Genetic approaches to understanding muscle development.

The analysis of both naturally occurring and experimentally induced mutants has greatly advanced our understanding of muscle development. Molecular biological techniques have led to the isolation of genes associated with inherited human diseases that affect muscle tissues. Analysis of the encoded proteins in conjunction with the mutant phenotypes can provide powerful insights into the function of the protein in normal muscle development. Systematic searches for muscle mutations have been made in experimental systems, most notably the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. In addition, known muscle protein genes from other organisms have been used to isolate homologs from genetically manipulatable organisms, allowing mutant analysis and the study of protein function in vivo. Mutations in transcription factor genes that affect mesoderm development have been isolated and genetic lesions affecting myofibril assembly have been identified. Genetic experiments inducing mutations and rescuing them by transgenic methods have uncovered functions of myofibrillar protein isoforms. Some isoforms perform muscle-specific functions, whereas others appear to be replaceable by alternative isoforms. Mutant analysis has also uncovered a relationship between proteins at the cell membrane and the assembly and alignment of the myofibrillar apparatus. We discuss examples of each of these genetic approaches as well as the developmental and evolutionary implications of the results.


Structural analysis of muscle development: transverse tubules, sarcoplasmic reticulum, and the triad.

Increased interest in the mechanism of excitation-contraction (E-C) coupling over the last few years has been accompanied by numerous investigations into the development of the underlying cellular structures. Areas of particular interest include: (1) the compartmentalization and specialization of an external and an internal membrane system, the T-tubules, and the sarcoplasmic reticulum, respectively; (2) interactions between the membrane proteins of both systems upon the formation of a junction, the triad; and (3) membrane-cytoskeletal interactions leading to the orderly arrangement of the triads with respect to the myofibrils. Structural studies using newly available specific molecular probes and a variety of in vivo and in vitro model systems have provided new insights into the cellular and molecular mechanisms involved in the development of the E-C coupling apparatus in skeletal muscle.


Myogenic cell lineages.

For many years the mechanisms by which skeletal muscles in higher vertebrates come to be composed of diverse fiber types distributed in distinctive patterns has interested cell and developmental biologists. The fiber composition of skeletal muscles varies from class to class and from muscle to muscle within the vertebrates. The developmental basis for these events is the subject of this review. Because an individual multinucleate vertebrate skeletal muscle fiber is formed by the fusion of many individual myoblasts, more attention, in recent times, has been directed toward the origins and differences among myoblasts, and more emphasis has been placed on the lineal relationship of myoblasts to fibers. This is a review of studies related to the concepts of myogenic cell lineage in higher vertebrate development with emphases on some of the most challenging problems of myogenesis including the embryonic origins of myogenic precursor cells, the mechanisms of fiber type diversity and patterning, the distinctions among myoblasts during myogenesis, and the current hypotheses of how a variety of factors, intrinsic and extrinsic to the myoblast, determine the definitive phenotype of a muscle fiber.


Epithelial-mesenchymal interactions in the outgrowth of limb buds and facial primordia in chick embryos.

The facial primordia in the chick embryo begin as rounded swellings that surround the primitive mouth and these grow out to form the beak. The control of proximodistal outgrowth is not well understood but may involve similar mechanisms to the limb bud. In order to test this hypothesis, combinations were made between epithelium and mesenchyme from facial primordia and limb buds. Signals from all three types of facial mesenchyme (frontonasal mass, mandibular, and maxillary) maintained the thickened apical ectodermal ridge of limb epithelium for up to 48 h. Combinations of tissues from the frontonasal mass mesenchyme and limb epithelium underwent substantial and correct morphogenesis. In contrast, poor development was observed in combinations with mandibular mesenchyme. Signals from frontonasal mass epithelium promoted outgrowth and morphogenesis of limb mesenchyme whereas mandibular and maxillary epithelium did not support joint morphogenesis. The results suggest that signals employed in the epithelial-mesenchymal interactions in facial primordia are similar but not identical to those signals used in the limb bud.


Effects of the steroid hormone, 20-hydroxyecdysone, on the growth of neurites by identified insect motoneurons in vitro.

During metamorphosis in the hawkmoth, Manduca sexta, identified larval leg motoneurons survive the degeneration of their larval targets to innervate new muscles of the adult legs. The dendrites and axon terminals of these motoneurons regress at the end of the larval stage and then regrow during adult development. Previous studies have implicated the insect steroid, 20-hydroxyecdysone (20-HE), in similar examples of dendritic reorganization during metamorphosis. The present studies were undertaken to test whether 20-HE acts directly on the leg motoneurons to regulate dendritic growth. Larval leg motoneurons were labeled with a fluorescent dye to permit their identification in culture following the dissociation of thoracic ganglia at later stages of development. Leg motoneurons isolated from early pupal stage animals (just before the normal onset of dendritic regrowth) survived in vitro and grew processes regardless of whether 20-HE was added to the culture medium. The extent of process outgrowth, however, as measured by the total length of all processes and the number of branches, was significantly greater for motoneurons maintained in the presence of 20-HE. The enhancement could be blocked by the addition of a juvenile hormone analog. By contrast, larval leg motoneurons that were isolated just before the normal period of dendritic regression did not show enhanced growth of neurites in the presence of 20-HE. The results suggest that 20-HE acts directly on the leg motoneurons to regulate the growth of processes during metamorphosis.


Interaction of Wnt and activin in dorsal mesoderm induction in Xenopus.

Both the activin and Wnt families of peptide growth factors are capable of inducing dorsal mesoderm in Xenopus embryos. Presumptive ventral ectoderm cells isolated from embryos injected with Xwnt8 mRNA were cultured in the presence of activin A to study the possible interactions between these two classes of signaling proteins. We find that overexpression of Xwnt8 RNA alters the response of ventral ectoderm to activin such that ventral explants differentiate dorsoanterior structures including notochord and eyes. This response is similar to the response of dorsal ectoderm to activin alone. When embryos are irradiated with uv light to inhibit dorsal axis formation, ectodermal explants differentiate notochord when they are induced by a combination of both signaling factors, but not when cells receive only one inducing signal (activin or Xwnt8). This result is further supported by the observation that goosecoid (gsc) mRNA, an early marker for dorsal mesoderm, is expressed in these explants only when they are injected with Xwnt8 mRNA followed by exposure to activin. Early morphogenetic movements of the induced cells and activation of muscle-specific actin and Brachyury (Xbra) genes also reveal a cooperation of activin A and Xwnt8 in mesoderm induction.


Sexually dimorphic expression of a laryngeal-specific, androgen-regulated myosin heavy chain gene during Xenopus laevis development.

Masculinization of the larynx in Xenopus laevis frogs is essential for the performance of male courtship song. During postmetamorphic (PM) development, the initially female-like phenotype of laryngeal muscle (slow and fast twitch fibers) is converted to the masculine form (entirely fast twitch) under the influence of androgenic steroids. To explore the molecular basis of androgen-directed masculinization, we have isolated cDNA clones encoding portions of a new Xenopus myosin heavy chain (MHC) gene. We have detected expression of this gene only in laryngeal muscle and specifically in males. All adult male laryngeal muscle fibers express the laryngeal myosin (LM). Adult female laryngeal muscle expresses LM only in some fibers. Expression of LM during PM development was examined using Northern blots and in situ hybridization. Males express higher levels of LM than females throughout PM development and attain adult levels by PM3. In females, LM expression peaks transiently at PM2. Treatment of juvenile female frogs with the androgen dihydrotestosterone masculinizes LM expression. Thus, LM appears to be a male-specific, testosterone-regulated MHC isoform in Xenopus laevis. The LM gene will permit analysis of androgen-directed sexual differentiation in this highly sexually dimorphic tissue.


Expression analysis of a Notch homologue in the mouse embryo.

The Drosophila Notch gene has been shown to be involved in the determination of fate in a number of different cell types. Similarly, Notch homologues in Caenorhabditis elegans are involved in cell decision-making steps. It is of interest to determine if a mammalian Notch homologue plays a role in cell fate determination. We have isolated cDNA from a mouse Notch gene using low-stringency hybridization with probes derived from the Xenopus Notch gene. Sequence analysis reveals that this gene possesses EGF repeats, Notch/lin-12 repeats, and CDC-10/SWI-6 repeats, characteristic of other Notch homologues. Northern analysis revealed that the transcript size was roughly 10 kb as has been found for the other Notch genes. We have studied the expression pattern of the gene by both conventional and whole mount in situ hybridization. Expression patterns were consistent with mouse Notch having a determinative role in the formation of mesoderm, somites, and the nervous system.


Colocalization of transforming growth factor-alpha and a functional epidermal growth factor receptor (EGFR) to the inner cell mass and preferential localization of the EGFR on the basolateral surface of the trophectoderm in the mouse blastocyst.

Results of previous studies suggested that responses of mouse blastocysts to TGF-alpha/EGF treatment are mediated by EGF receptors (EGFR) located on the apical surface of the trophectoderm (TE). We report here results of experiments using gold-labeled EGF that confirm the presence of these apically located EGFRs. In addition, immunoelectron microscopy (IEM) studies using anti-EGFR antibodies indicate that the receptor is preferentially distributed on the basolateral surface of the TE. Furthermore, the receptor is also present on the inner cell mass (ICM) and is likely to be functional, since treatment of isolated ICMs with TGF-alpha affects [35S]methionine uptake and incorporation into acid-insoluble material. IEM was also used to demonstrate that EGF, which is not synthesized by the mouse preimplantation embryo, is present in both the oviduct and the uterus. Maternally derived EGF is present in both ICM and TE cells in freshly isolated blastocysts, but is present in greatly reduced amounts following overnight culture of blastocysts in vitro. Last, IEM was also used to demonstrate that TGF-alpha is preferentially localized to the ICM and polar TE. The co-localization of TGF-alpha and functional EGFRs to the ICM and polar TE suggests potential autocrine, juxtacrine, and paracrine roles for TGF-alpha in blastocyst development.


Mechanisms of synaptic fatigue in a developing autonomic ganglion.

