Contrast-enhanced CT scanning in vivo for the quantification of hepatic metastases from a human colonic cancer in the nude rat. Hepatic metastases were induced in nude rats by intraportal injection of 2.5-5.0 x 10(6) cells from the human colonic cancer cell line LS 174 T. Quantification of tumour burden, expressed as relative metastatic area, was performed by contrast-enhanced computed tomography (CT) scanning in vivo (n = 14), contrast enhanced CT scanning post mortem (n = 21) and computer-based area calculation (CBAC) (n = 21). To determine the false-positive contribution to the estimated tumour burden by the evaluation procedures themselves, six rats without metastases were assessed. The quantification in the three different assessment groups was in close accordance in animals with an intermediate or extensive metastatic burden, but not in rats with a minor (< 4%) tumour burden. The results indicate that contrast-enhanced CT scanning can be used in this model to quantify hepatic metastases, except in animals with few and small lesions. Furthermore, the results suggest a potential for the assessment of therapeutic response by repeated contrast-enhanced CT scanning in vivo, as well as prospects for a corresponding evaluation in man.


Primary non-Hodgkin's lymphoma in ileal Crohn's disease. Non-Hodgkin's lymphoma in association with Crohn's disease is a rare occurrence. A case of high grade B-cell lymphoma arising in an isolated segment of ileal Crohn's disease is described and the mechanisms for its possible causal association are discussed.


Non-polypoid inflammatory fibroid polyps concomitant with early carcinoma in the stomach. A 69-year-old Japanese woman with early carcinoma of the stomach was treated. Postoperative pathological examination of the resected stomach disclosed a widely spreading early carcinoma and two concomitant submucosal lesions which were histologically compatible with inflammatory fibroid polyp (IFP). Neither IFP showed a polypoid appearance: instead they had a down-growth pattern. The covering mucosa was replaced by gastric carcinoma cells. The IFPs were located mainly in the submucosa, but reached invasively to the subserosa through the proper muscular layer. The boundaries of the lesions were well demarcated in the submucosa but poorly demarcated in the subserosa. This case is very rare because the two IFPs showed a down-growth pattern and were concomitant with early gastric carcinoma.


Subcutaneous implantation of liver metastasis after fine needle biopsy. A 50-year-old woman underwent percutaneous ultrasonographic fine needle biopsy of a suspicious liver metastasis of colon cancer. A subcutaneous metastasis developed at the site of the puncture 20 days after biopsy. Tumor recurrence in the needle biopsy track is rarely reported in the literature. The possible causes of this complication are discussed.


Subcutaneous tumor growth complicating the positioning of Denver shunt and intrapleural port-à-cath in mesothelioma patients. Patients with malignant ascites and malignant pleural fluid from abdominal or pleural mesothelioma underwent the positioning of Denver type peritoneovenous shunt or intrapleural catheter. They developed tumor growth in the subcutaneous tissue surrounding the devices throughout their courses. Neoplastic seeding is a potential complication of the positioning of shunts and catheters in cavities filled with fluid rich in tumor cells.


Rational choice of therapy in established open angle glaucoma. Currently, the standard treatment for primary open angle glaucoma favours initial medical therapy. Laser trabeculoplasty and filtering surgery are reserved for those patients who fail medical therapy. There is now, however, good evidence to suggest that perhaps this approach deserves reassessment.


Encapsulated filtering bleb. A selective review--new deductions. Filtering bleb encapsulation may, in some cases, be a severe complication following filtering surgery. The cause and mechanism of its development are not known. A selective review of data that might shed some light on these dilemmas, is presented. Based on these data, it is suggested that: (a) non-contractile collagen-producing fibroblasts play a major role in the process of bleb encapsulation, while in wound healing following filtering surgery, contractile fibroblasts are the major components; (b) the process of bleb encapsulation is less sensitive to the toxic effect of 5-Fluorouracil than would healing; (c) collagen-producing fibroblasts may be less sensitive to the destructive effect of 5-Fluorouracil than contractile fibroblasts; (d) inflammatory mediators are important triggers of bleb encapsulation.


