The role of the skeletal muscle ryanodine receptor gene in malignant hyperthermia. Malignant hyperthermia (MH) is an inherited, potentially lethal condition in which sustained muscle contracture with attendant hypermetabolism and hyperthermia is triggered in humans, heterozygous for the gene defect, by inhalational anaesthetics and skeletal muscle relaxants, and in pigs, homozygous for the defect, by stress. Because muscle contracture could result from a defective Ca2+ release channel, we have focussed our attention on the linkage of MH to defects in the gene (RYR1) encoding the skeletal muscle Ca2+ release channel. We have cloned and sequenced human RYR1 cDNA and found restriction fragment length polymorphisms (RFLPs) in the human gene. We also localized RYR1 to human chromosome 19q13.1. Studies of the cosegregation of MH with these RFLPs established RYR1/MH linkage on human chromosome 19q13.1 (lod score of 4.2; recombinant fraction 0.0). We then sequenced MH and normal porcine RYR1 cDNAs. Mutation of C1843 to T, leading to substitution of Cys for Arg615, was the sole amino acid change noted between MH and normal animals. Linkage of this mutation to MH was established in a study of 338 informative meioses (lod score of 102; recombinant fraction 0.0). We identified the corresponding mutation in 1 of 35 human MH families studied and found cosegregation of the mutation and MH. The combination of a high lod score with crossing of a species barrier supports the causal nature of this mutation. Future studies are aimed at finding the major human MH mutations and establishing assays for their accurate diagnosis.


The consequences of a constitutive expression of MyoD1 in ES cells and mouse embryos. A variety of differentiated cell types can be converted to skeletal muscle following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones tested, carrying single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 genes but not the endogenous MyoD1, MRF4, myf5, skeletal muscle actin or myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers was observed only when the transfected cells were allowed to differentiate, via embryoid bodies, in low mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and myf5 genes and in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of MyoD1 was not required for myogenesis. Interestingly, no preferential myogenesis was observed when the transfected ES cells were allowed to differentiate in vivo to teratocarcinomas. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function. Second, MyoD1 function in ES cells, even under environmental conditions that favour differentiation, is not dominant (incomplete penetrance). Third, that the exogenous MyoD1 transactivates the endogenous myogenin and MLC2 genes in ES cells. No live transgenic mice could be produced following microinjection of the beta-actin/MyoD1 gene into the pronuclei of fertilized eggs. Transgenic embryos died before mid gestation. The majority of tested embryos between 7.5 and 9.5 days, although retarded compared to control litermates, differentiated into tissues representative of all three germ layers. The expression of the introduced gene was detected in all ectodermal and mesodermal tissues but was absent in all endodermal cells. These results demonstrate again that MyoD1 is not a dominant regulatory factor.


Expression of muscle genes in the mouse embryo. Using isogene specific probes and in situ hybridization on sections, we have examined the expression of structural and regulatory genes in the mouse embryo during the formation of cardiac and skeletal muscle. The temporal and spatial information thus obtained about the onset of expression of muscle genes provides insight into the regulation of myogenesis in vivo. Actin and myosin sequences present in different compartments of the adult heart are initially all co-expressed in the cardiac tube (between 7-8 days). The process of spatial restriction to atrial or ventricular compartments of the heart takes place asynchronously later. In contrast, the onset of expression of actin and myosin genes in the first skeletal muscle, the myotome, which corresponds to the central compartment of the somite, as well as their subsequent down-regulation in different skeletal muscle masses, takes place very asynchronously. One might predict that factor(s) responsible for the transcriptional activation of these genes are present in sufficient quantity in the cardiac tube, whereas in skeletal muscle individual genes are responding to variable levels of factor(s). In fact the four myogenic regulatory sequences present in the mouse - MyoD1, myogenin, myf-5 and myf-6 - do show distinct patterns of expression during the development of skeletal muscle. None of these sequences have been detected in the heart. In the myotome there is no general correlation between the appearance of a particular myogenic sequence and the activation of a particular structural gene. A striking example of this is provided by the muscle isoform of creatine phosphokinase. We would propose that each muscle structural gene has a different threshold of activation, depending on the quantity and nature of the myogenic factor present. We have also examined the onset of expression of the X-linked dystrophin gene known to be expressed in adult heart and skeletal muscle. In the myotome dystrophin transcripts are first detected at the time when myosin heavy chains first accumulate and muscular contraction is initiated. In contrast in the cardiac tube dystrophin transcripts are not detected initially, at a time (from 8 days) when the heart contracts. This observation can be correlated with the pathology of the disease which points to a more essential role of dystrophin in skeletal muscle. No muscle structural gene examined is expressed in the somite prior to myotome formation. If the myogenic regulatory sequences are implicated in muscle cell determination then they should be expressed in the dermomyotome of the immature somite which gives rise to muscle precursor cells.(ABSTRACT TRUNCATED AT 400 WORDS)


Molecular analysis of protein sorting during biogenesis of muscle cytoarchitecture. Isolated, rod-shaped adult rat cardiomyocytes (ARC) were kept in long-term cell cultures and the changes of the cardiomyocyte structure were investigated by confocal microscopy. The cells round up and make contact with the substrate by very flat, foot-like structures. After prolonged culture the amorphous cells regenerate a cardiomyocyte-like cytoarchitecture and myofibrils reemerge. In the perinuclear region myofibrils form continuously while in other cells discontinuous myofibrillogenesis was observed, where short sarcomeric segments occur all over the cytoplasmic space. During the regeneration of myofibrils certain proteins like a smooth muscle actin sort to non sarcomeric region, while myomesin or heart C-protein localize on myofibrils with high specificity. This culture system combined with method of epitope-tagging of contractile proteins are ideally suited to monitor the intracellular localization sites of exogenously introduced constructs to different cytoskeletal, since ARC exhibit at the same time stress fiber-like filaments (SFLF) and nascent myofibrils. The molecular properties of the different members of the myosin light chain isoprotein family were investigated by transfection experiments using epitope-tagged myosin light chain (MLC) cDNA. The sorting of the different types of MLC was shown to be isoprotein specific and with chimeric constructs it was shown that the isoprotein-specific incorporation into myofibrils was dependent on the presence of the middle domain of MLC-1f/3f. These MLC isoproteins can be arranged into a sequence of increasing affinity to myofibrils. A hierarchical order of myofibrillar assembly is postulated based on the association affinity. Similar experiments with constructs containing alpha-cardiac, alpha-smooth muscle and gamma-cytoplasmic actins have shown that expression of epitope-tagged actins in ARC result in different epitope staining patterns. While the alpha-cardiac actin showed a marked preference for sarcomeres, the alpha-smooth muscle isoproteins had an intermediate specificity and could either be preferentially incorporated into stress fiber-like filaments (SFLF) and in some cells to a lesser extent into myofibrils as well. Most striking results were obtained with gamma-cytoplasmic actin carrying a 5 or 11-mer epitope. This actin gave rise to large cells, induced the formation of filopodia filled with the transfected actin and depletion of the transfected actin from the perinuclear myofibrillar region.


Cardiac troponin T gene expression in muscle. We have been analyzing the regulatory regions of the cardiac troponin T gene promoter as a mean toward understanding the mechanisms that govern the transcription of genes which are cross-expressed in cardiac and skeletal muscles during development. By analyzing the activities of mutant cardiac troponin T gene promoter by transient transfection of primary embryonic muscle cells, we showed that both common and distinct elements are required for activity of the cardiac troponin T promoter in these embryonic muscle cells. In skeletal muscle the minimal promoter sufficient to direct activity of the cardiac troponin T promoter is only 99 nucleotides upstream from the transcription initiation site. Within the distal half of this promoter are two tandem copies of a conserved hexanucleotide sequence (5'-CATTCCT-3') we termed the 'M-CAT motif'. Since mutation of either one of the M-CAT motifs abolishes promoter activity, we concluded that both M-CAT motifs are essential for activity of the promoter. The above minimal promoter is insufficient to confer promoter activity in embryonic cardiocytes. In these cells an additional 48-nucleotide region approximately 100 nucleotides upstream of the minimal promoter is needed for efficient promoter activity. We have named this region the 'cardiac element'. This element contains a conserved sequence motif found in other muscle gene promoter. The cardiac element can also act irrespective of orientation and is relatively independent of position, characteristics that are like transcriptional enhancers. This element alone, however, is insufficient to direct cardiac promoter activity. Activity of the 48-nucleotide cardiac element is dependent on either direct or indirect interaction with the downstream M-CAT motifs because mutation of either M-CAT motif also abolishes promoter activity in cardiac cells. The third regulatory region (nucleotide position -550 to -268) is not essential for promoter activity but can enhance activity of the cTNT promoter or a heterologous promoter in both cardiac and skeletal muscle cells by three to five folds. Within this region are sequences which show similarity to motifs found in other muscle gene enhancers. In vitro DNA-protein binding studies showed that the above three regulatory regions interact with nuclear factors. A direct correlation exists between promoter activity and sequence specific binding of a nuclear factor we termed the 'M-CAT binding factor' to the M-CAT motifs. Similar interaction of nuclear regulatory molecules with sequences within the cardiac element and the upstream enhancer region is likely to be the mechanisms which control the action of these regulatory regions.(ABSTRACT TRUNCATED AT 400 WORDS)


Regulation of myosin heavy chain and actin isogenes during cardiac growth and hypertrophy. Expression of myosin heavy chain (MHC) and actin multigene families changes in mammals during cardiac growth and hypertrophy, but whether or not there is a common regulatory pathway is unclear. To address this question, we have looked at the alpha- and beta-MHC, and at the alpha-skeletal and alpha-cardiac actin (alpha-skel act and alpha-card act) isomRNA transitions during development and senescence, both in rat and human hearts. Since the precise amounts of each isoactin mRNA were not precisely known in the above situations, we first analyzed the time- course of accumulations of the two sarcomeric transcripts by primer extension assays, which allow an umambiguous quantification of the ratios of the two actin transcripts. In rats, both isogenes are expressed in-utero. alpha-skel act represents 40% of the total one week after birth, remains constant for 3 weeks, decreases to less than 5% at two months and does not re-accumulate thereafter. In humans, in contrast, alpha-skel act represents < 20% in-utero and in neonates, increases to 48% during the first decade after birth and becomes the predominant isoform of adult hearts. In rats beta-MHC mRNAs accumulate at birth, become undetectable at 3 weeks and reaccumulate to as much as 80% during senescence, and in humans beta-MHC mRNAs predominate throughout all developmental stages. These data show that in both species, the multigene families encoding the major contractile proteins are not coordinately regulated during development and aging.(ABSTRACT TRUNCATED AT 250 WORDS)