Synaptic transmission in developing systems has often been noted to exhibit depression or failure at moderate frequencies of stimulation. While this is often presumed to be a transient, nonspecific inability of developing systems to meet the demands of synaptic transmission, this report demonstrates that such failure in the choroidal neurons of the embryonic ciliary ganglion is due to muscarinically mediated inhibition. Although the ganglion is composed of both choroid and ciliary neurons, only the choroid neurons exhibit the muscarinic depression, and only during embryonic development. The pharmacological properties of the relevant receptor are different from those of the muscarinic receptor involved in presynaptic inhibition in adult autonomic systems. Receptor-mediated, synaptic failure during development may serve to protect immature postsynaptic neurons from potentially toxic overstimulation.


Programmed nuclear death: apoptotic-like degradation of specific nuclei in conjugating Tetrahymena.

During conjugation in the ciliated protozoan Tetrahymena, new macronuclei differentiate from germinal zygotic micronuclei while parental (old) macronuclei are eliminated in two stages, condensation or pycnosis coincident with cessation of transcription followed by resorption. We show that pycnosis is accompanied by degradation of old macronuclear DNA into oligonucleosome-sized fragments, a hallmark of programmed cell death, or apoptosis, in a variety of eukaryotic systems. As expected, oligonucleosome formation does not occur in the new micro- and macronuclei, confirming the coordination of different developmental fates for different nuclei in a common cytoplasm. NULLI 3 conjugants have wild-type old macronuclei but lack chromosome 3 germinally and hence in the new macronucleus. In NULLI 3 conjugants, old macronuclear pycnosis and oligonucleosome fragmentation occur normally but the resorption step fails, and the pycnotic old macronucleus is retained, demonstrating that the two steps are genetically separable and thus distinct and implying that genes on chromosome 3 in the new macronucleus are required for the resorption step. Comparison of whole cell polypeptides synthesized during stages of macronuclear development in both wild-type and NULLI 3 crosses reveal similar profiles. However, a polypeptide (apparent M(r) of 53 kDa) synthesized during old macronuclear elimination is not observed in NULLI 3 conjugants; its role, if any, in elimination of the old macronucleus is unknown. The results show that the old macronucleus is selectively destroyed by a mechanism which is remarkably similar to apoptosis in other eukaryotes and that the zygotic genome is required for the resorption step.


Localization of a chymotrypsin-like protease to the perivitelline space of Xenopus laevis eggs.

A chymotrypsin-like protease is released from Xenopus laevis eggs at activation and is involved in conversion of the vitelline envelope to the fertilization envelope. To localize this enzyme in unactivated and activated eggs, we used the synthetic peptide substrate succinylalanylalanylprolylphenylalanyl-4-methoxy-2-naphthylamide whose product can be visualized using transmission electron microscopy. Protease product was localized within the perivitelline space of unactivated eggs, appearing as strings of beads. No protease activity was detected in activated eggs, which is consistent with the observation that the protease is released from the egg at activation.


Popliteal lymph node assay as a tool to predict drug-induced GvH-like reactions.

The popliteal lymph node (PLN) assay was proposed to study a panel of drug-induced clinical manifestations, e.g. lupus syndrome, serum sickness-like disease, lymphadenopathy, scleroderma-like reaction, the mechanism of which was suggested as being similar to a graft-versus-host (GvH) reaction, hence the term GvH-like reaction. This assay can readily be performed in either the mouse or rat. The commonest endpoint is PLN weight, but cellularity and lymphocyte-phenotype analysis can also be determined. Experiments so far show that most drugs and chemicals known to induce such complications in man can be detected by the PLN assay and false-positive responses are extremely few. Interestingly, preliminary results indicate that cimetidine, an immuno-enhancing agent, can increase the response to phenytoin, a positive reference compound in the PLN assay. Thus, in addition to predicting those drugs with the capacity to induce GvH-like reactions and investigating the mechanism involved, the PLN assay may also prove useful in the pre-clinical assessment of immunomodulating agents.


Assessment of the anti-viral effect of a short-term oral treatment of mice with live Saccharomyces cerevisiae cells.

For assessing the efficacy of antiviral treatments, influenza and herpes virus HSV-1 infections of varying degrees of severity have been produced. The infections proved to be reproducible with respect to both their course and death rate. These infections also exhibited a course slow enough to permit the assessment of treatments under conditions mimicking human infections and lent themselves to the choice of the best adapted strategy to treat an infection. A short-term oral treatment with live cells of S. cerevisiae was efficacious in protecting mice against mild influenza infection and partly but significantly against severe infection. On the other hand, it did not afford significant protection towards either mild or severe HSV-1 infections, but it significantly potentiated the effectiveness of the antiviral drug vidarabin. S. cerevisiae treatment induced the synthesis of IFN alpha but not that of TNF alpha.


Protodyne: an immunostimulatory protein component, prepared from gram-positive Bacillus subtilis.

A protein component derived from bacterial protoplasm, called Protodyne, increases the non-specific resistance to infections by bacteria and viruses. Here we show that Protodyne can be prepared not only from Gram-negative bacteria, but also from Gram-positive bacilli. Several preparations of Protodyne, prepared from Bacillus subtilis by phenol extraction or by ammonium sulfate precipitation, were evaluated for immunomodulatory activities in a variety of assays. Protodyne had a marked mitogenic activity on mouse spleen cells; it was a potent inducer of tumor necrosis factor (TNF) and stimulated production of interleukin-1 (IL-1) in human peripheral blood mononuclear cells; it increased the capacity of activated macrophages to undergo a respiratory burst, to produce intracellular killing of leishmanial parasite and extracellular lysis of mastocytoma cells; it also stimulated phagocytosis of latex particles, and prolonged survival of immunosuppressed mice infected with Pseudomonas aeruginosa. These activities were not inhibited by polymyxin B, indicating that the activity of Protodyne is not the result of contamination with exogenous lipopolysaccharide. It appears that Protodyne exerts its many immunomodulatory actions by inducing the release of soluble mediators, including TNF and IL-1.


Standardized mouse infection models as a way of evaluating the potency of anti-infectious agents.

Standardized bacterial and viral mouse infection models have been developed. Infections with extracellular bacteria (K. pneumoniae, S. pneumoniae, S. pyogenes A) were produced by either of two routes: via the intravenous route (i.v.) resulting in septicaemia and the intranasal route (i.n.) giving infections confined to the respiratory apparatus. Infections with intracellular bacteria (L. monocytogenes, S. typhimurium) were produced only by the i.v. route. Two types of viral infection, mild and severe, were produced. Infection with influenza virus was by aerosol and herpes virus HSV-1 by the intraperitoneal route. All infection models produced under strictly controlled conditions were shown to be characterized by a remarkable reproducibility regarding both the pattern of development and death rate. The infection models lend themselves to estimation of the efficacy of a drug as well as the designing of new therapeutic strategies.


A sequential multi-assay protocol for the preclinical assessment of natural product complex carbohydrate immunomodulators.

A major barrier to the understanding, development and utilization of natural product complex carbohydrate immunomodulators has been the lack of standardization during pre-clinical efficacy and safety testing. In addition, it has been our experience that no single assay system or model is adequate for assessing preclinical efficacy and safety of these agents. To address these important issues, our laboratory group has developed a sequential multi-assay protocol for the preclinical evaluation of natural product complex carbohydrate immunomodulators. This sequential multi-assay screening protocol is divided into four phases: 1) physiochemical characterization of the carbohydrate polymer; 2) evaluation of immune stimulatory activity; 3) assessing in vivo anti-microbial activity and anti-tumor efficacy and 4) preclinical safety evaluation. This sequential protocol provides an effective, reproducible and rational approach to the preclinical assessment of complex carbohydrate immunomodulators that, in our experience, is predictive of clinical safety and efficacy.


Evaluation of clinical trials of immunomodulators for prevention of recurrent respiratory infections in children.

Despite numerous clinical studies showing "significant differences" in favour of immunomodulators versus placebo in the treatment and/or prevention of recurrent respiratory tract infections, the efficiency of such treatments is still not readily acknowledged by a large part of the medical profession nor by governmental authorities in several countries. In an effort to analyze the causes of this scepticism we performed a retrospective study of 15 clinical trials, using a series of quality assessment criteria. From the results of our analysis we propose a series of recommendations for improving the quality of clinical trials with immunomodulators. Furthermore, in order to avoid suspicion of a publication bias, we recommend setting up a registry of all ongoing clinical studies with immunomodulators.


Immunomodulators and primary prevention of respiratory infections: methodological considerations.

Immunomodulators have been evaluated mainly for their efficacy as a secondary preventive treatment to protect sick children from the recurrence of infections. We investigated the efficacy of one such product, IMOCURR, as a primary preventive treatment in children attending daycare, who face a very high risk of recurrent infections. EPICUR is a double-blind trial which was designed to study the efficacy of IMOCURR versus placebo. In order to have sufficient statistical power, data collection was based on a daily follow-up procedure and all important concomitant risk factors, such as age, environment and season, were controlled at the study design stage. The sample size required was calculated to be at least 400 children and a total of 423 were actually included. The results obtained for the total follow-up period and those obtained for the treatment period only were very different. This finding raises important questions which have methodological implications. This paper discusses these implications.


Prediction of pulmonary immunodeficiency in chronic bronchitis by cutaneous hypoergy to multitest.

Multitest (Pasteur Mérieux) is a plastic disposable device used to assess delayed cutaneous hypersensitivity (DCH) to seven antigens. Hypoergy to Multitest is believed to reflect a status of compromised cell-mediated immunity. Among the many possible pathogenic factors of chronic bronchitis, immunodepression has been little investigated. Indeed, whether lower immunodefence is the cause or the consequence of chronic bronchitis is still a subject of debate. The aim of the study was to evaluate DCH in a large group of patients with chronic obstructive pulmonary disease (COPD) and in a control group, in order to relate DCH (expressed as score, mm) to clinical and functional impairment. One hundred patients with COPD in stable conditions and not undergoing treatment with immunomodulating drugs (mean age, yr, M +/- SD: 66.1 +/- 6.5) and 40 normal controls in the same age range were admitted to the study. Evaluated parameters were: symptoms scores, physical examination, lung function tests and Multitest. Fifty-three percent of the COPD patients were hypoergic compared with 14% of controls. The Multitest score was 10 +/- 10 (mm M +/- SD) in COPD compared with 17 +/- 9 in controls. The Multitest score was significantly related to age (r = -0.45) in the control group, but not in COPD patients. DCH to only three of the seven antigens may predict a response to Multitest, with a sensitivity of 90%.(ABSTRACT TRUNCATED AT 250 WORDS)


Peripheral blood specific antibody-forming cells after oral stimulation with a ribosomal vaccine.