The effect of stimulus duration upon the components of fluctuation in static automated perimetry. We studied the effect of varying the stimulus duration time on the components of fluctuation in static automated perimetry by testing 20 healthy subjects on the Humphrey Field Analyser within the central 20 degrees of vision. We found the total fluctuation at a stimulus duration of 0.065 s, to be 3.22 decibels (dB); 0.1 s, 3.22 dB; 0.2 s, 3.34 dB; 0.3 s, 2.95 dB; 0.4 s, 3.24 dB; and 0.5 s, 3.22 dB. Little difference was observed in the individual components (short-term, long-term homogeneous, long-term heterogeneous and inter-individual) or total fluctuation with an increasing stimulus presentation time when evaluated over the entire visual field or in a pointwise fashion. This study suggests that the stimulus duration within the range of times studied has little effect on the components or total fluctuation in healthy individuals.


Blue versus white stimuli in ocular hypertension with the Friedmann Mark 1 Visual Field Analyser. Fifty eyes of fifty patients with ocular hypertension had their visual fields tested on a Friedmann Mark 1 field analyser whilst wearing a Wratten 47B blue filter in a spectacle frame. All had normal visual fields to a white stimulus. Use of a scoring system with the blue field identified 11 patients (22%) with ocular hypertension as abnormal. The scores from this subgroup were indistinguishable from a group of subjects with early glaucomatous field loss, whilst the remaining scores were similar to normal subjects. These two subgroups of ocular hypertensive patients were similar in age and intraocular pressure. The use of a blue filter in front of the eye may offer a simple test to identify a subgroup of patients with ocular hypertension who are at increased risk of developing field loss.


Congenital and developmental myopia.

Congenital and developmental myopia. Both hereditary and environmental factors are important in the interactive growth of the ocular tissues responsible for determining the refractive state of the eye. Myopia has a low prevalence in otherwise healthy children and is seen with an increased prevalence early in life in many systemic and ocular diseases. Predicting how refractive error will change in any individual child after birth or at any stage of ocular development is not possible at present, although, trends can be seen in longitudinal studies. In the disease states associated with an increased prevalence of myopia, information regarding the time of onset of myopia and the specific values for the refractive components, is lacking, so that underlying mechanisms of myopia development and whether the myopia is congenital or developmental, are not known. In adults, three different types of myopia can be characterised on the basis of clinical characteristics which have prognostic significance for ocular disease. The mechanisms of ocular growth that lead to these forms of myopia are not clear. At present, treatments to prevent or slow myopic progression have been marginally beneficial and of questionable value.


The eye and liver disorders.

The eye and liver disorders. There are ocular changes associated with a wide spectrum of congenital, familial and acquired liver diseases and disorders. The early identification of ocular changes may aid diagnosis of the underlying liver condition. This is particularly important in conditions where there are effective treatments which can arrest hepatic damage. It is also of considerable value in patients who have untreatable disorders because identification of the correct diagnosis may offer prognostic information and spare the patient unnecessary invasive investigation. This article discusses the ocular findings in selected liver disease and reviews the current literature on the subject. The principles of investigation and diagnosis and treatment are described. Data on the incidence and prevalence of ocular involvement in liver conditions are included where such figures are available. The potential transmission of viral hepatitis following corneal grafting is discussed.


The results of ischaemia in chemical injuries. Severe burns of the anterior eye segment which include cornea, limbus and adjacent conjunctiva necrosis were accompanied by ischaemia. While necrotic conjunctival and subconjunctival tissue should be removed to eliminate its toxic influence, the opaque cornea and ischaemic sclera could not be removed. The ciliary body and the iris also suffer from ischaemic damage and under such conditions nutrient metabolites are deficient in the anterior segment of the eye. In the surrounding healthy tissues an inflammatory reaction begins after several days which induces an infiltration of the damaged tissues by leucocytes and an increase of N-acetylglucosaminidase and Cathepsin-D as demonstrated in model experiments and in patients. These destructive enzymes are apparently involved in the corneal and corneo-scleral ulceration and their activities may therefore be used to assess the severity of the ocular burn.


Eye injuries at work: a prospective population-based survey within the chemical industry. A population of 63,000 chemical industry workers was studied prospectively over approximately 10,000,000 man-hours of work. A total of 133 eye injuries were reported (8.4% of all injuries), of which 22% needed medical attention. Eye protection was often not worn, some protectors may have been wrongly recommended for a particular task, and some protectors may have failed to perform adequately. In this population, an eye injury occurred once every 75,000 man-hours of work on average (injury incidence 23:1000:year). Most injuries were avoidable. Revision of protection protocols at work, and improved education of personnel are needed to reduce the incidence of occupational eye injury.