Carbonic anhydrase 3 (CA3), a mesodermal marker. Carbonic anhydrase 3 (CA3) is an abundant muscle protein characteristic of adult type 1, slow-twitch, fibres. The protein plays an important role in facilitated CO2 diffusion and diverse processes involving H+ and HCO-3 transport. Nucleotide sequence comparisons have identified putative promoter and enhancer regions in the 5' flanking sequences of the CA3 gene. Functional assays show that 2.8kb of 5' flanking sequence efficiently promotes transcription of a reporter gene in a muscle specific manner. Removal of sequences 5' to -722bp leads to a major loss of activity and this result implies that the proximal promoter region which includes a GArG box and four potential MyoD1 binding sites is not adequate for maximal transcription. The longest CA3 promoter construct is also active in 10T1/2 cells, which are precursor mesodermal cells and do not normally express CA3. In situ hybridization to mRNA in developing mouse embryos reveals a pattern of expression in myotomes and pre-muscles masses of the limb buds which is consistent with the regulation of CA3 by myogenic determination factors. These studies also showed that CA3 expression is not confined to cells of the muscle lineage since it is expressed in primitive mesoderm prior to the onset of myogenesis. Later in embryogenesis CA3 defines a subset of mesodermal cell types which includes not only skeletal muscle but also notochord and adipocytes.


A molecular approach towards the understanding of early heart development: an emerging synthesis. In the past decade we have made an inventory of the changing three-dimensional patterns of expression of a number of key proteins involved in contraction, energy metabolism and conduction in developing and adult chicken, rat, bovine and human hearts. These integrated morphological and immunohistochemical studies were complemented with electrophysiological studies in developing chicken hearts and have resulted in a preliminary model of heart development, that explains how the embryonic heart can function without valves and without an atrioventricular conduction system that is indispensable for the adult heart. Cardiomyocyte-specific proteins are first expressed in the cardiogenic plate when 6 somites have developed, while electrical activity becomes detectable only slightly later. Development proceeds as follows: 1. Upon its formation 'primary' myocardium is characterised by anteroposterior gradients in gene expression. Therefore cardiogenesis resembles many other developmental processes in the embryo. It serves as source for endocardial cells and cells specialized in mechanical contraction and in impulse generation/conduction supporting the view that a single population of cells (the 'primary' myocardium) serves as a precursor for these distinct cell types. 2. 'Primary' myocardium is characterized by the expression of alpha and beta myosin, acetylcholinesterase and the absence of fast sodium channels and of connexin 43. It has a peristaltoid contraction form due to a relatively slow propagation of the impulse. 3. In the looping stage, two cardiac segments appear due to the development of atrial and ventricular working myocardium, that is characterized by the expression of either alpha or beta myosin, connexin 43, fast sodium channels, the disappearance of acetylcholinesterase and by a relatively fast conduction.2+ sinuatrial and atrioventricular nodes.


Reciprocal changes in myosin isoform expression in rabbit fast skeletal muscle resulting from the application and removal of chronic electrical stimulation. Chronic indirect electrical stimulation of adult mammalian skeletal muscle brings about a transformation from the fast-twitch to the slow-twitch type. Underlying this transformation there is a sequence of profound changes in the expression of proteins involved in all the major molecular systems of the muscle. These include qualitative changes in the expression of myosin light and heavy chain isoforms. The time course of these changes has been studied in some detail at the protein level, both during chronic stimulation and during the recovery process that follows the cessation of stimulation. Here we report on the use of cDNA probes to study corresponding changes in myosin heavy chain (MHC) and light chain (MLC) mRNAs in rabbit fast-twitch muscles during continuous electrical stimulation at 10 Hz and during the first 12 days of recovery after cessation of 6 weeks of stimulation. At an early stage of the response to stimulation, fast MHC mRNA is replaced by slow MHC mRNA. During recovery this process occurs in reverse but takes longer. Broadly similar changes are seen for MLC mRNAs, although the time course is somewhat different. These experiments contribute to a growing body of evidence that many of the protein changes induced by chronic stimulation are the result of regulatory events that take place at a pre-translational level.


Fast-to-slow transition in myosin heavy chain expression of rabbit muscle fibres induced by chronic low-frequency stimulation. An in situ-hybridization assay using a digoxigenin-labeled cRNA probe specific for the slow myosin heavy chain (HCI) was established. Type I fibres of normal rabbit muscles were stained with this probe. The reaction product was confined to the perinuclear regions of the subsarcolemmal space and extended along the I-bands into the fibre core region. Myosin HCI mRNA was also detected in transforming fibres of low-frequency stimulated rabbit fast-twitch muscles. Its intracellular distribution resembled that of normal type I fibres, but higher amounts of the message were present in fibres undergoing a fast-to-slow transition. The number of HCI mRNA-positive fibres in stimulated muscles increased in a time-dependent manner and correlated with the amount of myosin HCI protein in these muscles. These findings support the notion that enhanced transcription of the slow myosin HC gene leads to an increased translation of HCI mRNA during the stimulation-induced fibre transformation. Finally, the progressive increase in fibres expressing myosin HCI mRNA indicates that the fast-to-slow fibre conversion occurs in a sequential manner. The pre-existing type IIA fibres appear to transform first, whereas fibre types IIB and IID have to first reach the IIA state. Adult muscle fibers represent versatile entities and may be transformed in response to altered functional demands. Although the majority of normal muscle fibers express only a single myosin HC isoform (for review see Pette and Staron, 1990), the coexistence of two or more myosin HC isoforms has been shown in transforming adult muscle fibers (Staron, Gohlsch, Pette, 1987; Termin, Staron, Pette, 1989).(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of insulin-like growth factor 1 gene expression in skeletal muscle. Insulin-like growth factor 1 (IGF-1) is implicated in the growth processes of many tissues in the adult animal. This hormone can act in an endocrine manner or can be produced in the specific tissues in response to growth promoting stimuli to act in an autocrine/paracrine manner. We have examined, in the rat, changes in serum concentrations of IGF-1 and muscle IGF-1 mRNA levels in several studies in which muscle growth has been significantly altered. In the first study we examined the interactions of growth hormone (GH) and under-nutrition upon muscle growth. We observed that when GH was administered to hypophysectomised rats the anabolic effect of this hormone was independent of dietary intake. In a similar manner muscle IGF-1 mRNA levels were also elevated by GH but unaffected by food intake. In contrast serum IGF-1 levels were markedly reduced by under-nutrition. These data suggested that the anabolic action of GH on muscle could be mediated through the autocrine/paracrine action of the IGF-1 hormone. Similarly we observed in other studies that muscle hypertrophic stimuli of work-overload and passive stretch are associated with significantly increased muscle IGF-1 mRNA levels. In contrast insulin dramatically affected muscle protein synthesis rates but had no measurable effect upon muscle IGF-1 mRNA levels, which suggests that the anabolic action of this hormone is not mediated through the autocrine/paracrine action of IGF-1. These studies suggest that IGF-1 may mediate growth in muscle in response to variety of stimuli by autocrine/paracrine action or in response to certain stimuli possibly by endocrine action.


Activation of muscle-specific transcription by myogenic helix-loop-helix proteins. Myogenin is a muscle-specific transcription factor that acts as a molecular switch to induce myogenesis. Myogenin shares homology with MyoD and other myogenic regulatory proteins within a basic region and helix-loop-helix (HLH) motif that mediate binding to a conserved DNA sequence (CANNTG) present in the regulatory regions of numerous muscle-specific genes. Binding of myogenin and other members of the MyoD family to DNA can be augmented upon heterodimerization with the widely expressed HLH protein E12. We have used the muscle creatine kinase (MCK) enhancer as a target to study the mechanism whereby myogenin activates muscle-specific transcription. Full activity of the MCK enhancer requires cooperative interactions between myogenin (or other myogenic HLH proteins that bind the same site) and a complex array of ubiquitous and cell type-specific nuclear factors. To define the domains of myogenin responsible for sequence-specific DNA binding, activation of muscle-specific transcription, and cooperativity with other transcription factors, we have generated an extensive series of mutants by site-directed mutagenesis and domain swapping. These mutants have revealed strong transcriptional activation domains in the N- and C-termini of myogenin that rely on a specific amino acid sequence within the DNA binding domain for activity. Myogenin's ability to induce muscle-specific transcription is subject to negative regulation by growth factor and oncogenic signals. Mechanisms through which growth signals may repress myogenin function are discussed.


Positive and negative gene regulation in muscle. By the analysis of cis and trans-acting element involved in transcriptional regulation of chicken myosin alkali light chain genes, we have identified the MLC box, muscle specific enhancer element and negative regulatory element. The MLC box is an essential element for the expression of MLC genes located at approximately 100 bp upstream from mRNA start sites. The core sequence of MLC box is similar to the consensus of actin gene CArG box and SRE of c-fos oncogene. In vitro DNA-protein binding assay has revealed that the MLC box, CArG box and SRE might bind to a common or a similar protein complex. CMD1, cMyogenin, and cMRF4 transactivate the promorter with an intact MLC box, but not the promoter lacking MLC box, indicating that the MLC box itself is transactivated by the myogenic regulatory factors. This transactivation must have been due to the indirect effect of the myogenic regulatory factors, because chicken myogenic factors do not bind to the MLC box. A cis element identified at about 150 bp upstream from the cap site of cardiac MLC gene suppresses the cardiac MLC gene expression in skeletal muscle cells but not in cardiac muscle cells. The protein(s) bound to NRE might be identical with one of proteins bound to SRE. NRE may block the function of MLC box and resultantly inhibits the expression of cardiac MLC1 gene in skeletal muscle cells. Skeletal muscle enhancer at -2 kb of skeletal MLC1f gene is composed of two subelements P and D, cooperative action between them is required for sufficient enhancer activity. CMD1 and myogenin bind to the enhancer sequences of skeletal MLC1 gene and MCK gene and transactivate these genes preferentially in skeletal muscle cells. In addition to the CMD1 responsible enhancer, another cis-element is required for transactivation of the MLC1f gene by cMyogenin. An E-box adjacent to MLC box may co-work with the enhancer to increase the expression of MLC1f gene. Muscle specific and developmentally regulated expression of MLC gene family is regulated by the combination of these cis and trans-acting elements.