A double-blind study was performed in 12 healthy volunteers in order to determine whether circulating immunocytes are present after oral immunization. Sequential samples of peripheral blood were collected at various times after ingestion of a ribosome vaccine (D53) or placebo. Immunoglobulin-containing cells were identified in immunofluorescence and specific antibody-forming cells were detected in agarose ELISA-spot. Higher numbers of both types of cells were observed in the group of individuals receiving ribosomal extracts. An open study allowed a better approach to the kinetics of this phenomenon, related to the release of activated B-cells from Peyer's patches upon antigenic stimulation. This methodological approach has been described in animal models but seldom reported in humans.


Immunostimulant effects on granulocyte functions during an acute respiratory infection.

Treatment of healthy volunteers by Ribomunyl, whether or not in association with in vitro antibiotics (amoxicillin or josamycin), has been earlier demonstrated to be an efficient stimulant of PMN functions (adhesion and migration). This second double-blind study versus placebo was performed on 17 patients with acute respiratory infections (placebo group: eight subjects; Ribomunyl group: nine subjects). Ribomunyl treatment (four days) associated with antibiotherapy (amoxicillin) induced a significant increase of PMN adhesion (p = 0.014) with no significant effect of the placebo (p = 0.30) and no significant difference between the initial level of PMN adhesion of the two groups (placebo-Ribomunyl).


Recent progress in immunopharmacology and therapeutic effects of polysaccharides.

Lentinan, a (1----3)-beta-D-glucan with (1----6)-beta-D-glucopyranoside branches and its related polysaccharides have marked antitumour activity in allogeneic, syngeneic and autochthonous primary hosts, suppress chemical and viral oncogenesis, and prevent cancer recurrence or metastasis after surgery. Results of the clinical application of lentinan have proven prolongation of life-span of the patients with advanced and recurrent stomach, colorectal and breast cancer with only little toxic side effect. These polysaccharides also increase host resistance to various kinds of bacterial, viral and parasitic infections including AIDS. Lentinan appears to represent Host Defence Potentiators (HDPs), which can restore or augment the ability of responsiveness of the host to lympho-cytokines or other intrinsic bioactive factors through maturation, differentiation or proliferation of the important cells for host defence mechanisms. That is, HDPs might make the physiological constitution highly cancer and infection-resistant, which may be a concept in Oriental Medicine, the fundamental principle of which is to regulate homeostasis of the whole body and to bring the diseased person to his normal state. HDPs such as lentinan are the most appropriate drugs to prevent cancer recurrence, or the manifestation of AIDS symptoms in HIV carriers.


Long-term immunobiological effects of intravesical bacillus Calmette-Guérin against bladder carcinoma recurrences.

Intravesical bacillus Calmette-Guérin is a highly effective therapy against superficial bladder cancer recurrences and urothelial carcinoma in situ, the antitumoral effects of which seem to be linked to immunological effector mechanisms. To characterize further these mechanisms which contribute to the long-term effectiveness of BCG therapy, the local mononuclear cell subsets as well as the local cytokine production were assessed in the bladder wall of patients receiving intravesical BCG immunotherapy with special regard to long-term persisting changes, using immuno-enzymatical staining methods. All patients exhibited a significant increase after BCG in the local mononuclear cell population, with a predominance of the helper T-cell subset. Immunocompetent cells expressed activation markers for a prolonged period. Furthermore, a significant increase in the local cytokine production after the initial course of BCG was demonstrable, which persisted even after 12 to 21 months. These changes suggest that the BCG-induced activation of the local immune system persists over a long period after an initial course of intravesical therapy.


Standardized viability test required for preventive, immunomodulator and recombinant BCG vaccine.

The protective and immunostimulating effect of the living attenuated BCG vaccine cannot be expressed by bacterial mass (mg/dose). Colony forming units (CFU 10(6)/ml) and 95% confidence limit estimates as precise in vitro potency tests are required for the exact evaluation of the immunogenicity and reactogenicity of the BCG vaccine. The laboratory technique and computerized statistical analysis presented here ensure the precision and validity of the BCG viability test, permit the standardized BCG CFU 10(6)/ml unbiased point and confidence interval estimation for preventive and immunomodulator administration of the BCG vaccines and the evaluation of the genetic transformation frequency in recombinant BCG experiments.


Stability of BCG vaccine (intravesical) Theracys/BCG therapeutic ImmuCyst and its importance in clinical efficacy.

The manufacture of Therapeutic BCG can and should be a controlled process, to ensure adequate treatment doses are received by CIS patients. Standardization of production methods and CFU potency testing methods are essential to ensure continued successful treatment of CIS. Stability data can be used to validate assumptions about Therapeutic BCG potency used in clinical trials. Accelerated heat degradation studies of Therapeutic BCG should not be used for predictions of shelf life, but can be used for comparative purposes.


Evaluation of clinical data in bladder cancer immunotherapy with Connaught BCG (ImmuCyst).

Treatment of superficial bladder carcinoma in situ with Connaught BCG (ImmuCyst) significantly increased complete response rate and extended the disease-free interval when compared with Doxorubicin (DOX) chemotherapy. In 54 patients treated with ImmuCyst, 74% showed complete response compared with 42% of 60 patients treated with DOX. The median disease-free time was 48.2 months for BCG and 5.9 months for DOX treatment. Types and severity of adverse reactions were similar for both treatments and within tolerable ranges.


Oral administration of immunomodulators and the mucosal immune system.

Orally administered antigens reach the lymphoid tissue in Peyer's patches in the gut where they initiate an immune response with clonal expansion of antigen-specific T and B cells. Activated T cells migrate through lymph and blood to intestinal epithelium (intra-epithelial leukocytes) whereas activated B cells migrate to the lamina propria, other mucosae and exocrine glands where they differentiate into plasma cells secreting polymeric IgA1 or IgA2. These antibodies are transported across the epithelial cells after binding to a poly-Ig receptor, then excreted in the lumen as secretory IgA. Reciprocal interactions have been demonstrated between lymphoid and epithelial cells in the mucosae. Oral administration of antigens in different experimental models may induce the production of secretory antibodies and/or systemic unresponsiveness with suppression of delayed-type hypersensitivity or specific IgG and IgE antibody production or both. New strategies are currently being explored for the development of oral vaccines using recombinant antigens or viral vectors (e.g. pox-viruses, vaccinia virus, cholera toxin B subunit etc.). Conversely, immunomodulating compounds or procedures which could enhance specific oral tolerance in association with antigen would have considerable therapeutic applications in auto-immune diseases and allergy.


Synthetic lipopeptide immunomodulators derived from bacterial lipoprotein: tools for the standardization of in vitro assays.

For the evaluation of immunomodulators by in vitro assays, agents working reproducibly are difficult to obtain; conventional preparations of bacterial immunomodulators tend to vary for different preparations. Here we suggest that synthetic lipopeptide analogues derived from bacterial lipoprotein can be used as standards for various in vitro assays: studying B lymphocyte activation, lipopeptides act as potent mitogens and polyclonal activators inducing immunoglobulin synthesis. In monocytes/macrophages, lipopeptide stimulate the secretion of IL-1, IL-6, tumor necrosis factor (TNF) and nitrogen oxide (NO); they also induce tumor cytotoxicity. Lipopeptides also constitute potent immuno-adjuvants in vitro and in vivo, either in combination with or covalently bound to antigen. These activities are displayed in various species; in mice they are found in LPS non-responder and responder strains. The novel synthetic lipopeptides described here can be synthesized readily in gram amounts with high purity and reproducibility; they are non-toxic and can be stored for a long time even at room temperature. Thus, lipopeptides meet the requirements to serve as effective standards for a multitude of relevant biological assays.


IMOCUR stimulates production of immunoglobulins; preliminary results concerning a correlation between in vitro and in vivo experiments.

The bacterial extract IMOCUR is described as an in vivo stimulant of antibody production during animal testing and human clinical trials. Using a slightly modified procedure (13) dealing with in vitro immunoglobulin production by C57B1/6 mouse spleen cells, we have shown that IMOCUR potentiates spontaneous IgM production. In order to explore the putative relation between this in vitro activity and the current in vivo control test (stimulation of plaque-forming cell production after sheep red blood cell injection to Balb/c mouse), we have assayed 10 lyophilisates in vitro and in vivo before and after heat inactivation (80 degrees C, 7 days in a saturated water atmosphere). Results have shown that this treatment inhibits, respectively, totally and partially in vivo and in vitro activities. Thus the in vitro technique seems to be appropriate for the control of activity of the various batches of IMOCUR. Experiments are under way to clarify the mathematical correlation which may exist between the in vitro and in vivo experiments.


C-fos and c-myc modulation, mitogenic effect and Ia expression in the P388D1 murine macrophage line treated by immunomodifiers.

The aim of the present study was to investigate whether the early modulation of the c-fos and c-myc oncogenes could give some orientation to the impact of immunomodulators on the monocyte-macrophage lineage. In order to work in a homogeneous system we used the P388D1 mouse macrophage cell-line which is considered as an almost mature macrophage. When P388D1 cells were stimulated by LPS, interferon-gamma or the association of both compounds, no direct correlation could be found between the modulation of DNA synthesis and the early expression of the c-fos and c-myc oncogenes. The positive regulation of Ia antigen expression seemed to correlate with the absence of induction of c-fos oncogene.


Immunotoxicology of immunomodulators.

Immunotoxicology is the study of the immune system as a target organ for the toxicity of drugs and chemicals. As immunotoxicologists focused most of their interest on immunosuppression, the toxic consequences of immunomodulation are less clearly established. However, a number of immunopharmacologically-mediated side-effects including influenza-like symptoms, exacerbation of underlying diseases, e.g. autoimmune diseases, increase in hypersensitivity reactions against unrelated antigens and inhibition of hepatic drug metabolism have been described and may be used as a basis for further safety evaluation. Current pre-clinical assessment of immunotoxicity is usually performed in the rat during sub-chronic testing. The relevance of these protocols as far as immunomodulators are concerned is unknown. Careful attention should be paid to safety pharmacology testing, e.g. cytokine release, effects on animal models of auto-immune and hypersensitivity reactions, and hepatic drug metabolism. The role of post-marketing drug surveillance should also be emphasized. Advances in our understanding of the mechanism(s) of immunomodulation together with the development of more active immunomodulators are expected to improve the current pre-clinical safety assessment of these compounds.