Management of VIth nerve palsy--avoiding unnecessary surgery. Unresolved VIth nerve palsy that is not adequately controlled by an abnormal head posture or prisms can be very suitably treated by surgery. It is however essential to differentiate partially recovered palsies, which are amenable to horizontal rectus surgery, from unrecovered palsies, which must be treated initially by a vertical muscle transposition procedure. Botulinum toxin is a valuable tool in making this distinction. It also facilitates full tendon transposition in unrecovered palsies, which appears to produce the best functional outcome of all the transposition procedures, with a reduction in the need for further surgery. A study of the surgical management of 12 patients with partially recovered VIth nerve palsy and 59 patients with unrecovered palsy provides clear guidelines on how to attain a successful functional outcome with the minimum amount of surgery.


Non-traumatic mycotic keratitis.

Non-traumatic mycotic keratitis. Two patients presented with culture proven Paecilomyces corneal infection, and a further patient with histologic evidence of fungal infection, on deep corneal biopsy. In all three cases the corneal infection was macroscopically present only in the depth of the cornea and on the endothelial surface with an intact epithelium and no overlying stromal involvement. Repeated surgery with large corneo-scleral grafts in two cases, and with medical therapy and a small patch-graft alone in the third case, resulted in long-term eradication of the infection and preservation of the globes. Antecedent modulation with steroid and/or cyclophosphamide may well have delayed the diagnosis, however, as there was no history of trauma in any of these cases, we postulate that these infections were not exogenously derived.


Septicaemic infection with group B streptococci presenting with endophthalmitis in adults. Metastatic bacterial endophthalmitis is an uncommon and serious infection, that can be caused by a variety of bacteria. Group B streptococci have rarely been implicated. We report four cases of metastatic endophthalmitis in adults caused by group B streptococci. The organisms were isolated from the eyes and blood in each case.


Cataract as a complication of severe microbial keratitis. Complicated cataract results from local ocular disease; we report five cases that followed severe anterior segment infection. Three patients had Pseudomonas keratitis and two Acanthamoeba keratitis. All patients had severe keratitis and iridocyclitis. Mature cataracts developed after a mean of 5.5 months from the onset. Cataract formation with severe keratitis may be attributable to bacterial toxins, iridocyclitis and treatment toxicity. All these factors may cause cataract by interference with lens metabolism. One of our cases had no steroid treatment; the remaining four had between 7.7 and 28.14 mg of topical steroid (256-938 drops of Dexamethasone 0.1%). Cataract formation may result from severe microbial keratitis alone but is probably enhanced by concurrent treatment with high doses of topical steroid. The potential for cataract formation must be considered when managing microbial keratitis with the use of steroids and when planning surgical rehabilitation of the anterior segment.


Corneal neovascularisation in acute hydrops. Four cases of corneal neovascularisation complicating acute hydrops in keratoconus are presented. Three cases were atopic. In two cases high doses of topical steroids failed to inhibit the neovascular process. The pathogenesis of neovascularisation in this situation is uncertain. Risk factors may be the size of the hydrops and the proximity of oedematous cornea to the limbal vascular arcades, with an associated inflammatory reaction. The coexistence of atopic keratoconjunctivitis may be an additional factor. Patients with large peripheral hydrops should be observed closely for the development of new vessels, and systemic steroids prescribed if topical medication fails to inhibit a neovascular reaction. Consideration should be given to the possibility of removing the stimulus to neovascularisation by corneal grafting, although the technical difficulties and the patients atopic state may militate against this.


The relationship between central nuclear scatter and perinuclear retrodots in the human crystalline lens. Retrodots are common perinuclear features of age-related cataract. Clinical observation suggested that retrodots were associated with increased nuclear scatter. To test this hypothesis, retrospective data were analysed from 78 non-diabetic subjects (118 eyes), of at least 50 years of age, who had neither subscapsular nor axial spoke opacities. Retrodots were graded using the Oxford Clinical Cataract Classification and Grading system and central nuclear scatter was assessed by image analysis of Scheimpflug photographic negatives. Regression analysis in groups was performed according to the "intra-class correlation model" either considering retrodots as a graded feature (grades 0-5) or as a dichotomous variable (present or absent). After adjusting for age (treated as a co-variate), a highly significant association (P < 10(-6) was found between retrodots and increased nuclear scatter in each analysis. It is suggested that the co-existence of these two cataract types may reflect a common cataractogenic mechanism, possibly oxidative stress.