Cis regulating elements which control in vivo alternative splicing of the chicken beta tropomyosin primary transcript. The beta tropomyosin gene of the chicken contains a pair of alternatively spliced mutually exclusive exons the use of which is developmentally regulated. Exon 6A is used by non muscle and undifferentiated muscle cells (myoblasts) while exon 6B is exclusively used in differentiated skeletal muscle cells. A complex array of cis acting sequence elements are involved in the regulation of this alternative splicing process. Transfection assays of quail muscle cells in culture were used to define these cis acting elements. We show that, in undifferentiated muscle cells, exon 6B is skipped as a result of a negative control on its selection while exon 6A is spliced as a default choice. We provide evidence that this negative control involves a secondary structure of the primary transcript around the 5' end of exon 6B as well as intronic sequence elements located between the branch point and the acceptor splice site of exon 6B. In differentiated muscles, both exons are accessible to the splicing machinery and the preferential use of exon 6B depends on the existence of a competition between the two exons for the selection of the flanking splice sites. In particular, we show that the donor splice site of exon 6A is a weak splice site while the branch point associated with exon 6B is a strong branch point.


Regulation of myogenin expression in normal and transformed myogenic cell lines. The control of myogenin (Myf-4), one of the muscle-specific regulatory proteins, is particularly interesting since its expression appears obligatory in myoblasts at the onset of differentiation. We isolated the human Myf-4 (myogenin) gene and determined promoter elements which direct cell type-specific expression and are subject to transactivation by the muscle transcription factors Myf-5 and MyoD1 in fibroblasts. Extrinsic signals such as serum components and purified growth factors or potential intracellular signals such as cAMP down-regulate transcription of the myogenin gene. Constitutive expression of the catalytic subunit of PKA completely suppresses transactivation of the myogenin promoter by Myf-5 or MyoD1 suggesting that cAMP may act via phosphorylation by PKA. In contrast to normal myogenic cell lines in which differentiation and myogenin expression can be induced by the removal of serum components, retinoic acid (RA) is required for differentiation in the rat rhabdomyosarcoma cell line BA-Han-1C. This model system was utilized to investigate factors which influence the balance between the transformed state and differentiation. Administration of retinoic acid to BA-Han-1C cells leads to the accumulation of myogenin mRNA approximately 48 h after the addition of RA. This late induction requires ongoing protein- and DNA-synthesis suggesting that trans- and cis-acting factors may be involved in the control. The critical involvement of myogenin in the process of terminal muscle differentiation was also demonstrated in the rat L6 muscle cell line which has been blocked for differentiation by the transforming protein E1a of Ad5 adenovirus. In cells which stably express E1a, myogenin expression is completely suppressed while Myf-5 continues to be synthesized normally. However, E1a inhibits the transactivator function of Myf-5, as demonstrated on GAL4-Myf5 chimeric proteins. A possible interpretation of this result is that Myf-5 or factors activated by Myf-5 are required for the expression of myogenin and myogenin itself is necessary for the terminal differentiation of myoblasts.


Inhibition of in vitro muscle differentiation by the immortalizing oncogene py LT-ag. The interference of Polyomavirus (Py) early functions with in vitro myogenic differentiation is the object of this study. Single cell analysis of C2 myogenic Py infected cells showed a mutual exclusion between Py early functions and muscle gene expression. The morphological and biochemical analysis of clones obtained from C2 cells stably transfected with a plasmid carrying an ORI- Py genome, showed that myogenesis is blocked and cells display the transformed phenotype. By using plasmids separately encoding Middle T or Large T functions, the involvement of individual early viral gene products was determined. Py Middle T alone does not inhibit myotube formation even though cells are morphologically transformed. Myogenic differentiation, on the other hand, is inhibited by Py Large T. This inhibition, which is proportional to the level of Py Large T expression, does not entail to require alteration of cell growth properties and acts by blocking the expression of myogenin and terminal differentiation markers without altering the expression of the regulatory gene MyoD.


A cell division counter exists in the chick embryo myogenic lineage. A computer assisted timelapse video microscopy system was developed to study the behaviour of individual chick embryo myogenic cells in culture. With this system it is possible to monitor and photograph the development of 50-100 myogenic clones simultaneously. Photos were made with a frame grabber and image compressor and were stored in digital form on the computer's hard disc. Colonies derived from 10-day chick embryo myogenic were followed over a period of 3 days. The cultures were then fixed, reacted first with a mouse monoclonal antibody against sarcomeric myosin, then with peroxidase-labelled goat anti-mouse IgG, and stained with diaminobenzidine to detect antibody binding. The dishes were then replaced into the video system and the computer was used to find the originally photographed colonies. Cells which bound the antibody against sarcomeric myosin were identified in the last image stored for each colony. The computer was then used to retrieve the images from each colony in reverse order and to determine the lineage ancestry of every antibody-positive cell in each clone. Several hundred colonies and many hundreds of cell divisions have been examined thus far. In all but one clone, every antibody-positive cell was produced by a symmetrical cell division in which its sister was also antibody-positive (or died). Antibody-positive cells were generated by a sequence of no more than 4 symmetrical cell divisions. Thus, most colonies consisted of 1, 2, 4, 8 or 16 nuclei in terminally differentiated muscle cells. Those colonies in which cell numbers was not a power of 2 were always produced by cell death or detachment.(ABSTRACT TRUNCATED AT 250 WORDS)


Multimeric structures influence the binding activity of bHLH muscle regulatory factors. Sucrose gradients and molecular sieve chromatography were used to determine the native molecular weight of the basic HLH proteins myogenin, MyoD and E12. The muscle bHLH proteins not only formed dimers but also associated in a variety of higher order complexes. Although homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer, homotetramers and larger forms do not recognize this DNA sequence. Little evidence for complexes larger than dimers was found for the ubiquitous bHLH protein E12. Most of the myogenin remains in large complexes when myogenin and E12 are mixed. The same result was obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin was found to be present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein that contains only the myogenin HLH region fused to glutathione-S-transferase also forms large homomeric complexes. A model to explain these results is that each helix of the HLH motif can associate with a different subunit to form chains or ring structures. The presence of myogenin in nuclear extracts as both dimers that recognize known DNA sequences as well as higher order complexes that do not raises significant issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.


A simple and rapid method to study the association of the contact proteins of blood coagulation. Native and reduced SDS polyacrylamide gel electrophoresis on the automated PhastSystem (Pharmacia) were used to demonstrate protein-protein binding interactions and structural changes during proteolytic activations of the proteins involved in contact activation. The "mobility shift" assay in native gels has been used to visualize the kinetics of activation of factor XII by dextran sulfate as well as the formation of kallikrein-cleaved high molecular weight kininogen. It shows the formation of prekallikrein-high molecular weight kininogen complexes and factor XII-dextran sulfate complex for the first time in gels. The use of automation makes this procedure fast and reproducible using nanogram amounts of protein in relatively short time.


Protein C and protein S levels in uremic patients before and after dialysis. The inhibitory capacity of the natural protein C (PC)/protein S (PS) system was evaluated measuring both the functional activity and the antigen level of both these inhibitors in 30 uremic patients before and after a dialytic treatment and in 30 healthy normal volunteers. PC functional activity was determined by two methods, one clotting and one chromogenic. PS antigen level was measured both as free protein and as total content. Unlike previous authors, we found that PC functional activity and the antigen level were normal in patients before dialysis, with a significant increase after. PS functional activity and free and total antigen levels were all normal before dialysis, and all except free antigen showed a significant post-treatment rise.


Heparan sulfate as a venostatic endothelial stabilizing factor. A humoral transfer of a factor inducing a decrease of circulating endothelial cells (CEC) released during venostasis was demonstrated in rats. The possibility to block its activity by an in-vitro addition of protamine suggests its identity with an endogenous heparan sulfate possessing an inhibitory effect on endothelial turnover. This was supported by an analogous effect of intravenously administered heparan sulfate-related agent.


Increased phosphate content of fibrinogen in vivo correlates with alteration in fibrinogen behaviour. Fibrinogen was purified from five patients admitted for hip-replacement surgery the day before (day 0), the day after (day 2) and and one week after the operation (day 8). The behaviour of each patient's three fibrinogens was compared in thrombin gelation assays and plasmin degradation experiments to investigate whether the reported increase in protein-bound phosphate at day 2 and day 8 had any effect on the functional behaviour of fibrinogen as has been demonstrated in vitro. It was found that the thickness of the fibrin fibres produced by thrombin increased markedly at day 2 and declined thereafter. Susceptibility to plasmin appeared to decrease post-operatively by 50% and remained at that level on day 8 despite the phosphate content returning to normal. This has also been shown for fibrinogen phosphorylated in vitro. We conclude, after testing the fibrinogens with and without alkaline phosphatase pretreatment, that our data most resemble the published findings for in vitro phosphorylation of fibrinogen by casein kinase II.


Haemostyptic effects of batroxobin with regard to hirudin treatment. In contrast to thrombin the fibrinogen coagulant effect of the thrombin-like enzyme batroxobin in vitro and in vivo is not inhibited by the specific thrombin inhibitor hirudin. The haemostyptic effect of batroxobin has been studied in rats after bleeding had been induced by corresponding hirudin dosages. Dependent on batroxobin concentration bleeding time was shortened by local application of batroxobin containing solutions. Strong bleeding induced by i.v. injection of 5 mg r-hirudin/kg was stopped almost immediately when a batroxobin concentration of 40 BU/ml was used. Thrombin was less active to stop bleeding after r-hirudin administration than batroxobin.


The ideal anti-thrombotic drug.