18q- chromosomal abnormality in a phenotypically normal 2 1/2-year-old male with autism.

The authors report the case of a 2 1/2-year-old male with autism, with an 18q- chromosomal abnormality in the absence of phenotypical features of the 18q- syndrome. It is suggested that clinicians evaluating children with autistic disorders consider obtaining chromosome analysis, even in the absence of phenotypical abnormalities.


Peripheral and central mechanisms of hindfoot equinus in childhood hemiplegia.

A detailed clinical examination of 24 hemiplegic children at a mean age of 9.9 years showed that nine had an obligate toe-strike when walking at a self-selected speed and this was maintained throughout stance. None of the 24 children had a fixed equinus. No association could be found between this pattern of gait equinus and lower-limb atrophy, reduced ankle-joint range, muscle extensibilite, power of dorsiflexors and plantarflexors or actual muscle imbalance at the ankle joint. Gait equinus was independent of reduced compliance of the calf muscles, of a clinical diagnosis of tonic spasticity, of fine-motor dexterity of the toes, and of the side of the hemiplegia. Gait equinus cannot be explained merely in terms of a central paralytic foot-drop. A developmental model of equinus is advanced.


Calf-tendon jerks are altered in congenital equinovarus deformity.

Stretch reflexes of posterior tibial (PT) and triceps surae (TS) muscles were studied electrophysiologically in 27 children with unoperated unilateral congenital equinovarus deformity, in an attempt to evaluate changes in tone of the leg muscles as a possible pathogenetic factor. Significantly reduced latency of the PT responses on the affected side was recorded in a majority of patients. In most patients the amplitudes of the PT responses were asymmetrical; however, lateralization was inconsistent. The TS responses were asymmetrical in a smaller proportion of the patients, though still significantly different from the controls. The findings suggest an asymmetrical increase in excitability level of the PT motoneuron pool on the affected side.


The offspring of people with cerebral palsy.

To test a hypothesis that a placental deficit as a possible cause of CP might be transmitted, 88 people with congenital CP were contacted by questionnaire and letters. They had had 122 children, 93 per cent of whom were reported to be normal. The frequency of miscarriages and toxaemia was normal. 5 per cent of the mothers had had emergency caesarean sections. Three mothers also had minor malformations. The mean gestational age of the children was 38.8 weeks and their mean birthweight was 3295g. The mean birthweight of the mothers born preterm was 984g and that of their babies was 3244g. Two of the 122 children had diplegia, four had malformations, one had trisomy 18 and there was one stillbirth.


Care-load for children and young adults with severe cerebral palsy.

Care-load was analysed for 44 children and young adults (mean age 18.9 years) with severe spastic quadriplegia. All were enrolled in a residential school/hospital and used wheelchairs. The majority were in the moderate to borderline range of mental retardation. They were monitored for 24 hours a day over a seven-day period, and were timed in 10 major activities, including basic care (bathing, toileting, dressing, grooming, feeding), as well as sleep, leisure, education/vocation, counseling/psychosocial therapy, medical/nursing care, transportation, and at home. The findings replicated those of an earlier Swedish study, which suggests that the basic care needs of individuals with severe cerebral palsy may be predictable, regardless of the type of care being provided.


Technical aids for daily activities: a regional survey of 204 disabled children.

Home-care of even severely disabled children is strongly favoured today. However, the role of technical aids for daily activities has not been emphasised in the assessment of the needs of families with disabled children. This study evaluated the extent of help needed for daily living among 204 disabled children and the uses and role of and need for technical aids for eating, dressing, toileting and bathing. The children needed much help. Severely disabled children and their parents benefited most from the use of aids. The structured interview unexpectedly revealed many problems, including a considerable need for extra aids for dressing, bathing and toileting. The parents were ready to accept technical aids, but needed more information on their possibilities, benefits and therapeutic aspects. The authors conclude that the need for technical aids for daily activities is not readily recognised during outpatient or hospital visits.


Goldenhar syndrome and autistic behaviour.

Two girls with concomitant Goldenhar syndrome (oculo-auriculovertebral spectrum disorder) and autistic disorder are described. One was diagnosed as having Goldenhar syndrome in the first few weeks of life and as having autistic disorder in her fifth year; the other was diagnosed as having Goldenhar syndrome when she was referred for evaluation of autistic symptoms at seven years of age. The type of physical abnormalities encountered in Goldenhar syndrome suggests damage to neural structures in the second or late stages of the first trimester. The two cases described in this report suggest that autistic disorder sometimes can result from neural damage during the second trimester.


Is a fluoroscopic verification of the electrode position necessary in ambulatory intragastric pH monitoring?

To assess whether a fluoroscopic verification of the electrode position is necessary before and whether electrode displacement occurs during intragastric pH measurements, a crossover study was performed in 20 healthy male volunteers. The pH electrode was initially placed in the gastric corpus using pH readings and catheter length only, and the electrode tip was fluoroscopically located before and after the 24-hour study. Only in one study arm was an adjustment of the electrode position allowed, if fluoroscopy showed a position outside the gastric body. Thirty-seven (92%) of all 40 electrodes were in the corpus when placed by pH-metric methods alone. With fluoroscopic guidance, 2 of the 3 electrodes lying outside the corpus could be repositioned as desired. The median night, day or 24-hour gastric pHs measured in the groups with or without replacement of the electrode tip were identical. Displacement after 24-hour measurements did not occur with initially correctly positioned electrodes. Fluoroscopy is not necessary for the verification of the position of electrodes placed by pH-metric techniques in individuals with residual acid secretion, and electrode displacement is rare.


Functional and structural changes in the jejunum of the rat following cysteamine and stress-induced duodenal ulcer.

The effects of cysteamine and stress-induced duodenal ulcer on the functional and structural properties of the rat jejunum were investigated. The absorptive capacity of the jejunum was determined using alanine as the permeant solute and the single-pass perfusion technique. A statistically significant decrease (p < 0.01) in alanine absorption was observed after 8 h and 3 days of duodenal ulcer induction by stress and cysteamine respectively. However, alanine transport measured 7 days after cysteamine or stress ulcer induction showed no significant change from control values. Cysteamine and stress-induced duodenal ulcer did not show any significant change in water absorption across the jejunum when measured after 8 h, 3 and 7 days of ulcer induction. Microscopically, the jejunum of rats with 3-day cysteamine-induced ulcer exhibited diffuse type of apical derangements with excessive swelling of the villi and progressive degenerative changes. No such changes were noticed on the 7th day nor in the jejunum of the rats with stress-induced duodenal ulcer. The results suggest that cysteamine-induced duodenal ulcer produces an inhibition in the absorptive capacity of the jejunum which is time-dependent and reversible.


Continuous monitoring of sodium ion concentration in the human stomach--a new technique for the detection of duodenogastric reflux.

Measurement of sodium ion concentration in gastric juice offers a simple means of detecting duodenogastric reflux (DGR). Using aspirated gastric juice, we measured sodium ion concentration, bile acid levels and phospholipase A2 activity and found that sodium ion levels increase along with these other standard markers during DGR. Following this, using a sodium ion selective electrode and a portable data logger, continuous recordings of sodium ion concentration were made in the human stomach. These studies demonstrated the ability of sodium ion monitoring to detect major DGR episodes in the body of the stomach.


Cellular immune responses of peripheral blood mononuclear cells to HBV antigens during chronic and acute HBV infection.

Patients with acute self-limited and chronic HBV infection were studied to determine their in vitro cellular immune response to HBV antigens. In interferon-gamma production cultures which were evaluated as an indicator of cellular immune response, peripheral blood mononuclear cells from patients with HBeAg-positive chronic hepatitis showed elevated response to HBcAg, while those from HBeAg-positive asymptomatic carriers revealed no response to either HBcAg or HBeAg. HBeAg-stimulated interferon-gamma production was higher in anti-HBe-positive chronic hepatitis and asymptomatic carriers than that in HBeAg-positive patients. Interferon-gamma production against HBcAg was shown to be HLA class II restricted by blocking assay using monoclonal antibodies. HBsAg with or without pre-S2 did not amplify interferon-gamma production of patients with chronic hepatitis. In acute hepatitis B, both envelope and nucleocapsid antigens induced greater interferon-gamma production than in chronic HBV carriers. These results indicate that the patients with acute hepatitis responded more to HBV antigens compared with chronic HBV carriers. Furthermore, enhanced cellular immune responses, particularly to HBeAg, were observed in anti-HBe-positive patients compared to HBeAg-positive patients during chronic HBV infection, suggesting that the poor response to HBeAg in HBeAg-positive patients may account for the failure to clear HBV.


Low incidence of ulcerative colitis and proctitis in Bangladeshi migrants in Britain.

To assess the incidence of ulcerative colitis and proctitis in a defined migrant population, a retrospective, epidemiological community study was performed in the London Borough of Tower Hamlets from 1972 to 1989. The population of 164,000 includes 28,000 Bangladeshis. Potential cases were identified from hospital departments of pathology and medical records. There were 107 cases of ulcerative colitis in Europeans and 5 in Bangladeshis. There were 74 and 2 cases of proctitis in these communities, respectively. The mean standardised incidence of ulcerative colitis in Bangladeshis (1.8 cases/10(5)/year) was marginally lower than in Europeans (6.2 cases/10(5)/year, Z = 0.7, n.s.). The mean standardised incidence of proctitis in Bangladeshis was 0.6 cases/10(5)/year and in Europeans 3.2 cases/10(5)/year (Z = 0.6 n.s.). Anatomical extent of colitis was similar in all ethnic communities, although complications were less likely in minority groups. These findings suggest that the incidence of ulcerative colitis and proctitis in Bangladeshis, in Britain, is amongst the lowest in the world.


Fat and gastric acid secretion.

To evaluate the importance of the terminal carboxyl group of the oleic acid molecule in the inhibition of gastric acid secretion, 6 normal persons were stimulated twice with duodenal perfusates containing either 20 mM oleic acid or 20 mM oleyl alcohol. Oleic acid significantly inhibited the gastric acid secretion stimulated by pentagastrin (100 ng/kg/h) and increased the levels of secretin in plasma. The effect of oleyl alcohol was insignificant. It is concluded that the carboxyl group of the fat molecule has an important role in the inhibition of gastric acid secretion, and the effect could in part be attributable to the release of secretin into plasma.