Glare testing in pseudophakes with posterior capsule opacification. We measured Snellen visual acuity under standard and glare conditions using the Allergan Humphrey 570 autorefractor in 46 pseudophakes (46 eyes) undergoing Neodymium-YAG laser posterior capsulotomy before and after treatment. All the patients studied had a best corrected vision of at least 6/12 but complained of visual disability and had clinically observable posterior capsule opacification. The patients did not have other known causes of glare disability. Following posterior capsulotomy, 30 (65%) patients showed an improvement in standard Snellen visual acuity and 44 (97%) an improvement in Snellen visual acuity under glare conditions. This difference was statistically significant (p < 0.001). Of the 16 patients whose standard Snellen visual acuity did not improve after posterior capsulotomy, 14 showed a reduction in glare disability. Glare testing was clinically useful in evaluating pseudophakes with posterior capsule opacification who complained of visual disability in spite of good Snellen visual acuity under standard testing conditions.


Pigment epitheliopathy with serous detachment of the retina following intravenous iron dextran. We report a case of pigment epitheliopathy with serious detachment of the retina in a 23-year-old man following a total dose infusion of iron dextran. Initial impairment of visual acuity resolved spontaneously, along with changes in the electro-oculogram and fundus fluorescein angiogram. Ocular features of systemic iron toxicity are reviewed and the pathophysiology discussed. Practitioners using total dose iron infusion should be aware of the possible ocular complications.


Erythrocyte stearic to oleic acid ratio in patients with ocular melanoma. The main characteristics of malignant cells include increased metabolism and capacity for division which are associated with high membrane fluidity. Membrane fluidity is in turn controlled by multiple factors which include the ratio of saturated to unsaturated fatty acids in the cell wall i.e. the Saturation Index (SI). The SI is known to be reduced in neoplastic cells as well as in the circulating erythrocytes of patients with some malignant tumours but not others. It has been suggested that the SI may be useful as a marker of malignancy.


Cellular automata: retinal cells, circulation and patterns. Cellular automata modelling is a useful mathematical technique for simulating complex biological systems. An area to be studied is broken into a lattice of adjacent cells depicted by picture elements on a computer screen. The initial tissue pattern evolves on the computer screen, directed by a rule that considers the state of each cell and its neighbours in the lattice. Simulations of wound repair, cell proliferation, retinal circulation and pigment aggregation serve to illustrate the potential value of cellular automata modelling in ophthalmic research and practice.


Expression of HLA-DR molecules and some other differentiation antigens within early human fetus. Leukocyte membrane markers have been examined within cryostat sections and cell suspensions of human fetuses aged between 4 and 18 weeks of gestation using a panel of monoclonal antibodies. The HLA-DR molecule was present on a population of erythroid cells and macrophages in liver sinuses at 34 days and periarteriolar splenic macrophages at 60 days of gestation. CD5, CD15 and CD45 antigens were detected at 6 weeks of gestation, whereas CD4, CD8, CD10, CD11c, CD14 and IgM were not expressed at this stage. These findings and ultrastructural examination of embryonic liver confirm early differentiation of macrophages in the pre-lymphatic developmental period.


Osteoblastic and chondroblastic response to a variety of locally administered immunomodulators in mice. Administration into mouse shank muscles of various immunomodulators which directly or indirectly activate lymphocytes (BCG, Con A, Carrageenan IV, Dextran, PHA-M, PWM, lipopolysaccharides from Corynebacterium) have a dual effect on local bones, stimulating both periosteal bone formation and bone resorption, the former being dominant. These effects vary in frequency and magnitude, the most potent stimulation of periosteal bone formation being observed after administration of Con A and BCG in complete Freund adjuvant. BCG was also a strong inducer of bone resorption in vivo. Concanavalin A and, to a lesser degree, other immunomodulators applied, when administered subcutaneously into the pinna, also have induced perichondrial chondrogenesis. These novel effects of immunodulators could be applied in the field of skeletal tissues regeneration.