The ideal anti-thrombotic drug. An ideal antithrombotic drug should inhibit thrombosis without affecting hemostasis. It should have a long half life. It should be absorbed after oral administration; it should be safe and it should have a wide therapeutic range. These criteria are discussed using new antithrombotics as examples.


Calcium signaling mechanisms in the gastric parietal cell. Gastric hydrochloric acid (HCl) secretion is stimulated in vivo by histamine, acetylcholine, and gastrin. In vitro studies have shown that histamine acts mainly via a cAMP-dependent pathway, and acetylcholine acts via a calcium-dependent pathway. Histamine also elevates intracellular calcium ([Ca2+]i) in parietal cells. Both gastrin and acetylcholine release histamine from histamine-containing cells. In humans, rats, and rabbits, there is considerable controversy as to whether or not gastrin receptors are also present on the parietal cell. We utilized digitized video image analysis techniques in this study to demonstrate gastrin-induced changes in intracellular calcium in single parietal cells from rabbit in primary culture. Gastrin also stimulated a small increase in [14C]-aminopyrine (AP) accumulation, an index of acid secretory responsiveness in cultured parietal cells. In contrast to histamine and the cholinergic agonist, carbachol, stimulation of parietal cells with gastrin led to rapid loss of the calcium signaling response, an event that is presumed to be closely related to gastrin receptor activation. Moreover, different calcium signaling patterns were observed for histamine, carbachol, and gastrin, Previous observations coupled with present studies using manganese, caffeine, and ryanodine suggest that agonist-stimulated increases in calcium influx into parietal cells do not occur via voltage-sensitive calcium channels or nonspecific divalent cation channels. It also appears to be unlikely that release of intracellular calcium is mediated by a muscle or neuronal-type ryanodine receptor. We hypothesize that calcium influx may be mediated by either a calcium exchange mechanism or by an unidentified calcium channel subtype that possesses different molecular characteristics as compared to muscle, nerve, and certain secretory cell types such as, for example, the adrenal chromaffin cell. Release of intracellular calcium may be mediated via both InsP3-sensitive and -insensitive mechanisms. The InsP3-insensitive calcium pools, if present, do not appear, however, to possess ryanodine receptors capable of modulating calcium efflux from these storage sites.


Acid secretion and the H,K ATPase of stomach. The regulation of acid secretion was clarified by the development of H2-receptor antagonists in the 1970s. It appears that gastrin and acetylcholine exert their effects on acid secretion mainly by stimulation of histamine release from the enterochromaffin-like (ECL) cell of the fundic gastric mucosa. The isolated ECL cell of rat gastric mucosa responds to gastrin/cholecystokinin (CCK), acetylcholine, and epinephrine with histamine release and to somatostatin and R-alpha-methyl histamine by inhibition of histamine release. Histamine and acetylcholine stimulate the parietal cell by elevation of cAMP or [Ca]i by activation of H2 or M3 receptors, respectively. These independent pathways converge to activate the gastric acid pump, the H+,K+ ATPase. Activation is a function of the association of the ATPase with a potassium chloride transport pathway that occurs in the membrane of the secretory canaliculus of the parietal cell. Hence the secretory canaliculus is the site of acid secretion, the acid being pumped into the lumen of the canaliculus. The pump is composed of two subunits, a large catalytic and a smaller glycosylated protein. This final step of acid secretion has become the target of drugs also designed to inhibit acid secretion. The target domain of the benzimidazole class of acid pump inhibitors is the extracytoplasmic domain of the pump that is secreting acid, and the target amino acids are the cysteines present in this domain. The secondary structure of the pump can be analyzed by determining trypsin-sensitive bonds in intact, cytoplasmic-side-out vesicles of the ATPase, and it has been shown that the alpha subunit has at least eight membrane-spanning segments. Omeprazole, the first acid pump inhibitor, forms a disulfide bond with cysteines in the extracytoplasmic loop between the fifth and sixth membrane-spanning segment and to a cysteine in the extracytoplasmic loop between the seventh and eight segments, preventing phosphorylation of the pump by ATP. As a result of the effective and long-lasting inhibition of acid secretion by the acid pump inhibitor, superior clinical results have been found in all forms of acid-related disease.


The gastric H3 receptor: a review.

The gastric H3 receptor: a review. Previous in vivo and in vitro studies from our laboratory have revealed a line of pharmacological evidence supporting histamine H3 receptor(s) involvement in the control of gastric acid secretion. We have recently extended our studies to the human gastric tumoral cell HGT-1. This cell was found to contain an H3 receptor inhibiting basal and carbachol-stimulated inositol phosphate formation. Furthermore, we were able to solubilize and affinity-purify this receptor in the form of a single 70 kDa protein. These findings are the first biochemical description of the H3 receptor subtype and the first direct demonstration that this subtype can occur on a non-neural cell. Furthermore, they provide a molecular basis to explain its suggested inhibitory role in gastric physiology.


Small GTP-binding proteins in parietal cells: candidate modulators of parietal cell membrane dynamics. The stimulated fusion of intracellular H/K-ATPase-containing tubulovesicles with a target canalicular membrane surface is central to the process of acid secretion. A super-family of small GTP-binding proteins (smGTPBPs) has been implicated in many aspects of intracellular dynamics and vesicle membrane trafficking. We have investigated the presence of smGTPBPs in isolated rabbit parietal cells. Parietal cells possess a number of smGTPBP species with molecular masses of 18-28 kDa. One 23 kDa smGTPBP has been localized to tubulovesicles and identified immunochemically as rab2. Rab2 redistributes during stimulation in concert with the movement of the H/K-ATPase. The results demonstrate that specific smGTPBPs are associated with the parietal cell secretory apparatus. Small GTP-binding proteins are important candidate regulators of parietal secretory membrane dynamics.


Pathophysiology and clinical relevance of Helicobacter pylori. Considerable knowledge has recently accumulated on the mechanism by which Helicobacter pylori (H. pylori) induces chronic gastritis. Although H. pylori is not an invasive bacterium, soluble surface constituents can provoke pepsinogen release from gastric chief cells or trigger local inflammation in the underlying tissue. Urease appears to be one of the prime chemoattractants for recruitment and activation of inflammatory cells. Release of cytokines, such as tumor necrosis factor alpha, interleukin 1 and 6, and oxygen radicals, leads to a further tissue inflammation accompanied by a potent systemic IgA and IgG type of immune response. Chronic inflammation and antigens on glandular epithelial cells lead to a progressive destruction with loss of the epithelial barrier function. Within the gastric mucosa, patches of intestinal metaplasia develop, which may be a risk factor for subsequent development of gastric carcinoma. Hyperacidity in duodenal ulcer patients induces gastric metaplasia in the duodenal bulb, which represents a target for H. pylori colonization and ulcer formation. H. pylori can be detected in the majority of patients with peptic ulcers and, compared to age-matched healthy people, it is also found more often in patients with dyspepsia and gastric carcinoma. Although H. pylori can be detected in healthy people, the marked reduction of the ulcer recurrence rate by eradication of H. pylori (80 percent versus 20 percent relapse within one year) suggests that H. pylori is a major risk factor for duodenal ulcer formation. The potential role of H. pylori in non-ulcer dyspepsia and carcinogenesis is under investigation. Current regimens aimed at eradicating H. pylori use a combination of several drugs that are potentially toxic. Since the risk of complications may exceed the potential benefit in most patients, eradication treatment should be limited to clinical trials and to patients with aggressive ulcer disease. New drug regimens, e.g., the combination of proton pump inhibitors with one antibiotic, may provide less toxic alternatives. Beyond ulcer treatment, effective and well-tolerated eradication regimens may have a place in prophylaxis of gastric carcinoma.


Progress with proton pump inhibition.

Progress with proton pump inhibition. The proton pump, a H+/K(+)-ATPase located on the secretory canalicular membrane of the parietal cell, forms the final pathway for gastric acid secretion. Omeprazole is concentrated in the secretory canaliculus, where it is converted to its active form, which binds covalently with the H+/K(+)-ATPase, thus inhibiting acid secretion arising from any stimulus. Meta-analysis has defined the primary determinants for peptic ulcer healing as the degree of acid suppression, the duration of suppression over 24 hours, and the length of treatment. The longer duration of acid suppression with omeprazole, particularly during the day, when food is ingested and H2-receptor antagonists are less effective, is reflected in the clinical superiority for symptom relief and ulcer healing and especially for the treatment of erosive esophagitis. Extensive clinical experience has proved omeprazole to be safe, and concerns over hypergastrinemia, ECL-cell hyperplasia, and carcinoid formation have not been substantiated in humans. Recent evidence has shown that omeprazole suppresses Helicobacter pylori and, in combination with antibiotics, can eradicate this organism in a substantial proportion of patients. This effect may result from enhancement of antibiotic bioavailability and optimizing host defense mechanisms.


Pathobiology and management of hypergastrinemia and the Zollinger-Ellison syndrome. Gastrin is both stimulatory and trophic to the cells of the gastric fundus--parietal and peptic cells, and enterochromaffin-like (ECL) cells which are major intermediaries of the gastrin effect. Gastrin (from the antrum) and acid (from the fundus) represent the interactive positive and negative limbs of a feedback loop. The nature and extent of sub-loops, perhaps involving the vagus, acetylcholine, histamine, and other peptides and cell products are at present unclear or unknown. Loss of either gastrin or acid has predictable consequences. Absent acid, as in pernicious anemia or as a result of omeprazole, leads to hypergastrinemia. In rats, such hypergastrinemia (gastrin > 1,000 pg/ml) causes fundic ECL hyperplasia and, eventually, carcinoids; in humans with pernicious anemia, hypergastrinemia causes ECL-cell hyperplasia, which may progress to carcinoids that are reversible upon withdrawal of gastrin, illustrated by three cases described here. Loss of gastrin by antrectomy for duodenal ulcer leads to fundic involution and marked reduction in basal acid output, maximal acid output, and fundic histamine. An uncontrolled excess of gastrin, as from a gastrinoma outside the negative feedback loop, causes acid and pepsin hypersecretion with upper GI mucosal damage, the Zollinger-Ellison syndrome. This paper summarizes the abnormal regulation of gastrin and the biology, natural history, diagnosis, and management of ZE syndrome by medical and surgical means.