Effects of ethanol and wine on hepatic arterial and portal venous flows in conscious dogs.

The effects of ethanol and wine on hepatic arterial and portal venous flows were examined in conscious dogs. Ethanol was given intravenously or intragastrically, and red wine (ethanol: 14%) was given intragastrically over 30 min. Intravenous ethanol (0.8 g/kg) and intragastric ethanol (14% vol/vol) increased hepatic arterial flow, which remained elevated for 60 min after the cessation of ethanol administration. Ethanol also increased portal venous flow. Portal venous flow returned gradually toward basal levels after the cessation of intravenous ethanol infusion, whereas it remained elevated even after the cessation of intragastric ethanol. Intragastric wine increased hepatic arterial and portal venous flows. In contrast to intragastric ethanol, hepatic arterial flow continued to rise after the cessation of intragastric wine infusion, while portal venous flow returned toward basal levels. We conclude that, though both ethanol and wine increase hepatic blood flow, the responses of hepatic arterial and portal venous flows differ substantially among intravenous ethanol, intragastric ethanol and intragastric wine.


Sonographic imaging of extracorporeal shock wave effects in the liver and gallbladder of dogs.

During extracorporeal shock wave lithotripsy, changes in tissue echogenicity are observed by ultrasound. Their significance is not known. An experiment was performed in which 3,000 extracorporeal shock waves were applied under sonographic observation to the gallbladder wall of 6 dogs. No stones had been implanted, but transient shadows appeared simulating jumping stone fragments in the bladder. Echoes within the bladder lumen occurred in 3 dogs and were associated with hemorrhage into the lumen; in an additional ex vivo experiment, shock waves generated echoes in bile only after injection of a small amount of blood. In the liver, a transient increase in echogenicity was noted after a few shocks; it coincided with the region of tissue damage. Intense focal echoes occurred in the liver of 2 dogs at sites where a hematoma was found at autopsy. It is concluded that an increased focal echogenicity is an indicator of tissue damage by shock waves. The sonographic changes are thought to be caused by the transient generation of gas bubbles. The interaction of shock waves with gas bubbles is an established powerful mechanism which could explain the generation of tissue damage by shock waves.


Increased luminal release of hyaluronan in uninvolved jejunum in active Crohn's disease but not in inactive disease or in relatives.

Recently obtained data suggest that there is a subclinic inflammatory activity in the apparently uninvolved intestinal mucosa in Crohn's disease (CD). As CD is characterized by an activation of connective tissue and fibrosis, we investigated the extent to which hyaluronan (HA), an essential component of the connective tissue, was released into the lumen of an isolated jejunal segment in CD patients and in relatives. Patients with active CD of the terminal ileum (CD activity index, CDAI, > 150; n = 14), patients with CD in remission (CDAI < 150 n = 10), first-degree relatives of the CD patients (n = 21) and healthy controls (n = 43) were orally intubated with a catheter allowing occlusion and perfusion of a segment of the proximal jejunum. The jejunal fluid concentration of HA was 65 +/- 45 micrograms/l in patients with active CD in the terminal ileum, significantly higher than the value for 43 healthy controls (42 +/- 23 micrograms/l; p < 0.05), and the corresponding values for patients in remission (42 +/- 23 micrograms/l) and for first-degree relatives of the CD patients (53 +/- 52 micrograms/l), were not increased compared to the control group. To localize HA in the tissue, small bowel biopsies were taken during surgery from patients with CD and from controls and affinity stained for HA. There was an intense staining for HA in the lamina propria of the villi, both in biopsies from patients with CD and from controls, but no staining in the epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)


Severe exacerbation of chronic active hepatitis B during interferon alpha therapy.

The case of a 52-year-old male with chronic active type B hepatitis in whom severe exacerbation of liver disease was associated with interferon alpha treatment is described. It seems that patients with very active forms of chronic hepatitis B may experience severe and symptomatic exacerbation of liver disease during interferon treatment.


The location and expression of fibroblast growth factor (FGF) in F9 visceral and parietal embryonic cells after retinoic acid-induced differentiation.

It is well-established that fibroblast growth factors (FGFs) participate in mesoderm formation and patterning in the developing embryo. To identify cells in mammalian embryos that produce and/or respond to FGFs, we utilized the F9 teratocarcinoma cell system. Undifferentiated F9 cells resemble inner cell mass (ICM) cells of the mouse blastocyst by several criteria including having a characteristic high nuclear to cytoplasmic ratio and by their expression of stage-specific embryonic antigens. F9 stem cells differ from ICM cells by their low spontaneous rate of differentiation and their differentiation potential. ICM cells are heterogeneous with a proportion of the cells maintaining totipotency. In contrast, F9 stem cells appear capable of forming only endodermal derivatives. Retinoic acid (RA) treatment of F9 stem cells is required for them to differentiate, and under different culturing conditions the F9 cells will form either extraembryonic parietal or visceral endoderm. We have previously shown that FGF is synthesized by F9 parietal endoderm, but not by F9 stem cells. Our present study demonstrates that F9 aggregate cultures that contain visceral endoderm cells produce cell-associated-heparin-binding mitogens for 3T3 and endothelial cells, factors with characteristics of FGFs. Furthermore, our studies detect endothelial cell-mitogens within the extracellular matrix (ECM) of F9 parietal endoderm cells, not detected within F9 stem cell 'matrices'. Parietal endoderm cell matrix mitogens could be removed by prior treatment of the ECM with buffers containing heparin or 2 M NaCl, and could be neutralized by basic FGF antibodies.


The influence of type I collagen on the growth and differentiation of the human colonic adenocarcinoma cell line HT-29 in vitro.

HT-29 Human colonic adenocarcinoma cells when grown on a plastic substratum were anaplastic in appearance and failed to express any morphological or biochemical features that were characteristic of intestinal differentiation. Growth of HT-29 cells subcutaneously in the flank of immune deprived mice gave rise to morphologically heterogeneous tumors which were poorly differentiated but contained approximately 11% of cells with an intestinal phenotype: these showed features typical of cell polarization with well-developed microvilli, tight junctional complexes and desmosomes between adjacent cells. The transfer of cells from plastic onto either a fixed (designated 'non-released') or floating (designated 'released') type I collagen gel induced some morphological features typical of intestinal differentiation; for example goblet-like cells were observed after 9 days, but biochemical markers of differentiation were expressed only modestly. The continued subculture of HT-29 cells on collagen type I gels, which were either attached to the plastic or floating in the medium, induced some morphological features of intestinal differentiation and changes in the activity of brush border-associated enzymes. Alkaline phosphatase activity was enhanced from 1.3 x 10(-3) mumoles/mg/min for cells cultured on plastic substrata to 2.1 x 10(-3) mumoles/mg/min when gels were non-released, and 2.9 x 10(-3) mumoles/mg/min when gels were released after 12 days of culture. This was confirmed by electron microscopical visualization of alkaline phosphatase activity. Elevated levels of aminopeptidase activity were also observed on day 12 (plastic = 26 milliunits/mg; non-released gel = 41 milliunits/mg; released gel = 36 milliunits/mg). Similarly, changes occurred in the secretion of carcinoembryonic antigen from 0.96 x 10(-2) micrograms/mg/48 hours by cells cultured on plastic to 2.3 x 10(-2) micrograms/mg/48 hours by cells cultured on floating collagen gels. The effects of permitting HT-29 cells to undergo polarization were tested by culture on inert filter inserts: morphological features of intestinal differentiation were observed although this did not occur until after 21 days. These studies show that optimization of the growth conditions of anaplastic cells in vitro may provide cultures more representative of the tumor in vivo. This model system may be useful for cell biological and pharmacological studies of colon carcinoma.


Examination of the role of the proteolytically-activated form of protein kinase C in the differentiation of human haemopoietic cells.

In neutrophils, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the translocation of the Ca(++)- and phospholipid-dependent protein kinase, protein kinase C (PK-C) from the soluble to the particulate fraction. At the same time there was a corresponding increase in the amount of Ca(++)- and phospholipid-independent protein kinase activity recovered in the soluble fraction. This soluble Ca(++)- and phospholipid-independent protein kinase presumably reflects proteolytic activation of the particulate associated PK-C. Bone marrow and undifferentiated HL-60 cells also translocated PK-C to the particulate fraction in response to TPA but did not accumulate the soluble Ca(++)- and phospholipid-independent form of the enzyme. Similar results were obtained using HL-60 cells induced to differentiate with dimethyl sulphoxide (DMSO), recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) or 1 alpha,25-dihydroxyvitamin D3. There was also no significant change in either the number or time of expression of differentiation-specific cell surface antigens observed on HL-60 cells induced to differentiate with either DMSO, 1 alpha,25-dihydroxyvitamin D3 or TPA in the presence of cyclosporin A, an agent reported to inhibit the proteolytic breakdown of PK-C to the Ca(++)- and phospholipid-independent form. Likewise, cyclosporin A did not affect the rate of extent of differentiation of primary bone marrow cell cultures. These results suggest that the proteolytically activated and phospholipid-independent form of PK-C is probably not involved in haemopoietic cell differentiation.


Chemical allergy: molecular mechanisms and practical applications.

Allergic reactions can be defined as the adverse, tissue-damaging, and sometimes fatal consequences of specific immune responses, usually to exogenous antigens. In the context of toxicology it is allergic reactions resulting from immune responses to chemicals and drugs which are of greatest relevance. The allergy may take a variety of forms including contact hypersensitivity (allergic contact dermatitis), respiratory hypersensitivity (with symptoms ranging from mild rhinitis to severe asthma), and various types of comparatively ill-defined reactions which in many respects resemble autoimmunity. Of these contact hypersensitivity is the most frequently encountered health problem resulting from the interaction of chemicals with the immune system. A wide variety of chemicals are able to induce contact sensitization. Some of these are, in addition, known to cause respiratory hypersensitivity, a less frequent, but no less important, form of chemical allergy.


Further validation of an in vitro method to reduce the need for in vivo studies for measuring the absorption of chemicals through rat skin.