Stable DNA-polypeptide complexes from eukaryotic nuclei purified on heparin Sepharose. A novel simple method using affinity chromatography on Heparin Sepharose CL-6B is described for purification of stable DNA-polypeptide complexes from preparations of eukaryotic nuclear DNA. These complexes resist RNase A and proteinase K treatment and copurify with DNA on phenol extraction. The content of heparin-binding complexes amounted to about 20% of the total DNA quantity and 60 to 80% of nitrocellulose-retained DNA, being similar in preparations of DNA from calf thymus, chicken erythrocytes and cauliflower inflorescence. This content was influenced by the size of DNA fragments and the presence of dithiothreitol. The heparin-binding fraction was shown to represent a definite type of complexes which is different from the other(s) retained on nitrocellulose.


Effects of fermentation on product consistency. A variety of different fermentation processes has been successfully employed to produce consistent protein-based biopharmaceuticals from genetically engineered animal cells. Chinese hamster ovary (CHO) cells were genetically modified to produce recombinant human soluble CD4, tissue plasminogen activator (tPA) or erythropoietin (EPO). Soluble CD4 was collected from extended perfused fermentations of several months' duration, during which some quantitative loss of DNA copy level, mRNA expression level, and fermentation titer were observed. In one extended run, a novel contaminant appeared in intermediates purified from later harvests. However, in all cases, the final soluble CD4 product was consistent in terms of purity and potency. Evaluation of genetic stability for tPA examined both biological traits at the cellular level as well as potency, purity and structure of product derived from cells at various levels of in vitro age; no significant cell age effects were observed. Similarly, evaluation of the EPO product showed that genetically-determined and process-determined traits such as potency, tryptic peptide mapping, and sialylation were consistent from lot to lot. These data exemplified how process design, process validation, and in-process and quality control assays can be used effectively to ensure the consistency of recombinant products derived from cell culture fermentations.


Peptide mapping for detecting variants in protein products. The genetic stability of biotechnology production systems is of importance in evaluating the safety and efficacy of protein products. The analysis methods currently available for assessing the genetic stability of such systems are limited by the complexity of the eukaryotic genome and by their inability to predict the effects of post-transcriptional and post-translational events, such as glycosylation or host cell protein level changes, on the protein produced. Therefore, it is important to focus attention on the protein product itself in evaluating product consistency rather than on nucleotide sequence analysis for genetic stability of the production systems involved. Peptide mapping has arisen as the best method for the analysis of proteins as a surrogate measure of genetic stability. This method uses known chemical reactions or enzymes to cleave the protein at specific sites, resulting in discrete peptides whose structure and composition can be readily compared to that of the theoretical protein coded for by the DNA sequence of its gene. Peptide mapping, which is capable of detecting single amino acid changes in a molecule, can also be used for determining glycosylation sites. Peptide mapping requires sophisticated chromatographic techniques and equipment, and is limited to molecules of m.w. of c. 100,000. The method also requires careful validation to limit the potential for misinterpretation of artifactual peaks. Despite its limitations, no better method currently exists to evaluate product consistency and, thereby, the genetic stability of recombinant products.


Changing issues of quality control: diploid and non-diploid cell lines. Early viral vaccines were prepared from primary monkey kidney cell cultures. However, primary cell cultures can be highly variable and also present a risk of contamination by endogenous viruses. The development of human diploid cell lines for vaccine production provide greater assurance of consistency and freedom from contaminating viruses. Continuous cell lines present considerable advantages in the preparation of proteins for therapeutic use. The perceived risks associated with the use of these cell lines include those of retroviral contamination, oncogenic DNA and consistency of final product during the extended life in culture. Advances in molecular biologic and analytical chemistry procedures have enabled extensive characterisation not only of the cells, but also of the product itself. The degree of testing which can now be performed on the product, to ensure its safety and consistency, should lead to a reappraisal of testing regimes applied to the cells themselves.


Introduction to the issues: detection of retroviruses and associated risks. A brief overview of retroviral contamination as it relates to biological products produced in cell substrates is presented, including currently used methods of detection and potential risks. The presence of infectious retrovirus in a cell substrate represents what may be a potential risk to recipients of the final product prepared from such cell banks, and it is the approach that can be taken to minimize this risk that is addressed.


Retroviral DNA integration.

Retroviral DNA integration. This overview summarizes the properties and mechanism of retroviral DNA integration and the consequences of the integration process to the infected cell and host. Integration of viral into cellular DNA is a regular and irreversible event, essential for virus replication, whose mechanism is becoming well understood. Many pathogenic consequences of retrovirus infection are due to side-effects of the integration process, including gene disruption, activation of proto-oncogenes, acquisition of oncogenes, and inappropriate expression of viral gene products. The properties and risks associated with these events are reviewed along with overall conclusions as to the importance of these to possible retroviral contaminants of biologicals.


