injections of glibenclamide (2 mg/kg). Specimens of the pancreas were taken for insulin extraction and quantitation by radioimmunoassay (RIA), ultrastructural analyses and immunocytochemistry. The sections were treated either by polyclonal or monoclonal anti-insulin serum at various concentrations and peroxidase-antiperoxidase (PAP) technique eventually followed by silver amplification. The B-cell insular fraction or relative B-cell area (RBA) was measured with an automatic image analyser. The reference value for the relative B-cell area was established in control rats and corresponds to the ratio of the insular area occupied by immunostained B cells to the islet area. Its value was confirmed by calculation of the area occupied by non-B cells. RIA indicates a decrease of 83% of the insulin content in treated rats while the number of B granules decreases by 72% at the ultrastructural level. In usual conditions (polyclonal serum, 1/1500) the degranulation leads to a 16% underestimation of the B-cell insular fraction. This underestimation increases when monoclonal antibodies are used and further increases when higher dilutions are tested. The silver amplification does not prevent this underestimation and, in this particular model, exclusively acts by increasing the contrast. The only means of restoring the correct level of detection is to use the serum at a higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)


Sialoglycoderivatives of bovine submandibular gland identified in situ by histochemical techniques combined with lectins. We employed sialidase procedures followed by lectin stainings combined with oxidizing and deacetylating agents to visualize the distribution and sequentiate sialoglycoconjugates in the bovine submandibular gland. In particular we evidenced in acinar and ductal cells the dishomogeneous presence of sialic acids acetylated in the polyhydroxy side chain (C7, C8, C9), whereas O-acetyl substituents at position C1 and/or C4 were not found. Sialoglycoderivatives were also differentiated by the occurrence of penultimate sugars; indeed the dimers sialic acid-(alpha 2----3,6)-beta-galactose and sialic acid-(alpha 2----6)-alpha-N-acetylgalactosamine were identified. Using such technique we supported further the possibility to develop methods for the identification of the positions of O-acetyl groups and the reconstruction of terminal disaccharides within surface and cytoplasm glycoconjugates. in situ the distribution of different O-acylated sialoglyco-derivatives in the bovine submandibular gland. To this purpose we employed oxydizing and deacetylating agents combined with sialidase digestion and lectin binding.


Immunohistochemical co-localization of glycogen phosphorylase with the astroglial markers glial fibrillary acidic protein and S-100 protein in rat brain sections. Immunofluorescence double-labelling and immunoenzyme double-staining methods were used to examine the location of glycogen phosphorylase brain isozyme with the astrocyte markers glial fibrillary acidic protein (GFAP) and S-100 protein in formaldehyde-fixed, paraffin-embedded slices from adult rat brain. Astrocytes in the cerebellum and the hippocampus, which express GFAP or S-100 protein immunoreactivity, show glycogen phosphorylase immunoreactivity. Regional intensity and intracellular distribution of the three antigens vary characteristically. In ependymal cells, glycogen phosphorylase immunoreactivity is co-localized with S-100 protein immunoreactivity, but not with GFAP immunoreactivity. These findings confirm that glycogen phosphorylase in the rat brain is exclusively localized in astrocytes and ependymal cells. All astrocytes, as far as they express GFAP or S-100 protein, do contain glycogen phosphorylase.


LR Gold embedding of nervous tissue for immunoelectron microscopy studies. A major drawback of all acrylic resins commonly used for post-embedding immunocytochemical studies of the central nervous system is the disruption of the ultrastructural morphology, due to the high lipid content of neural tissue. We have investigated the suitability of the acrylic resin LR Gold, which has been employed recently for immunogold labeling studies in several non-neural tissues. Optimal preservation of both antigenicity and ultrastructure of nervous tissue was obtained after en bloc staining with uranyl acetate, followed by total dehydration in acetone and curing at low temperature. Cell membranes and myelin sheaths, which are usually lost with other acrylic resins, were well maintained. The degree of antigenicity of LR Gold-embedded tissues was comparable to that of LR White-embedded one, but the morphologic detail was much better preserved. The use of LR Gold is particularly advantageous for studying neurodegenerative disorders such as Alzheimer disease.


Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning. Analysis of the secretory organelle matrix of Paramecium trichocysts. Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at greater than or equal to 0 degree C, such as "progressive lowering of temperature" (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.