Current requirements for the registration of agrochemicals, particularly in the U.S.A., often require the provision of dermal absorption data. In this process the rat is often used and complex in vivo studies, using large numbers of animals, are performed. We have compared the data obtained from in vivo and in vitro dermal absorption studies using eight pesticides with a range of physicochemical properties. Measurements were made of the 14C-labeled pesticides which could be washed from the skin, were associated with (on/in) skin, or absorbed through the skin following dermal applications in vivo and in vitro at various time points over a 24-hr exposure period. Good agreement was found between the amounts washed from and associated with the skin in vivo and in vitro. Over the time period 4-24 hr after application the in vitro experiments predicted the in vivo absorption within a factor of 2-3. These results show that, with a range of pesticide molecules, the in vitro method accurately predicted in vivo absorption supporting the utilization of the in vitro method for risk assessment from exposure to pesticides and other chemicals.


A comparative study of the rates of in vitro percutaneous absorption of eight chemicals using rat and human skin.

In vitro percutaneous absorption studies were carried out for eight chemicals using full thickness rat skin and human stratum corneum. The purpose of the studies was to compare the rates of absorption for the two species. For each of the chemicals, the observed rate using full thickness rat skin was greater than that observed for human stratum corneum. The ratios of the rates (rat/human) varied from 1.7 to 5.8 with a mean value of 3.1. The chemicals tested were tritiated water, 2-ethoxyethyl acetate, diethylene glycol monobutyl ether, urea, di(2-ethylhexyl) phthalate, 2-ethylhexanol, ethyl 3-ethoxypropionate, and 2-propoxyethanol. The chemicals were chosen to represent a wide range of physical properties and permeability constant values. It was concluded that rat skin was more permeable than human skin for each of these eight chemicals. This conclusion is supported by similar findings from studies in other laboratories and suggests that results from studies in the rat overestimate skin absorption in man.


Rate and route of oxidation of acrylic acid to carbon dioxide in rat liver.

Results of in vivo metabolism studies with acrylic acid (AA) have indicated that 60-80% of the administered dose is excreted as CO2 within 2-8 hr of oral dosing of rats; however, the pathway of AA metabolism to CO2 in mammals has not been determined. To define this route, rat hepatocytes were isolated and incubated with [1-14C]AA in a sealed vial modified to trap evolved 14CO2. Rapid oxidation of AA to CO2 was observed. Similar incubations conducted with rat liver homogenates fortified with ATP, ADP, coenzyme A, carnitine, and malate also resulted in oxidation of AA. Mitochondria isolated from liver homogenates were incubated with AA under the same conditions and yielded higher rates of AA oxidation than homogenates. Addition of equimolar amounts of propionic acid, 3-hydroxypropionic acid, or 3-mercaptopropionic acid significantly inhibited the oxidation of AA by mitochondria. HPLC analysis of the mitochondrial incubation mixtures indicated that a single major metabolite, which coeluted with 3-hydroxypropionate, accumulated in the solution. The results indicate that AA is rapidly incorporated into a mitochondrial pathway for propionic acid catabolism that results in the release of CO2 and possible bioincorporation as acetate. This pathway appears to be the principal route of detoxification of AA in mammals.


The developmental toxicity of 2-ethylhexanoic acid in Wistar rats.

The developmental toxicity of 2-ethylhexanoic acid (2-EHA), a wood preservative and a mammalian metabolite of di-(2-ethylhexyl) phthalate was examined in Wistar rats (20-21 pregnant females/dose). Mated animals were exposed to 2-EHA in their drinking water at doses of 100, 300, or 600 mg/kg/day on Days 6-19 of gestation. Control animals received vehicle water. The fetuses were examined (on Gestational Day 20) for external, visceral, and skeletal malformations and variations. 2-EHA was marginally toxic to the dams at 600 mg/kg, but not at lower doses, since the mean near term body weight was reduced by 11%. This dose level was also slightly fetotoxic as indicated by a 5 to 8% decrease in the mean fetal body weight both in males and females. No treatment-related effects were observed in the number of implantations or live fetuses. At doses of 100 mg/kg and above, 2-EHA caused skeletal malformations (clubfoot, absence of fibula, polydactyly), while the development of visceral tissues was less affected. The number of affected fetuses increased in a dose-dependent way (4.9, 8.9, and 15.3% of treated offspring at 100, 300, and 600 mg/kg/day, respectively, vs 2.4% control). These results indicate that 2-EHA is teratogenic in rats already at doses which are not yet maternally toxic. The skeleton appears to be the main target of 2-EHA in developing rats.


Carcinogenicity of oral cadmium in the male Wistar (WF/NCr) rat: effect of chronic dietary zinc deficiency.

The effect of chronic dietary zinc deficiency on the carcinogenic potential of dietary cadmium was assessed in male Wistar (WF/NCr) rats. Groups (n = 28) of rats were fed diets adequate (60 ppm) or marginally deficient (7 ppm) in zinc and containing cadmium at various levels (0, 25, 50, 100, or 200 ppm). Lesions were assessed over the following 77 weeks. Zinc deficiency alone had no effect on survival, growth, or food consumption. Cadmium treatment did not reduce survival or food consumption and only at the highest doses of cadmium (100 and 200 ppm) was body weight reduced (maximum 17%). The incidence of prostatic proliferative lesions, both hyperplasias and adenomas, was increased over that seen in controls (1.8%) in both zinc-adequate (20%) and zinc-deficient rats (14%) fed 50 ppm cadmium. The overall incidence for prostatic lesions for all cadmium treatment groups was, however, much lower in zinc-deficient rats, possibly because of a marked increase in prostatic atrophy that was associated with reduced zinc intake. Cadmium treatment resulted in an elevated leukemia incidence (maximum 4.8-fold over control) in both zinc-adequate and zinc-deficient groups, although zinc deficiency reduced the potency of cadmium in this respect. Testicular tumors were significantly elevated only in rats receiving 200 ppm cadmium and diets adequate in zinc. Both zinc-deficient and zinc-adequate groups showed significant positive trends for development of testicular neoplasia with increasing cadmium dosage. Thus, oral cadmium exposure is clearly associated with tumors of the prostate, testes, and hematopoietic system in rats, while dietary zinc deficiency has complex, apparently inhibitory, effects on cadmium carcinogenesis by this route.


In vivo and in vitro percutaneous absorption and skin evaporation of isofenphos in man.

Studies were done to determine the percutaneous absorption of isofenphos in human volunteers from whom informed consent had been obtained. In vivo absorption in man was 3.6 +/- 3.6% of applied dose for 24-hr exposure and 3.6 +/- 0.5% for 72-hr exposure. Skin wash recovery data show that isofenphos evaporates from in vivo skin during the absorption process; the surface dose is minimal (< 1%) by 24 hr. Skin stripping showed no residual isofenphos in stratum corneum. This explains the similar absorption for 24 and 72-hr dose prewash exposures. Skin surface recovery in vivo with soap and water was 61.4 +/- 10.4 for the first dosing time (15 min). Time-recovery response declined with time to 0.5 +/- 0.2% at 24 hr. In vitro absorption utilizing flow-through diffusion methodology with human cadaver skin and human plasma receptor fluid gave 2.5 +/- 2.0% dose absorbed, an amount similar to in vivo studies. An additional 6.5 +/- 24% was recovered in the skin samples (total of 9%). Skin surface wash at 24 hr recovered 79.7 +/- 2.2% and skin content was 6.5 +/- 2.4% (total dose accountability of 88.7 +/- 4.6%). Thus, isofenphos was available for absorption during the whole dosing period. Neither in vitro absorption nor in vitro evaporation studies predicted the potential skin evaporation of isofenphos. Published dermal studies in the rat had predicted isofenphos absorption at 47% of applied dose (12-fold greater than actual in man). Subsequent toxicokinetic modeling predicted possible concern with the use of isofenphos.(ABSTRACT TRUNCATED AT 250 WORDS)


Subchronic toxicity of barium chloride dihydrate administered to rats and mice in the drinking water.

Barium Chloride dihydrate (BaCl2.2H2O) was given for 92 days to B6C3F1 mice and Fischer 344/N rats in their drinking water at levels of 0, 125, 500, 1000, 2000, and 4000 ppm. The no-effect level for this study was 2000 ppm BaCl2.2H2O in the drinking water. At 4000 ppm, daily consumption for mice was 436 to 562 mg/kg barium, up to four times more chemical than rats. Mortality ranged from 60 to 70% in mice and from 10 to 30% in rats in the 4000 ppm groups. Deaths in mice were associated with a treatment-related renal toxicity. Renal lesions in rats were much less severe than in mice and did not contribute to the treatment-related deaths seen in the high dose group. Body weights of both species and sexes in the 4000 ppm groups were lower than controls at 92 days. Male and female rats in treated groups exhibited higher serum phosphorus than controls. Serum sodium, potassium, and calcium levels in rats were unchanged by barium treatment, as were hematological values. In both species at 4000 ppm, motor activity, grip strength, and thermal sensitivity were marginally affected. These effects were probably secondary changes resulting from BaCl2 toxicity observed at this dose level. In a mating trial, no anatomical effects on offspring of rats or mice were seen. Rats receiving 4000 ppm exhibited marginal reductions in pup weights. No effects were seen on reproductive indices.


Investigation of factors influencing urinary porphyrin excretion in rats: strain, gender, and age.

Urinary porphyrin excretion rates were evaluated in male rats of the Sprague-Dawley, Fischer 344, and Osborne-Mendel strains, and in female Fischer 344 rats, in order to identify an appropriate rat model for long-term studies of chemical-induced porphyria. Sprague-Dawley rats displayed a wide interanimal range in total 24-hr porphyrin excretion rates, from 0.9 to 59.6 nmol/24 hr (64-fold). Additionally, individual Sprague-Dawley rats varied by up to 5-fold in total porphyrins excreted from week to week over a 4-week period. Osborne-Mendel rats displayed a 12-fold interanimal difference in 24-hr total porphyrin excretion rates (ranging from 6.2 to 71.1 nmol/24 hr) and up to 4-fold individual variation from 1 week to another. In contrast, rats of the Fischer 344 strain displayed no significant differences in porphyrin excretion rates either among different animals or for individuals from week to week. Determination of individual porphyrin concentrations in the urine demonstrated that inconsistencies in total porphyrin concentration were attributable principally to differences in coproporphyrin concentrations. Frequency analysis of 24-hr coproporphyrin excretion levels among Sprague-Dawley rats demonstrated a widespread range encompassing low levels comparable to those of Fischer rats, high levels similar to those of most Osborne-Mendel rats, as well as intermediate levels. The mean total porphyrin excretion rate among female Fischer 344 rats was 1.2 +/- 0.51 nmol/24 hr, compared to 2.1 +/- 0.50 nmol/24 hr among male Fischer rats. Maturation and aging were found to have little influence on porphyrin excretion rates in male Fischer 344 rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Prenatal dexamethasone exposure in rats: effects of dose, age at exposure, and drug-induced hypophagia on malformations and fetal organ weights.