Current concepts in gastric microcirculatory pathophysiology. When the barrier to acid back-diffusion is disrupted, there is a protective increase in gastric mucosal blood flow to help remove the back-diffusing acid. Only recently has the mechanism for calling forth this protective hyperemia been determined. The gastric mucosa and submucosa are innervated by many capsaicin-sensitive sensory nerve fibers containing vasodilator peptides. The gastric mucosal sensory neurons monitor for acid back-diffusion, and, when this process occurs, signal for a protective increase in blood flow via release of calcitonin gene-related peptide from the submucosal periarteriolar fibers. The endothelium-derived vasodilator, nitric oxide, plays an important role both in the maintenance of basal gastric mucosal blood flow and in the increase in blood flow that accompanies pentagastrin-stimulated gastric acid secretion. It also interacts with the capsaicin-sensitive sensory nerves in the modulation of the microcirculation to maintain mucosal integrity. Finally, it has been shown that neutrophils play an important role in various forms of mucosal injury. The leukocytes adhere to the vascular endothelium and contribute to injury by reducing blood flow via occlusion of microvessels, as well as by releasing mediators of tissue damage.


Roles for transforming growth factor-alpha in gastric physiology and pathophysiology. Transforming growth factor alpha (TGF alpha) is a 5.6 kd single-chain polypeptide that acts through binding to the epidermal growth factor receptor (EGFR). TGF alpha is produced in a wide range of normal as well as embryonic and neoplastic cells and tissues. TGF alpha and EGFR, but not EGF, are expressed in normal gastric mucosa. We have identified the following biological roles for TGF alpha in the stomach, using a variety of primate and rodent models: inhibition of acid secretion; stimulation of mucous cell growth; protection against ethanol- and aspirin-induced injury. This last effect is associated with a time- and dose-dependent increase in levels of insoluble gastric mucin. Based on these known biological actions of TGF alpha, we have examined TGF alpha production in Ménétrier's disease, a disorder characterized by foveolar hyperplasia, hypochlorhydria, and increased gastric mucin content. In four patients with Ménétrier's disease, there was enhanced TGF alpha immunostaining throughout the gastric mucosa. Furthermore, metallothionein (MT)-TGF alpha transgenic mice which overproduce TGF alpha in the stomach exhibit histopathological and biochemical features characteristic of and consistent with the diagnosis of Ménétrier's disease. Thus locally produced TGF alpha may mediate a number of biological processes in the stomach, and its altered production may participate in the pathogenesis of selected pathological states.


Function and regulation of gastrin in transgenic mice: a review. The gastrin gene is expressed in fetal pancreatic islet cells, but in the adult is expressed mainly in the gastric antrum. To study the regulation of the gastrin promoter, we created several transgenes containing the human and rat gastrin 5' flanking regions joined to the coding sequences of the human gastrin gene. The human gastrin transgene contained 1,300 bp of 5' flanking DNA, while the rat gastrin transgene contained 450 bp of 5' flanking DNA. The human gastrin transgene was expressed in fetal islets, but was not expressed in adult gastric antrum. In contrast, the rat gastrin transgene was expressed in adult antral G cells, but no expression was observed in fetal islets. To study the possible role of gastrin as an islet growth factor, a chimeric insulin-gastrin (INS-GAS) transgene was created, in which the expression of the human gastrin gene is driven from the rat insulin I promoter. These INS-GAS mice were mated with mice overexpressing TGF alpha, transcribed from a mouse metallothionein-transforming growth factor alpha (MT-TGF alpha) transgene. While overexpression of gastrin or TGF alpha alone had no effect on islet mass, overexpression of both transgenes resulted in a twofold increase in islet mass. In conclusion, these data indicate that (1) gastrin can interact synergistically with TGF alpha to stimulate islet growth; (2) the human gastrin transgene contains the islet specific enhancer; (3) the rat gastrin transgene contains the antral specific enhancer.


Therapeutic potential of growth factors and their antagonists. This article describes studies with four peptides, epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha), gastrin-releasing peptide/bombesin (GRP), and gastrin. The mitogenic and anti-secretory activities of EGF/TGF alpha appear to be mediated by a single class of high-affinity membrane receptors but may involve different signal transducing mechanisms. Biological activity of EGF resides in the N-terminal 42 amino acid fragment with the C-terminal undecapeptide determining binding affinity. A parenteral depot formulation of an EGF-related peptide or a small molecule agonist of the EGF receptor could have utility in treating various ulcerative disorders of the gut. Although antagonism of EGF (and thus TGF alpha) receptors and/or transducing mechanisms is frequently cited as a potential therapeutic approach to hyperproliferative diseases, blocking the action of TGF alpha, GRP, or gastrin with neutralizing antibodies or receptor antagonists did not influence the growth of a wide range of solid tumors in nude mice. These findings suggest that, unless tumor growth displays absolute dependency on one particular mitogen, antagonism of a specific growth factor is unlikely to have great effect in cancer therapy.


Structure, regulation, and pathophysiology of tight junctions in the gastrointestinal tract. The tight junction, or zonula occludens, forms an intercellular barrier between epithelial cells within the gastrointestinal tract and liver and, by limiting the movement of water and solutes through the intercellular space, maintains the physicochemical separation of tissue compartments. The paracellular barrier properties of junctions are regulated and quite different among epithelia. The junction also forms an intramembrane barrier between the apical and basolateral membrane domains, contributing to segregation of biochemically distinct components of these plasma membrane surfaces. Here we briefly review three rapidly developing areas of medically relevant basic knowledge about the tight junction. First, we describe the presently incomplete knowledge of the molecular structure of the tight junction as a framework for understanding its functional properties. Second, we consider experimental evidence defining how the barrier properties of junctions are physiologically regulated and, third, how barrier properties are specifically altered in, and contribute to, pathologic processes affecting epithelia.


Praomys (Mastomys) natalensis: a model for gastric carcinoid formation. The gastric carcinoid tumors of Praomys (Mastomys) natalensis have been reviewed with respect to histogenesis, development, biochemistry, and morphological properties. Multicentric gastric carcinoids frequently develop in the oxyntic mucosa of aging Mastomys. The development of these tumors can be significantly enhanced by drug-induced hypergastrinemia, e.g., histamine2-receptor blockade. Spontaneous and drug-induced gastric carcinoids are endocrine in nature, as evidenced by their argyrophilic staining properties and chromogranin A content. They are also rich in histidine decarboxylase activity and produce large amounts of histamine, although other hormones, such as peptide YY and enteroglucagon, have also been demonstrated in these tumors. Ultrastructurally, gastric carcinoids are composed of tumor cells with typical secretory granules resembling those of enterochromaffin-like (ECL) cells. A close examination of the gastric carcinoids in Mastomys reveals striking similarities with gastric carcinoids developing in humans suffering from chronic atrophic gastritis type A or from the Zollinger-Ellison syndrome in combination with multiple endocrine neoplasia type 1 (MEN-1). Both these conditions are associated with hypergastrinemia and a higher risk for developing multi-centric gastric carcinoids of ECL-cell origin. The Mastomys tumor model therefore appears to be a significant experimental model in which induction and formation of gastric carcinoid tumors can be studied.


The biology and pathobiology of the ECL cells. The enterochromaffin-like (ECL) cells represent the predominant endocrine cell population in the acid-producing part of the stomach of both experimental animals and man. These cells actively produce and store histamine in addition to an anticipated but as yet unidentified peptide hormone and are under the control of gastrin. An acute gastrin stimulus causes exocytosis of the cytoplasmic granules/vesicles (and release of histamine and activation of the histamine-forming enzyme, histidine decarboxylase), while a more sustained gastrin stimulus causes first hypertrophy and then hyperplasia of the ECL cells in the rat (at most, a fivefold increase in the cell number). These effects can be demonstrated following infusion of gastrin or following an increase in the concentration of circulating gastrin of endogenous origin. The growth of the ECL cells reflects an accelerated self-replication rate. As studied in the rat, the self-replication rate is accelerated quite soon after induction of hypergastrinemia (blockade of acid secretion), the rate is maximally elevated within two weeks and then declines to control values at ten and 20 weeks despite the sustained hypergastrinemia. Lifelong hypergastrinemia in rats is associated not only with ECL-cell hyperplasia but also with an increased incidence of ECL-cell carcinoids. Recently, we could show that alpha-fluoromethylhistidine, which is a suicide inhibitor of histidine decarboxylase, effectively depletes the ECL cells of histamine and that the histamine-depleted ECL cells respond to gastrin with hyperplasia in a manner identical to normal ECL cells. Other factors beside gastrin seem to participate in the control of ECL-cell function and proliferation. Although exogenous somatostatin is known to suppress the activity of the ECL cells, we have failed to obtain evidence that the somatostatin cells in the oxyntic mucosa play a role in the physiological control of the ECL cells. The vagus, however, is important for the ability of the ECL cells to respond to gastrin. This conclusion is based on the observation that vagal denervation suppresses the hyperplastic response of the ECL cells to gastrin. Porta-cava shunting, on the other hand, greatly enhances the responsiveness of the ECL cells to gastrin. The mechanism behind this effect is unknown.


The pathobiology of the human enterochromaffin-like cell. The significance of the enterochromaffin-like (ECL) cell as a critical endocrine regulator of gastric fundic mucosal function has only recently been recognized. Although the percentage of these cells present in the human fundic mucosa is less than that in rodents, the observation that they secrete histamine and are probably important modulators of parietal cell function has resulted in their attaining some considerable biological significance. The further identification of gastrin and somatostatin receptors on the surface of the ECL cells has suggested that other neurohormonal influences may be significant in the regulation of parietal cell function, utilizing the ECL cell as an intermediate modifier. While abnormalities of ECL cells in the human stomach (hyperplasia/neoplasia) have been mostly confined to observations in patients with pernicious anemia and atrophic gastritis, the recent recognition of hyperplasia in pharmacotherapeutically induced achlorhydric or hypochlorhydric states has excited considerable interest. It has been proposed that the generation of luminal hypo- or achlorhydria by powerful acid inhibitory pharmacotherapy may result in hypergastrinemia. This condition is responsible initially for the development of hyperplasia and, subsequently, possibly even neoplasia of the ECL system of the fundic mucosa. This phenomenon seems to be prevalent in rodents but has so far been only rarely observed in humans, e.g., pernicious anemia, atrophic gastritis. In particular, patients with the gastrinoma component of the multiple endocrine neoplasia type I syndrome exhibit ECL-cell hyperplasia and neoplasia after exposure to acid inhibitory pharmacotherapy. It is therefore likely that an underlying genomic phenomenon is necessary prior to the induction of hyperplasia and subsequent neoplastic transformation. The scientific evaluation of the relationship between gastrin, ECL-cell function, and the development of hyperplasia and neoplasia may provide some important information in regard to the molecular evolution of gastrointestinal neuroendocrine disease states. It is possible that the future pharmacotherapy of acid secretory disease may require regulation not only of parietal cell but of ECL-cell function.