PCR and other test systems in human gene therapy. Three protocols using retroviral-mediated gene transfer are currently undergoing clinical trials. The uniqueness of these protocols, which were the first approved experiments in which genetically engineered cells were administered to human beings, necessitated the development of novel biological and physical safety testing procedures. In addition to standard testing for contamination with exogenous pathogens and toxins, specific tests were developed to assay for the presence of recombinant helper virus in both retroviral vector preparations and in the engineered human cells. Two new assays were developed, an enhanced S+/L-virus assay called 3T3 amplification, and a specific polymerase chain reaction protocol of great sensitivity. The results of these safety tests, along with pre-clinical primate safety studies, and the accumulating clinical data, suggest that retroviral-mediated gene transfer does not pose any serious short-term safety problems for patients enrolled in gene therapy protocols.


Introduction to the issues: recently developed methods for characterizing cell lines. Cell line characterization is an integral part of establishing quality control over the manufacturing process of a biological product. Characterizing the general and unique features of the continuous cell line and the absence or presence of contaminants establishes a fundamental knowledge about the raw material from which the biological product is being derived. Such information can be used for cell line selection, design of the purification scheme and extent of ensuing validation studies. Developing the selected cell line into a cell bank system allows for a constant source of starting material and decreases the likelihood of contamination. Characterization of the cell bank system may include a) identification of the cell line species of origin, b) identification of unique reference markers, and c) pre-testing for cross-contamination and adventitious agents. This paper introduces issues surrounding present and upcoming methods used for authenticity and characterization testing of cell banks.


Applicability of statistical reliability methods to the validation process. This paper discusses some statistical considerations for the reliability of validation processes. An underlying theme is assessment of sources of variability for validation data. Confidence intervals and other statistical methods can clarify the impact of such variability. Statistical strategies for strengthening the design of systems for evaluation of validation processes are outlined.


Philosophy and goals of validation for biotech products and the relevance of scale. A model for validation of viral clearances in Biopharmaceutical processes is developed from an analysis of validation of terminal sterilization processes. The model develops the role for small-scale experiments in the validation of large-scale production processes. The steps for process validation are: identify or discover the basic general physical, chemical, or biological laws governing the process; identify the critical process control parameters for the specific application under investigation; and demonstrate the ability of the production equipment to maintain critical parameters with specified limits. For concurrent validation, this final step is seen as equivalent to demonstrating that a scaled-down model experiment is an appropriate representation of the production process.


Methods for authenticating cell lines.

Methods for authenticating cell lines. Methods for authentication of cell lines include tests for microbial contamination, verification of the species of origin, documentation of particular characteristics or functions and the absolute identification of individual cell lines. Experience indicates that mycoplasma contamination is still a serious problem in the cell culture field, with estimates on frequencies of infection varying from 10% upwards. The utilization of pre-screened reagents and antibiotic-free cultivation, plus the application of improved procedures, such as fluorescent dyes and molecular probes for detection, provide effective means to avoid mycoplasma infection and facilitate control. Viral contamination is perhaps more problematic, especially where no overt cytopathic effect results. The potential exists for the introduction of viruses from the cell culture technician or reagents used. Representative problem viruses are listed with standard procedures for screening. Detailed studies on animal cell cross contaminations have been performed and published. The frequency of detection of problem culture varied from 17-36% in studies performed in the USA. Both interspecies and intraspecies contaminations have been involved. Awareness of the potential for this problem plus the application of several characterization procedures are key factors for control. For example, fluorescent antibody staining, iso-enzyme analysis, cytogenetic evaluation and DNA finger-printing using molecular probes are needed for quality assurance on seed stocks. The critical importance of generating well-characterized reference cell stocks for use over the years is emphasized.