Glucocorticoids cause stunting and cleft palate in rodents. The aim of this study is to identify fetal organs and developmental periods sensitive to stunting induced by maternal exposure to dexamethasone (DEX). DEX (0.2 or 0.4 mg/kg) or saline was given sc to pregnant CD albino rats on Gestation Days (GD) 9-14 or 14-19. On GD 20 dams were euthanized. Fetuses were weighed and examined for cleft palate. Eight fetuses/litter were randomly selected, and weights were obtained. Fetal skeletons were examined for abnormalities, and long bone measurements were taken. A dose-related decrease in maternal and fetal body weights occurred at both exposure periods. Developmental stage-specific malformations were noted in the high-dose group on GD 9-14 (cleft palate) and on GD 14-19 (wavy ribs). A dose-response in stunting occurred in all organs except cerebellum in at least one exposure period. Across both exposure periods the brain, heart, testes, and long bones were relatively resistant to DEX. Sensitive organs included thymus, spleen, adrenals, lungs, liver, and kidneys. DEX substantially reduced maternal food intake and increased water intake in some dams. Pair-feeding experiments suggested that the hypophagic effect of DEX was not responsible for the noted malformations and had little impact on growth stunting. The present findings have identified fetal organs, skeletal regions, and developmental periods sensitive to DEX exposure.


Immunogenic properties of structurally modified human tissue plasminogen activators in chimpanzees and mice.

Immunogenic properties of second generation human tissue plasminogen activator (tPA) derivatives were examined in chimpanzee and mouse systems. Five species of modified tPAs (mtPAs) (designated 2660, 2663, 2810, 8000, and 9200), recombinant native tPA or bovine serum albumin (BSA) as a positive control were subcutaneously injected nine times at suitable intervals into chimpanzees, genetically the closest species to man. These animals were tested for antigen(Ag)-specific antibodies to the corresponding proteins by means of enzyme-linked immunosorbent assay and Western blot analysis. Neither 9200, one of the five mtPAs tested, nor tPA was immunogenic, although BSA and the other four mtPAs were immunogenic under these conditions. Thus, an antigenic determinant was not exposed by the modification on 9200 and this modified tPA is expected not to be immunogenic in humans. In the mouse studies, mice were immunized with mtPAs. Serum samples from these animals were tested for antibodies to the mtPAs which did not concomitantly recognize native tPA by immune adsorption of the antibodies to tPA. The amount of such antibodies after the elimination of native tPA-reactive antibodies was little or none when the serum samples from 9200 and from the other mtPAs, except 8000, were tested. Taking into consideration the results of the chimpanzee studies, it can be concluded that Ag-specific antibodies are dominantly produced to unchanged epitopes present in modified proteins in the mouse system, in which the native protein is immunogenic.(ABSTRACT TRUNCATED AT 250 WORDS)


Subchronic toxicity of benzyl selenocyanate and 1,4-phenylenebis(methylene)selenocyanate in F344 rats.

Chemopreventive agents benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) were fed in NIH-07 diet to male and female F344 rats (4, 2, and 0.5 mg/kg/day for BSC and 20, 10, and 5 mg/kg/day for p-XSC) for 13 weeks. Weight gains were depressed for male and female rats fed 4 and 2 mg/kg/day BSC, females fed 0.5 mg/kg/day BSC, and male rats fed 20 and 10 mg/kg/day p-XSC. At necropsy, no clear treatment-related lesions were noted, but dose-dependent hepatomegaly was observed in both sexes of BSC and p-XSC groups. Plasma transaminases AST and ALT were elevated in the higher dose groups, while hemoglobin, HCT, and RBC were reduced in most BSC and some p-XSC treatment groups. Plasma glucose was reduced in BSC-treated males. Significant histologic findings included moderate to severe hepatic centrilobular hypertrophy with fatty change in all males and females in the 4 mg/kg/day BSC groups and in 9/15 males and 3/15 females in the 2 mg/kg/day BSC groups. Dose-dependent, mild centrilobular hypertrophy with minimal fatty change was observed in the mid- and low-dose BSC groups and in all p-XSC groups. Mild to moderate renal tubular and interstitial nephritis occurred in the 4 mg/kg/day male BSC group. Dietary maximum tolerated dose levels for chemoprevention studies are 0.5 mg/kg/day (3.0 ppm Se) for BSC and 5 mg/kg/day (32.5 ppm Se) for p-XSC, compared to literature values of 2-3 ppm Se for Na2SeO3.


Suppression of the murine gut mucosal IgA response to cholera toxin with oral cyclosporine.

The gut mucosal immune system may be a primary target for many ingested chemicals. Methods have been developed to examine the effects of chemicals on the systemic humoral immune response; however, studies to evaluate various methods of assessing the local gut mucosal immune response in a toxicology assay have been limited. The objectives of this study were to examine the effects of the known immunosuppressive compound, cyclosporine (CYS), on the generation of a cholera toxin (CT)-specific gut mucosal IgA response and evaluate the methods used to measure the gut IgA response. Groups of female B6C3F1 mice were left untreated or were treated daily, p.o., with corn oil (vehicle) or CYS at doses of 10 and 50 mg/kg for 20 days. On Days 3 and 13, mice were sensitized p.o. with CT. On Day 21, mice were terminated, gut washings were collected, and lamina propria lymphocytes were extracted from gut tissue with collagenase treatment. Cholera toxin-specific IgA in the gut washings was measured by an ELISA. The numbers of CT-specific IgA (CT-IgA) and total IgA antibody-forming cells (spot-forming cells, SFC) obtained from the lamina propria were determined by the ELISPOT method. A dose of 50 mg/kg CYS produced a significant decrease in the amount of CT-IgA in gut washings. This dose also decreased the number of cells recovered from the lamina propria by at least 50%. The amount of CT-specific SFC/million lamina propria cells decreased with a dose of 10 mg/kg CYS, whereas 50 mg/kg CYS did not alter the response.(ABSTRACT TRUNCATED AT 250 WORDS)


Interspecies differences in the phagocytic activity of pulmonary macrophages subjected to acidic challenge.

Examining interspecies differences in response to ambient pollutants is an essential component of risk assessment. The potential hazard to public health from the inhalation of acid sulfate aerosols is of current concern. A significant biological target is the pulmonary macrophage, which provides a primary defense of the respiratory region of the lungs. One essential function of these cells is phagocytosis of particles. This study assessed the effects of acidic environments on the phagocytic activity of pulmonary macrophages obtained by lavage from humans and three species of laboratory animals commonly used in acid aerosol toxicology studies, namely, rats, rabbits, and guinea pigs. Cells were incubated with polystyrene latex particles in media acidified by addition of sulfuric acid. The percentage of cells which were phagocytic, as well as the relative number of particles ingested by these cells, was found to decrease with increasing acidity for all species. The ranking of response in order of decreasing sensitivity to acidic challenge was as follows: guinea pig > rat > rabbit > human.


Comparison of the effects of Wyeth-14,643 in Crl:CD BR and Fisher-344 rats.

Wyeth-14,643 (WY) belongs to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation and hepatocellular carcinoma in rodents. Based on a review of bioassay data, a relationship appears to exist between peroxisome proliferating compounds and Leydig cell adenoma formation. Most rat bioassays with peroxisome proliferators have been conducted in the Fisher-344 (F344) rat, which has a high spontaneous incidence of Leydig cell adenomas. Thus, it was necessary to use an alternative animal model to investigate this relationship further. Therefore the Crl:CD BR (CD) rat, which has a low spontaneous Leydig cell adenoma incidence, was chosen for a 2-year feeding study with WY. Before initiating this 2-year feeding study, it was necessary to investigate whether any strain differences existed between CD and F344 rats with respect to WY-induced peroxisome and cell proliferation. In this study, male CD and F344 rats were fed diets containing 0, 50, or 1000 ppm WY for 21 days. Peroxisome proliferation in the liver and testis was determined biochemically by measuring beta-oxidation activity and was confirmed ultrastructurally. Serum hormone levels and cell proliferation rates in the liver and testis were also measured. In addition, basal beta-oxidation activity and cell proliferation rates were compared between the CD and F344 rats. A significant decrease in final body weight was observed in the 1000 ppm WY groups for both CD and F344 rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Enhanced inflammatory response to acute ozone exposure in rats during pregnancy and lactation.

Experimental evidence from several studies suggests that pregnant animals and women are more susceptible to oxidants than nonpregnant controls. In the study reported here, we sought to determine whether pregnant rats are more sensitive than age-matched virgin females to the inflammatory effects of ozone, a gaseous oxidant of considerable environmental significance. Rats at several stages of pregnancy and lactation, as well as age-matched virgin females, were exposed to 1 ppm ozone for 6 hr. Controls were sham-exposed to pure air for an identical period of time. Bronchoalveolar lavage was performed 24 hr after the beginning of exposure, and components of the lavage fluid considered to be indicators of inflammation were used to assess the severity of pulmonary inflammation. The results of this experiment showed that significantly enhanced sensitivity to ozone-induced pulmonary inflammation develops during pregnancy, is maintained during lactation, and disappears following lactation. Implicit in this pattern of differential sensitivity in rats is the possibility of a similar pattern of inflammatory response in analogous groups of humans as well as the potential for applicability to other oxidative pollutants.


Female family doctors: their work and well-being.