The malaria control program in Papua New Guinea: epidemiological surveillance and a look into the future. The four major phases of the malaria control program in Papua New Guinea are briefly described. Routine indicators utilized during Phases II and III (the periods when residual indoor spraying was used) are presented and their limitations outlined. The new epidemiological indicators developed during Phase IV and now included in the National Health Plan (1991-1995) are presented. Their future use and implications for the malaria control program are analyzed and discussed.


The diagnosis of malaria: traditional and contemporary approaches. There have been many recent developments in techniques for the diagnosis of malaria. The quantitative buffy coat (QBC) system and immunological and molecular biological methods are discussed, and compared with the present slide microscopic methods for the diagnosis of malaria. The suitability of each of these techniques for use in the field is reviewed.


Malaria in pregnancy.

Malaria in pregnancy. In its booklet 'Tropical Diseases 1990' the World Health Organization (WHO) Division of Control of Tropical Diseases (CTD) said, "Malaria remains the most important of the tropical diseases--widespread throughout the tropics, but also occurring in many temperate regions". The size of the malarial scourge was estimated as: number of people infected 267 million; clinical cases 107 million/year; mortality 1-2 million/year; number of people at risk 2100 million. Immunity to malaria is reduced in pregnancy. This makes the disease particularly dangerous for pregnant women, especially for nulliparae. Malaria causes a significant portion of the large number of perinatal and maternal illnesses and deaths in the tropical countries. It is the leading cause of indirect obstetric deaths. Even so, it is impossible to measure its full impact in this respect. It may masquerade as anaemia. A maternal death may be attributed to obstetric haemorrhage, when in fact it was malarial anaemia which tipped the scales. Fortunately, the disease can be prevented through chemoprophylaxis and personal protection. This is one of the most rewarding functions of antenatal care.


Distinct patterns of chronic gastritis associated with carcinoid and cancer and their role in tumorigenesis. A series of 60 gastric endocrine tumors comprised 44 body-fundus argyrophil carcinoids, of which 23 arose in a background of hypergastrinemia and type A chronic atrophic gastritis (A-CAG), mainly with histologic patterns suggestive of an autoimmune process. Only 22 percent (compared with 19 percent of 58 tumor-free A-CAG cases) of 36 carcinoids and 21 percent of 19 A-CAG carcinoids investigated had Helicobacter pylori (HP) colonization, against 50 percent of 14 CAG-associated neuroendocrine carcinomas or mixed endocrine-exocrine tumors, 84 percent of 150 cases with early gastric cancer (p < 0.001 versus carcinoids), mostly with B- or AB-type CAG, 76 percent of 97 tumor-free AB-CAG, and 95 percent of 151 tumor-free B-CAG cases. Secondary hypergastrinemia and local mechanisms activated by chronic autoimmune gastritis are among factors involved in the pathogenesis of relatively indolent CAG-associated carcinoids, whereas active HP gastritis in cooperation with environmental carcinogens may likely cause more severe epithelial transformation, leading to ordinary cancer and, possibly, to neuroendocrine carcinomas or mixed endocrine-exocrine tumors.


Hyperplastic proliferations of the ECL cells. Enterochromaffin-like (ECL) cells are the dominant endocrine cell type in the oxyntic mucosa. Normally regarded as histamine-producing cells, they are exquisitely sensitive to the trophic action of gastrin and undergo a hyperplastic increase in a variety of hypergastrinemic conditions. A hyperplasia-neoplasia sequence of ECL-cell proliferations has been recently proposed, following the realization that increasingly severe degrees of ECL-cell hyperplasias over a period of several years can progress to ECL-cell carcinoids. Such carcinoids arising in patients with chronic hypergastrinemia differ both in their clinical and pathologic profiles from the sporadic carcinoids that occur in normogastrimenic individuals and, therefore, need to be distinguished from them. This distinction is particularly important for their clinical management, since antrectomy appears to be of benefit in ECL carcinoids of hypergastrinemic patients.


Malaria in children in Papua New Guinea. Malaria is a major problem in children in many parts of Papua New Guinea; it has a high mortality and, if observations from other countries are applicable, significant long-term morbidity. Standard treatment regimens are the backbone of management of malaria throughout the country, but these must continue to be reviewed and updated in the face of changing patterns of parasite drug sensitivity. Diagnosis of malaria will for practical purposes remain primarily clinical for the foreseeable future. There is a need for uniform reporting of malaria statistics using the established definitions of uncomplicated, treatment failure and severe malaria, in order to assess incidence and outcome throughout the country, and to compare malaria patterns and outcome in Papua New Guinea with those in other parts of the world. Congenital malaria should always be considered in a febrile or ill neonate. Awareness of the complications of severe malaria, particularly hypoglycaemia, needs to be increased. Methods for the diagnosis and facilities for treatment of hypoglycaemia should be widely available. The characteristics of those children most at risk from hypoglycaemia need elucidation, and the current standard management regimen for severe malaria needs to be assessed for its propensity both to cause and to prevent hypoglycaemia. It would be reasonable to incorporate phenobarbitone prophylaxis of convulsions into the standard management regimen for cerebral malaria.


A new look at an old drug: artemesinin and qinghaosu. It is said that William Withering's discovery of digitalis arose out of curiosity engendered during a stage-coach journey, by witnessing an old woman collecting foxgloves by the side of the road. Whilst we are not aware of an analogous stroke of genius reported from ancient China, the story of qinghaosu has certain parallels. Just as foxgloves had been used traditionally for centuries to treat 'afflictions of the heart', the plant Artemesia annua has been used as a treatment for fever in China for almost two thousand years. Artemesia annua, also known as 'sweet wormwood', is found in many parts of the world, but it was not until the early 1970s that Chinese scientists recognized its potential for treating malaria and isolated the active principle, artemesinin or qinghaosu. This paper describes the evidence for the efficacy of this drug and some of its derivatives in the treatment of malaria and the potential of these drugs for the standard management of malaria in Papua New Guinea and elsewhere.


The brain in cerebral malaria: a pathological study of 24 fatal cases in Papua New Guinea. The pathological features of cerebral malaria in 24 fatal cases are described. The cases included 18 adults aged from 16 to 45 years and 6 children aged from 1 to 15 years. All were unconscious before death. Gross pathological findings in the brain included cerebral oedema, vascular congestion and petechial haemorrhages. Little clinical and laboratory information was available on these patients. Histological examination revealed parasitized red blood cells in all, ring haemorrhages in 6, hyaline material in the wall of medium-sized blood vessels in 4, and vascular thrombosis in 4.


Chemoprophylaxis against malaria in Papua New Guinea: a trial of amodiaquine and a combination of dapsone and pyrimethamine. A placebo-controlled chemoprophylaxis trial was carried out in 1980 in 318 semi-immune school children in the Madang area of Papua New Guinea, where there was a high prevalence of strains of Plasmodium falciparum resistant to 4-aminoquinolines. Since prophylaxis with amodiaquine at 5 mg/kg weekly had failed, amodiaquine at a dose of 10mg/kg weekly and Maloprim (half a tablet or one tablet depending on body weight, which gave ranges of dapsone of 1.7-3.3mg/kg and pyrimethamine 0.2-0.4 mg/kg) weekly were tried. Neither regimen was completely successful in preventing parasitaemia, though after 13 weeks of prophylaxis the slide positivity rate was 16% for the amodiaquine group and 2% for the Maloprim group, which was in each case significantly lower than the normal baseline rate in the controls of 42%. Amodiaquine was completely successful in suppressing Plasmodium vivax infections. Breakthrough parasitaemia occurred, with either P. falciparum or P. vivax, in 5% of subjects on Maloprim at some time during the 13-week period of prophylaxis. Significantly more children in both the amodiaquine and Maloprim groups than in the placebo group showed a reduction in spleen size. All groups showed an unexplained fall in haemoglobin level over the study period but the fall was significantly less in both the prophylaxis groups. There was no adverse effect on white cell counts by either drug regimen. Chemoprophylaxis as a component of an integrated malaria control program should not be overlooked, provided that compliance can be maintained. However, in this particular case the principal purpose of the study had been to evaluate the proposed chemoprophylactic regimens in school children before embarking on an intervention study in young children. As a result of this study it was decided not to go ahead with the chemoprophylactic intervention in young children but to adopt an approach based on early presumptive treatment.


The Malaria Vaccine Epidemiology and Evaluation Project of Papua New Guinea: rationale and baseline studies. The range of possible malaria vaccines, against different species of Plasmodium and various stages in the life cycle of the parasite in both human host and mosquito vector, is reviewed. The importance, in a malaria-endemic area, of protection by a malaria vaccine against disease rather than infection is emphasized, and the ways by which disease prevention may be achieved are discussed. Mechanisms of production and presentation of vaccines are considered, including the importance of appropriate and more effective adjuvants. The variety of immune responses to malaria is set out and linked to both human and plasmodial genetic factors. Host genetics may also modify susceptibility to malaria through mechanisms which are not immunological. There is a need for entomological studies of the Anopheles vectors, especially but not only in preparation for transmission-blocking vaccines. This overall complexity justifies a multidimensional approach to epidemiology and field-site preparation. An iterative procedure is proposed for initial field evaluation, through adult male volunteers to community studies in immune adults and then to semi-immune school children, before evaluation in the principal target population of nonimmune young children. The outcome variables for epidemiological evaluation are specified. After this brief review of malaria vaccines, the baseline studies being undertaken by the Malaria Vaccine Epidemiology and Evaluation Project of the Papua New Guinea Institute of Medical Research in the Wosera area of East Sepik Province are discussed in some detail, and their rationale linked to the range and complexity of the malaria vaccines that have been reviewed. These studies are described under the headings of their principal components of epidemiology, parasitology, immunology, genetics and entomology.