Ontogeny of glucagon messenger RNA in the rat pancreas. The synthesis of proglucagon mRNA was studied in rat pancreas from day 11 of fetal gestation (E11) to maturity. Proglucagon mRNA was first detected on E11, the time that the pancreatic bud forms in developing rats. The synthesis of proglucagon mRNA and its translation product at this early time point in pancreatic development suggests an early differentiation of A cell function. Between E17 prenatally and day 10-14 postnatally, pancreatic proglucagon mRNA abundance was higher than in adult pancreas. Regulation of the abundance of pancreatic proglucagon mRNA therefore appears to underlie the previously documented increases in serum and pancreatic glucagon immunoreactivity in the late fetal and perinatal periods. By day 20 postnatally, pancreatic proglucagon mRNA declined to levels found in adult pancreas. Prenatally between E17 and E21, changes in proglucagon mRNA abundance did not parallel previously reported developmental changes in relative mass of proglucagon-producing pancreatic A cells. This suggests that changes in proglucagon mRNA abundance during these times may be attributed to changes in proglucagon gene transcription or proglucagon mRNA stability per cell. In contrast between E21 and maturity, changes in proglucagon mRNA abundance paralleled previously reported changes in relative A cell mass, suggesting no major changes in proglucagon gene transcription or mRNA stability per cell during these times.


Cytochemical characterization of glycoproteins in the developing acrosome of rats. An ultrastructural study using lectin histochemistry, enzymes and chemical deglycosylation. The composition and distribution of rat acrosomal glycoproteins during spermiogenesis have been investigated at light and electron microscopic level by means of a variety of morphological techniques including the application of lectins conjugated to peroxidase, digoxigenin and colloidal gold, enzyme and chemical deglycosylation procedures and conventional histochemistry. Results obtained with lectin histochemistry in combination with beta-elimination reaction and endoglucosaminidase F/peptide N-glycosidase F digestion suggest that glycoproteins of mature acrosomes contain both N- and O-linked oligosaccharides. N-linked chains of acrosomal glycoproteins contain mannose and external residues of N-acetylglucosamine and galactose. They also have fucose residues linked to the core region of the oligosaccharide side chains. O-linked oligosaccharide chains contain external residues of both galactose and N-acetylgalactosamine. Mannose, fucose, galactose and N-acetylglucosamine residues were detected in acrosomes at all steps of spermiogenesis. N-acetylgalactosamine residues were only observed in the late steps of the spermiogenesis. N-acetylneuraminic acid residues were not detected throughout the acrosomal development. At initial stages of acrosome formation, glycoproteins were preferentially distributed over the acrosomic granules. In cap phase spermatids, lectin binding sites were homogeneously distributed throughout the acrosomes; however, in mature spermatozoa, glycoproteins were predominantly located over the outer acrosomal membrane.


[Acoustic rhinometry: the bat principle of the nose]. All cross-sectional areas of the upper airway can be measured by an acoustic signal using the acoustic reflection technique, or acoustic rhinometry. The plane of the cross-sectional areas measured was determined in nasal models. The isotemporal layers were found to be nearly parallel to the nasal valve. The acoustically measured cross-sectional areas correlated with the cross-sectional areas of cuts from nasal models. After digitizing these cuts, a CAD software calculates cross-sectional areas in all orientations and at all distances. The difference between the measured and calculated cross-sectional areas is up to 3% in the nasal cavity and up to 17% in the nasopharynx. The hypothesis that the cross-sectional areas measured lie nearly parallel to the nasal valve was confirmed. The normal rhinometric curve shows the minimal cross-sectional area (I-notch) to lie at the nasal isthmus. The second narrowest segment of the nasal cavity lies at the head of the inferior concha and septal concha (C-notch). Characteristic examples of patients with turbinate hypertrophy, choanal atresia, enlarged adenoids, and septal deviations are presented. Acoustic rhinometric curves can only be interpreted in combination with the rhinoscopic findings because different pathological conditions can produce similar curves. Recording of reliable and reproducible data by acoustic rhinometry demands that the connection between the rhinometer and the nose does not distort the valve area. When we used two different nose pieces (1.2 and 1.5 cm outer diameter) the cross-sectional areas in the anterior third of the nose of only 28% of the patients was measured correctly.(ABSTRACT TRUNCATED AT 250 WORDS)