The role of female physicians, which often involves combining medicine, marriage, and motherhood, may cause stress for some women. The purpose of this research was to identify correlates of depression, stress, and self-esteem among a sample of female Canadian family physicians. All female family physicians in London, Ontario, who met study eligibility criteria were asked to participate in a questionnaire study. Sixty-two physicians were surveyed and asked to complete four psychological survey instruments: the Cognition Checklist, the Coopersmith Self-Esteem Inventory, the People in Your Life Scale, and the Work Response Questionnaire. The response rate was 83.3%. Sixty-five percent of subjects felt overloaded or overwhelmed at least once per week. These women, on average, worked more hours than subjects who reported not feeling overwhelmed (r = .48). Subjects had lower depression scores than did other populations tested with similar survey instruments. Subjects' self-esteem was similar to that found in other populations and was positively correlated with opportunities to acquire new skills (r = .34) and perceived lack of difficulty in developing lasting relationships (r = .41). Depression appears to be less common among female family physicians than among other populations, but nearly two thirds of female family physicians feel overloaded or overwhelmed by their multiple responsibilities, as often as once per week.


The primary care research fellowship: an early assessment.

To address the continuing need for research-oriented faculty in primary care, the Bureau of Health Professions in 1988 funded 10 National Research Service Award (NRSA) Primary Care Fellowship Program grants. We report an early evaluation of these programs. Descriptions of fellowship programs were obtained from grant proposals. Fellows with at least one year of training completed a mailed questionnaire that included both structured and open-ended items. All programs emphasized research and gave mentorship explicit attention. Nine offered degree programs; six offered a third year of study. Twenty-seven of 32 eligible fellows returned the survey. They reported a median of three research projects during the fellowship. Sixty percent of fellows had extramural funding; 48% submitted manuscripts for publication; and 22% had work accepted for publication. In open-ended responses, fellows emphasized the importance of mentors' guidance on projects, accessibility, and emotional support; rarely did they mention help in establishing professional networks or in securing project funding. Fellows most appreciated the collegiality and protected atmosphere of fellowship programs, although they felt that both human and material resources were less than optimal. Fellowship environments are generally conducive to productivity, and fellows undertake relevant primary care research. Programs should devote more attention to mentoring, especially regarding networking and obtaining funding. Funding agencies should increase support for training-related expenses in lieu of research grants that subsidize NRSA training in other specialties.


Family medicine in Lebanon: the 10th anniversary.

Developing a family practice residency program was a logical step in addressing the needs for primary care in Lebanon. The American University of Beirut initiated a family practice residency program in 1979. The curriculum was structured following the guidelines of the American Academy of Family Physicians. Since its establishment, the program has participated in the design, launching, and maintenance of the family practice residency in Bahrain. Future plans include establishing other programs in the region and increasing community participation.


Medical school financial support, faculty composition, and selection of family practice by medical students.

This study examines the relationship between the choice of family practice by medical students in the 1986-1987 academic year and nine variables: ownership of school, weeks of required family medicine clinical training, age of school, and six variables related to medical school financial support and faculty composition. In multivariate analysis, three variables were found to be significantly related to choice of family practice (R2 = .441): weeks of required family medicine clinical training, proportion of faculty in family practice, and ownership of the school. A cluster analysis was performed using nine variables. Along with the multivariate analysis, the resulting nine clusters demonstrate that additional variables need to be identified to explain the variance in percentage of students choosing family practice at different schools.


Religious variables are infrequently reported in clinical research.

More than 200 published studies have found some relationship between an individual's religion and health, yet reference to religious variables rarely appears in published empirical studies. We performed a manual search of all articles reporting on human subjects in the 1989 issues of seven major medical journals to record and analyze religious and other demographic variables. Only 12 of the 1,066 articles (1.1%) mentioned religion, and eight of these mentioned only denominational affiliation. Most clinical investigators do not report on the religious variables of their subjects and thus may be missing important correlations.


Participation by sedentary elderly persons in an exercise promotion session.

Little is known regarding the dynamics of elderly persons' participation in health promotion programs. We studied factors influencing the participation of 337 sedentary elderly persons in an exercise promotion session. All study participants were members of a health maintenance organization. Baseline data were obtained on 337 persons age 65 and older who exercised irregularly or not at all. These individuals were then offered a class emphasizing individualized exercise activities for elderly persons. Participation in the exercise program was used as the dependent variable, and the baseline data were analyzed to identify factors associated with participation. There was no difference between participants and nonparticipants for age and sex. Baseline physical activity, as measured by the Paffenbarger index and self-report of physical activity, also did not differentiate participants from nonparticipants, nor did frequency of self-reported medical conditions. Participants were more likely to be nonsmokers, but the two groups did not differ with respect to other preventive health practices. Participants were characterized by more social interactions, as reflected by attendance at at least two meetings per month. Participants also were more likely to report three or more close friends. Perceived quality of life and self-reported health status were not significantly different between the two groups. Participants rated their current health slightly worse than nonparticipants. In a logistic regression model controlling for age and sex, lower current health perceptions, greater number of friends, prior expressed intent to participate in an exercise program, being a nonsmoker, and having received an influenza vaccination in the past year were significantly associated with participation. Participation in this exercise program may have appealed to persons with higher levels of social interaction. In this population, age, sex, and the number and type of self-reported medical conditions at baseline were not predictive of participation.


Voices from family medicine: cooperation across disciplines. Interview by William B. Ventres, John J. Frey and Alfred O. Berg.

A variety of individuals lent their efforts to the establishment of family medicine as an academic discipline. Many had careers outside general or family practice, yet because of their interests and active involvement, they helped forge the way toward a rational and comprehensive education for family physicians in this country. In this transcript, edited from interviews conducted in 1992, four contributors from other disciplines discuss their perspectives on the history of family medicine.


Hospital charting of substance use.

Recent evidence suggests that physicians can help patients reduce or even discontinue cigarette smoking. Physicians may also be able to help patients decrease their use of alcohol or illegal drugs. A prerequisite for helping patients decrease substance use is for physicians to ask patients about their use of cigarettes, alcohol, and illicit drugs. We performed a retrospective study of 212 medical records to measure physicians' documentation of patient use of alcohol, cigarettes, and illicit drugs in an inpatient setting. The study compared residents and attending physicians in family practice and internal medicine. Physicians charted cigarette use in 73% of medical records, alcohol use in 70%, and drug use in only 12.5%. Family physicians charted substance use more frequently than internal medicine physicians for alcohol and illicit drugs (P < .05 by Pearson's chi-square analysis) and for cigarettes (P = .07). In each specialty, residents charted substance use more often than attending physicians (P < .01). Attending family physicians (P < .05) charted substance use more often in male than in female patients. This was not found among internal medicine physicians. The results of the study suggest that the stage of medical career, medical specialty, and sex of the patient affect the frequency of charting substance abuse.


Can indirect cost funding be improved?

Like the tax laws many possible loopholes are in these proposed changes and therefore they have to be considered very carefully. If there is such a thing as a collective wisdom, it should be brought to bear on this problem. With strong and well-considered actions, which would be implemented in a controlled way, the mechanisms for funding research could be improved to encourage efficient use of funds.


Regulation of glucose transport in skeletal muscle.

The entry of glucose into muscle cells is achieved primarily via a carrier-mediated system consisting of protein transport molecules. GLUT-1 transporter isoform is normally found in the sarcolemmal (SL) membrane and is thought to be involved in glucose transport under basal conditions. With insulin stimulation, glucose transport is accelerated by translocating GLUT-4 transporters from an intracellular pool out to the T-tubule and SL membranes. Activation of transporters to increase the turnover number may also be involved, but the evidence is far from conclusive. When insulin binds to its receptor, it autophosphorylates tyrosine and serine residues on the beta-subunit of the receptor. The tyrosine residues are thought to activate tyrosine kinases, which in turn phosphorylate/activate as yet unknown second messengers. Insulin receptor antibodies, however, have been reported to increase glucose transport without increasing kinase activity. Insulin resistance in skeletal muscle is a major characteristic of obesity and diabetes mellitus, especially NIDDM. A decrease in the number of insulin receptors and the ability of insulin to activate receptor tyrosine kinase has been documented in muscle from NIDDM patients. Most studies report no change in the intracellular pool of GLUT-4 transporters available for translocation to the SL. Both the quality and quantity of food consumed can regulate insulin sensitivity. A high-fat, refined sugar diet, similar to the typical U.S. diet, causes insulin resistance when compared with a low-fat, complex-carbohydrate diet. On the other hand, exercise increases insulin sensitivity. After an acute bout of exercise, glucose transport in muscle increases to the same level as with maximum insulin stimulation. Although the number of GLUT-4 transporters in the sarcolemma increases with exercise, neither insulin or its receptor is involved. After an initial acute phase, which may involve calcium as the activator, a secondary phase of increased insulin sensitivity can last for up to a day after exercise. The mechanism responsible for the increased insulin sensitivity with exercise is unknown. Regular exercise training also increases insulin sensitivity, which can be documented several days after the final bout of exercise, and again the mechanism is unknown. An increase in the muscle content of GLUT-4 transporters with training has recently been reported. Even though significant progress has been made in the past few years in understanding glucose transport in skeletal muscle, the mechanisms involved in regulating transport are far from being understood.


Regulation of transferrin gene expression.

Transferrin (Tf) is the iron-transport protein of vertebrate serum. It is essentially synthesized in the liver, but lower amounts are also produced in other organs, such as testis and brain. A number of studies have been done to characterize the transcriptional elements implicated in the regulation of Tf gene expression in different organs. The results of these studies support the hypothesis that the Tf gene makes use of different combinations of nuclear proteins in different subsets of cells to achieve tissue-specific expression. It is interesting to point out that this occurs in tissues arising from different embryological origin.


SAPA/trans-sialidase and cruzipain: two antigens from Trypanosoma cruzi contain immunodominant but enzymatically inactive domains.

Trypanosoma cruzi, the parasitic protozoan that causes the American trypanosomiasis, or Chagas disease, contains a number of antigenic molecules, some of which have tandems of amino acid repeats. One of these molecules, SAPA (shed acute phase antigen), contains a so-far unique trans-sialidase activity that is essential for penetration of the parasite into mammalian cells. The enzyme consists of two different domains, one presumably enzymatic, which contains four copies of an amino acid motif conserved in bacterial neuraminidases, and the other highly antigenic, consisting of the repeats. Another enzyme that seems to be involved in the host-parasite relationship, the cysteine proteinase cruzipain, is also made up in its mature form of a catalytic domain with high homology to cathepsin L and a COOH-terminal domain that is highly antigenic in vivo. These bifunctional molecules may have arisen by incorporation of a highly antigenic domain to an essential enzyme in order to attract the immune response, thus protecting the enzyme activity.