An algorithm for the clinical differentiation of malaria and typhoid: a preliminary communication. The objective of this study was to determine which clinical features of typhoid and malaria are most helpful in distinguishing the two diseases among Papua New Guinean highlanders. In a study of 35 patients with culture-positive typhoid and 49 with blood-slide-positive malaria (Group 1), the odds of typhoid were increased most in patients with altered bowel habit, an illness of more than 2 week's duration, tremor or the presence of typhoid facies. The odds of typhoid were lowest in patients with pallor or jaundice. These findings were used to derive a clinical diagnostic algorithm, which was then evaluated in a further group of 34 typhoid patients and 41 malaria patients (Group 2). The sensitivity of the algorithm in diagnosing malaria was 91% in Group 1 and 71% in Group 2, with specificities of 85% and 79% respectively. For typhoid, the sensitivity of the algorithm was 85% and 79% for Groups 1 and 2, respectively, and the specificities were 91% and 71%. We conclude that the algorithm merits further evaluation in a primary health care setting and may prove useful in making an earlier diagnosis of typhoid.


Prenatal immune hypersensitization to malaria: Plasmodium falciparum-specific IgE antibody in paired maternal and cord sera from Papua New Guinea. In a study of malaria and pregnancy in East Sepik Province of Papua New Guinea 45 maternal and cord serum pairs were tested for Plasmodium falciparum-specific IgE antibody. There were 17 positive sera: 6 cases of maternal serum alone, 5 cases of cord serum alone and 3 pairs of maternal and cord sera. IgE antibody positivity rates in the mothers increased with parity, whereas placental parasitaemia rates decreased. Cord serum positivity was not affected by parity. Immunoblots of the sera revealed a diversity of IgE antibodies to specific antigens of the P. falciparum lysate, but an IgE antibody to a 48kd antigen was present in all positive maternal and cord sera.


Travel to the coast by highlanders and its implications for malaria control. Three groups of highland subjects were questioned about malaria and their visits to coastal areas: patients admitted to Goroka Base Hospital with malaria, patients admitted with diagnoses other than malaria who had visited the coast within the previous six months, and health staff working in Goroka. Nearly a third in all groups reported having had two or three attacks of malaria. 82% of malaria patients had visited the coast in the previous 4 weeks compared to 26% of patients without malaria. Most malaria seen in Goroka is imported from the coast. Most patients in the survey came from rural areas and were uneducated. However, health workers also failed in most cases to take adequate precautions when they visited the coast. It is suggested that a malaria prophylaxis station should be set up at the gateway to the highlands on the Highlands Highway, where malaria education and the means for chemoprophylaxis and protection from mosquitoes could be made available for all travellers.


Quinine blindness.

Quinine blindness. A young women was treated with intravenous quinine and chloramphenicol for suspected severe malaria and/or typhoid fever. On the second day of quinine therapy (after 2.25 g of quinine) she suddenly developed total bilateral loss of vision. Both drugs were stopped and cyclandelate therapy was started. She showed slight improvement in vision but on referral her visual acuity was limited to seeing waving hand movement only; visual fields were constricted and colour vision was absent. Both pupils were fixed and dilated. The fundi showed macular oedema and attenuated retinal arteries. She was treated with dexamethasone, cyclandelate, vitamin B complex and vitamin C. Colour vision was completely recovered after 5 days of treatment. Full recovery of the direct light reflex occurred after 10 days. Visual acuity improved slowly over a period of one month to 6/15 vision in both eyes. At this time macular oedema and retinal arteriolar attenuation were still present but less severe. In the context of this case report the condition of quinine blindness is briefly reviewed and the management discussed.


[Laparoscopic treatment of gastroesophageal reflux]. Authors describe laparoscopic treatment of gastroesophageal reflux disease in 12 patients. Particular aspects related to intra-operative complications are referred. Moreover the reasons that make laparoscopic procedure advantageous are discussed.


[Laparoscopic surgery: planning program]. Performing laparoscopic surgery requires an initial training program. A well-planned organization is essential and the surgeon has to become first familiar with the new procedures; the choice of the necessary equipment is the second step. Upkeep of surgical instruments and a careful consideration of legal aspects are the next important steps. Several areas of a planning program are evaluated on the basis of the authors' experience.


[Anesthesia for laparoscopic cholecystectomy]. Alterations in the serological, pulmonary and cardiocirculatory parameters, potentially determined by CO2 pneumo-peritoneum during laparoscopic cholecystectomy, require careful intraoperative evaluation. With the aim of verifying the real entity of these alterations, an experience is reported relating to 76 of the 303 patients undergoing laparoscopic cholecystectomy during 14 months. During surgery, besides an obvious increase in pO2, a slight hypercarbia and a slight decrease in pH were observed, which required pharmacological intervention in only 5 cases. In no case did the respiratory and haemodynamic alterations observed necessitate the conversion into laparotomy. This initial experience gives us reason to conclude that careful monitoring of the respiratory parameters leads to an adequate anaesthesiological conduct, and thus facilitates the completion of laparoscopic procedures.


[Reflection on 19 months of laparoscopic cholecystectomy]. The introduction of laparoscopic cholecystectomy (LC) has aroused enthusiasm on the part of the mass media and perplexity on the part of many medical workers. In an attempt to reconcile the two points of view, the video-laparoscopic activity in a surgical ambient was audited over 19 months. 450 LC were carried out during this period. The number of cholecystectomies performed by this method has risen significantly in the recent past thanks to the high esteem in which it is held by the public in general and by gastroenterologists in particular. The average age of patients is lower since young people accept more readily a type of surgery involving a lesser degree of traumatism, a relatively short convalescence and less aesthetic damage. After a brief initial period of scepticism, the method has also been received favourably by anaesthetist, and by operating theatre and ward nursing staff. Morbidity and mortality rates are not different from those noted after traditional cholecystectomy, which confirms the method as being safe and thus meriting the success it has widely achieved.


[The importance of preoperative study of cholelithiasis during the laparoscopic era]. Preoperative x-ray examination of the biliary tree has in the last decade been entrusted to ultrasonography. The diagnostic procedure was completed with an accurate evaluation of the main biliary tract by means of intra-operative cholangiography. However, recently-developed methods such as operative endoscopy and laparoscopic surgery require essential pre-operative information about the state of the biliary tract which ultrasonography is not able to provide. In our view, the most useful examination for this purpose is i.v. cholangiography; the results of this study, relating to 303 patients undergoing laparoscopic cholecystectomy, of whom 39 affected by cholecysto-choledochal lithiasis, would seem to confirm these view.


[Interaction between laparoscopic surgery and operative endoscopy. Results of an experience]. Nowadays it is possible to treat complex biliary disease such as cholecysto-choledochal stones with mini-invasive methods. In this field, laparoscopic surgeons and gastroenterologic endoscopists can collaborate not only in the planning of endo-laparoscopic sequential treatment of biliary stones but also in the successful treatment of complex cases having either surgical or endoscopic complications. Here are presented the results which 14 months of collaboration between laparoscopists and endoscopists have brought about in the diagnostic and therapeutic fields. In particular, the results are documented of the sequential endo-laparoscopic treatment of cholecysto-choledochal calculi proposed for 39 patients, of inverse laparo-endoscopy carried out in 2 cases, and of therapeutic procedure used to treat an iatrogenic perforation of the biliary tree by mini-invasive methods. The experience reported is an example of how technological progress is gradually pushing aside the barriers still existing between medicine and surgery.


[Laparoscopic cholecystectomy: a need to drain?]. The authors summarize their experience about the use of a drain after laparoscopic cholecystectomy. After an initial period without drainage, the drain is now routinely used by the Authors and their clinical experience suggests that it is probably very useful during the initial training and probably prevented some reoperations when biliary leakage and/or small hemorrhage from the gallbladder bed were present. Therefore the opinion of the Authors is to always drain after laparoscopic cholecystectomy, specially during the initial experience or after a particularly difficult operation.


[The endoscopic management of pancreatic pseudocysts: a case report]. The Authors present a case of post traumatic pseudocyst of the pancreas added to their observations and treated with an endoscopic cytogastric deviation. The incidence of such pathology has increased during the last few years thanks also to the improvement of the diagnostic techniques (TAC, U/S, Rm and eco-endoscopy) on hard today; in the second place the technological evolution has allowed, improving the diagnostic definition, the ability to heat pseudocysts, in selected patients, with minor surgery techniques with result equal to those of conventional surgery, but with complications and mortality decisively reduced. The morbidity and mortality rates of internal deviation surgery has respectively worsened varying between 21% and 50% and from 5% to 12%. The results of endoscopic deviations, even if the cases are less, report positive results from 80% to 100%, with complications and mortality greatly reduced. To be able to propose an endoscopic indication certain requirement are necessary of which one is indispensable: the distance between the two adjacent lumen must not be more than one centimeter. The Authors are of the opinion that such methods, in expert hand an on selected patients, should be the first surgical choice for the treatment of pancreatic pseudocysts.


[Videothoracoscopic treatment of spontaneous pneumothorax]. In the period between May and October 1992 we have admitted in our department 14 patients (M/F: 11/3) with recurrent spontaneous pneumothorax, being the entity more than 40% in 8 of them. All the patients were treated with video-assisted thoracoscopic pleurectomy, beginning from the 2nd to the 6th rib, completing by abrasing the further ribs below. Whenever the causative bullae or blebs were found, those were resect using and endoGIA. At the end of the procedure a pleural drain (no. 24) was positioned and maintained on suction until a complete lung reexpansion was ensured for at least a couple of days. Neither postoperative nor delayed complications and no recurrences have been observed. Our results of such an approach, once indicated the pleurodesis for the treatment of spontaneous pneumothorax, are undoubtedly encouraging both for the technical simplicity and the reduction in hospitalization time.


[Videothoracoscopic wedge resections of the lung]. The recent development in thoracoscopic instrumentation has widely improved their surgical applications so to allow lung resection whether for diagnostic of therapeutic purpose. From June to December 1992, we performed 14 segmental atypical lung resections in 12 patients. The indications were: recurrent spontaneous pneumothorax due to bullae or blebs in 7 cases, solitary peripheric lung nodule in 6 cases, for diagnostic purposes in suspicious interstitial lung disease in one case. Lung resection were carried out using stapling device (EndoGIA). In the cases of solitary lung nodule, the frozen section confirmed the presence of metastases in 4 subjects and thus it was proceeded towards an exploratory mini-thoracotomy (3 cm long) so to allow a further palpatory video-assisted evaluation of the lung surface. The remaining 2 patients were diagnosed to have hamartomas. The wedge resection in the patient with interstitial lung disease allowed diagnosis of sarcoidosis. There were no postoperative complications, nor deaths. Video-thoracoscopic lung wedge resections are indicated for treating bullae and blebs while performing pleurectomy for spontaneous pneumothorax, for removing benign and metastatic peripheral lung nodules. The procedure is substantially diagnostic in case of interstitial lung diseases. It's important to underline that the therapeutic value of thoracoscopic approach for metastatic lung nodules has not proved to be less or more efficient than the thoracotomy one and for this reason further investigations are required.


[Videothoracoscopy in diagnosis and treatment of chronic pleural effusions]. Thoracoscopy is the most important method for the diagnosis, and in particular cases for the treatment, of chronic pleural effusion of unknown aetiology. The Authors report their experience of 4 cases, 2 males and 2 females, that underwent a thoracoscopy from June to December 1992. The method was done in 3 cases in general anesthesia and in one case in local anaesthesia.


[Operative pelviscopy: notes on the technique]. The authors illustrate the present possibilities of operative pelviscopy with particular regard to its indications and to the surgical technique. The endoscopic technique can today be applied in around 70% of benign affections of the female genital apparatus. Most authors agree that ovarian cysts, adhesions, ectopic pregnancies, and myomas of the uterus are, within certain limits, treatable electively with endoscopic technique; reservations still prevail in the case of hysterectomy and serious doubts in that of pelvic lymphadenectomy. Pelviscopy has proved to be both useful and safe; indeed, out of 372 operations carried out by the authors, only three had to be converted, and no complications were ever observed requiring either a laparotomy or a further pelviscopy.


[Tension-free laparoscopic hernioplasty]. Hernia recurrence after traditional "open" hernioplasty is an observed event. The tension undergone by anatomical structures of the area is believed to be responsible in large part for these failures. Thus, techniques involving "tension free" hernioplasty have been developed, some of which involve laparoscopic access. Here an experience is reported regarding laparoscopic hernioplasty carried out to repair groin hernias, first of all on an animal model (pigs: 7 cases) then in the clinical field. The technique chosen was the endo-peritoneal positioning of a PTFE prosthesis. The results revealed several advantages over the traditional methods, basically the possibility of reinforcing the entire inguinal floor at the same time as repairing the hernia, and the decrease in groin area discomfort and less time off from work for the patient.


[Laparoscopic treatment of varicocele].

[Laparoscopic treatment of varicocele]. Varicocele is frequently a contributing factor in male infertility. We performed laparoscopic treatment of varicocele in 6 patients. This procedure can be carried out through three ports and allows not only ligation and excision of the dilated testicular veins, but those veins branching through the back of the inguinal canal lower down. It is a quick operative procedure and we completed it within 30 minutes. All the patients have gone home on the 2 days and, to date, there have been no complications. During a follow-up period of 3-6 months it was possible to observe the disappearance of varicocele. It is possible to conclude that the laparoscopic procedure is simple and effective as more traditional methods.


[Elective and emergency diagnostic laparoscopy]. A report is presented concerning 31 patients treated by laparoscopic approach under both elective and emergency conditions. The mini-invasive approach to an abdominal disease allows both the making of a direct visual diagnosis and the carrying out of biopsy or actual therapy without the need for laparotomy. Elective diagnostic laparoscopy has proved efficacious in the screening of patients affected by abdomino-pelvic neoplasms, determining the extension of the latter, staging them and judging their operability. Under emergency condition this method lends itself in particular to the study and the treatment of some cases of traumatic haemoperitoneum, intestinal obstructions resulting from adhesions and peritonitis of dubious aetiology. Explorative laparoscopy thus constitutes a valuable diagnostic and therapeutic aid, its possibilities being destined to increase with the improvement of laparoscopic techniques.


[Videolaparoscopy and conservative treatment of splenic injuries]. The treatment of lesions due to blunt abdominal trauma has changed considerably in the last decade. With a view to the prevention of immediate and later complications of splenectomy, conservative surgical technique have been put forward, or else, when the haemodynamic conditions of the patient permit, techniques of non-operative monitoring of splenic lesions. Laparoscopy today constitutes an approach to the diagnosis and treatment of blunt abdominal trauma which decisively favours conservative treatment. The experience presented bears witness to the potential usefulness of laparoscopy in these cases. Technological progress will widen the possibilities of this method and more detailed studies will define its exact indications.


[Experimental intestinal laparoscopic resection]. At present, there is a debate in the literature regarding the possibility of extending the laparoscopic approach to intestinal resection. Some techniques have been developed, but certain imperfections and problems remain. This experimental contribution suggests answers to some technical problems arising in the course of a small-bowel laparoscopic resection on a pig.


[A simultaneous study of intestinal permeability and the test of H2 in the expired breath of celiac children]. Permeability test with sugars (PTS) as a non-invasive diagnostic tool is of special interest in pediatric patients. This study evaluated the relationship between PTS and antigliadin antibodies (AA) with the intestinal biopsy and studied whether an altered expired hydrogen test (EHT) could interfere in this relation in celiac patients. Thirty children (13 girls, 17 boys) with diagnosis of celiac disease (ESPGAN criteria) were divided into three groups according to this histopathology: Group A (n = 23) normal biopsy; Group B (n = 7) biopsy grade III or IV; Group C (n = 25) controls. The intestinal permeability test showed significative differences (p < 0.01) of Group B (0.159 +/- 0.03) with Group A (0.048 +/- 0.005) and with the control Group C (0.039 +/- 0.002). The cut-off for the Youden maximum index (0.75) was 0.092 and ROC SE 0.87 +/- 0.7. The results of the AA were related to those of the PTS. The variance analysis showed that the results of the expired hydrogen test do not interfere statistically on the good correlation of PTS with the histology. The PTS is a good indicator of the status of intestinal mucosa. There is a good correlation between PTS and AA in celiac children. A pathological expired hydrogen test seems not to interfere with the correlation found between PTS and histological damage in our series.


[Endoscopic sphincterotomy. Its complications and their follow-up]. Between April 1981 and April 1991, there were 547 EPT performed, seventy-five of which had in situ gall bladder. They were performed in two groups, who were similar from the technical-anatomical point of view, and sociocultural background. The early morbidity showed 31 complications (5.6%). The most frequent ones were:perforation in 8 cases and haemorrhage in 5. Medical treatment, which included the endoscopic technique in 20 cases (3.7%) and surgical procedures in 11 cases (2.0%) was used. The mortality rate was 1.8% (10 cases), perforation (4) and cholecystitis (3) being the main causes. There were three deaths (0.5%) not related to the method. The morbimortality was not significantly increased by the complementary methods. After analysing the complications related to the cause of EPT performance, it was observed that they were more frequently produced in choledochal stones: 20 (3.6%), followed by choledochal and gall bladder stones: 7 (1.3%). However the mortality was 0.7% in the former and 0.5% in the latter. The morbimortality of the last two years is compared to that of the preceding ones. The follow-up was done in 114 (20.8%) of the cases with a marked difference between the high sociocultural group (76 cases) and the low one (36). Eighteen pathological cases were found (3.3%). The treatment of each complication in long term is shown.


[The correlation of IgA-class antigliadin and antiendomysial antibodies (AGA-IgA--EmA-IgA) with the intestinal histology in celiac disease (CD)]. The aim of this work was to establish the diagnostic and follow up value of IgA-class antiendomysium (IgA-EmA) and IgA-class antigliadin (IgA-AGA) antibodies in celiac disease. Correlation with the intestinal histology at the different stages of the disease was evaluated, as well as its therapeutic monitoring ability. Fifty six children, twenty seven girls and twenty nine boys, aged six months to twelve years old, were studied. Thirty nine celiac children were all different diagnostic stages of the disease. Seventeen children with malabsorption symptoms and with normal intestinal histology were used as controls. Sixty blood samples were obtained simultaneously with the small intestinal biopsy. IgA-AGA (ELISA method) and IgA-EmA (immunofluorescent test performed over lower third Rhesus monkey esophagus) were determined in every blood sample. In 34 serum samples from patients with total or subtotal villous atrophy, two were negative for IgA-AGA and only one was negative for IgA-EmA. In 26 samples from patients with normal intestinal histology, two were positive for IgA-AGA and four were positive for IgA-EmA. The results for IgA-EmA had sensitivity 97%, specificity 84.6%, positive predictive value 89.2% and negative predictive value 95%. In the case of IgA-AGA were: sensitivity 94.1% specificity 92.3%, positive predictive value 94.1%, negative predictive value 92.3%. IgA-AGA and IgA-EmA showed a high correlation with intestinal histology and are in combination powerful tools for the diagnosis and follow up of celiac patients. Besides, they provide a useful aid in the indication of a jejunal biopsy and in close monitoring of the dietary treatment compliance.(ABSTRACT TRUNCATED AT 250 WORDS)


[Symptomatic malignant melanoma of the small intestine]. A 46-year-old woman presented with abdominal pain, nausea vomiting and abdominal distention. Small bowel x-rays and CT scan of the abdomen revealed small bowel obstruction due to malignant melanoma. The diagnosis of cutaneous melanoma was performed 8 years prior to admission on one lesion in the back. Patient received surgical treatment. Completed resection of an involved jejunal [correction of ileal] segment was performed. Three tumor masses were found at laparotomy. Metastasis from malignant melanoma at the gastrointestinal tract occurs frequently though rarely are these intestinal lesions symptomatic. The efficacy of surgical treatment for symptomatic metastatic melanoma is justified to relief symptoms and prolonged survival.