Filovirus contamination of cell cultures. The filoviruses Marburg and Ebola comprise a newly recognized family of viruses. The first filovirus to be isolated was Marburg virus in 1967. This virus was imported in shipments of African green monkeys from Uganda and infected several cell-culture technicians, with serious illness resulting. The rarity of Marburg and Ebola virus transmission, decreasing use of imported African monkeys, and quarantine efforts have presumably been responsible for the lack of additional episodes until 1989, when a new filovirus related to Ebola was isolated from quarantined monkeys in Reston, Virginia. This virus was imported on multiple occasions from a Philippine supplier of cynomolgus macaques as a consequence of an epidemic of acute infections in the foreign holding facility. While quarantine procedures prevented the use of any of these animals in research and the three human infections that occurred were asymptomatic, this episode emphasizes that these little understood viruses have considerable potential for mischief. The finding of antibodies reacting with Ebola viruses in many biomedically important Old World primates, including colonized monkeys in the U.S., emphasizes the need for more research to understand the specificity of the antibodies, spectrum of filovirus strains in nature, potential hosts, and true distribution of the family. The filoviruses grow well in primary and established cell strains and cell lines, and cytopathogenic effects may be absent or require several days to be manifest, leading to the possibility of occult contamination. The known viruses are readily detected by polyclonal and monoclonal antibody staining of cells and by electron microscopy; nucleic acid probes exist to develop more sensitive techniques if warranted.


An overview of bovine spongiform encephalopathy (BSE) in Britain. Since 1985 cases of BSE have occurred in increasing numbers in the UK, particularly in dairy herds. Scientist, clinicians and administrators co-operated in showing that this was due to exposure to ruminant-derived meat-and-bonemeal products. The measures used to contain the epidemic and protect biological medicinal products are described. The epidemic will require continued control measures and monitoring until it finally declines.


Cell line issues related to specific expression vectors: retrovirus vectors. Retroviruses have received attention as vectors for gene transfer because they can infect a variety of cell types, they integrate stably into the genome of the target cell and they have simple, well-defined structures. Insertion of the gene of interest into a retroviral vector results in the formation of replication-defective particles that can be produced in "packaging" cell lines. Due to the nature of the present generation of vectors and packaging cell lines, multiple recombination events would be needed to produce replication-competent particles. However, the concern still exists that replication-competent virus could arise either in the production of retroviral vector stocks or following their introduction into target cells. The current use of retrovirus vectors and the challenge they present to testing programmes are discussed.


Effect of baculovirus on selected vertebrate cells. Risk assessment of viral pesticides is required by federal statute. Subdivision M of the Pesticide Testing Guidelines includes mammalian cell culture studies for viral agents. Using these tests we have demonstrated baculovirus toxicity to human fibroblast and lung cells, as well as monkey kidney cells. However, virus was not toxic to toad cells. Furthermore, toxicity was associated with the virus particle and was not removed by dialysis of the preparation, gradient purification or psoralen inactivation of the virus. However, heat-inactivated virus was not toxic.


Insect cells: adventitious agents.

Insect cells: adventitious agents. The varied associations between insects and micro-organisms provides ample opportunity for the contamination of insect cell cultures. As with cell cultures from vertebrates, the addition of serum or serum products into cell culture media risks the introduction of mycoplasma and bovine viruses into cell cultures. Rarely do these organisms persist or multiply in insect cultures; however, there have been reports of the growth of Acholeplasmas introduced with fetal bovine serum. The contamination of cultures with plant or mammalian pathogens that are transmitted by insects is more common. The micro-organisms that are associated with insects, either as pathogens or commensals, frequently contaminate primary cell cultures and may also contaminate cell lines.


Cell line characterisation by DNA fingerprinting; a review. DNA fingerprinting using multi-locus probes has become established as the most powerful technology for the identification and characterisation of mammalian cell lines. However, care must be taken in the technical preparation and statistical evaluation of the DNA banding patterns.


Tumorigenicity testing: past concerns, future problems. Tumorigenicity testing has been an integral part of cell substrate characterization. With the combination of recombinant technology and traditional cell culture, we have entered an era in which many of the principles developed with vaccine production may need to be modified and new ones developed. Models of tumorigenicity are reviewed in the light of current knowledge; the applicability and necessity of such testing in new areas such as somatic cell therapy will also be presented.


Cell line issues: historical and future perspectives. The initial decision to use only primary cell cultures for the production of human biological products was challenged in the late 1960s by the introduction of human diploid cells (HDCs), and again in the 1980s by continuous cell lines (CCLs). The history of the HDC controversy is reviewed and lessons from that era that are relevant to the use of CCLs are pointed out. With the introduction of recombinant DNA technology in the 1980s, and the potential usefulness of CCLs in product development, the issue of cell acceptability became more urgent, and several attempts were made to reach a consensus on regulatory issues. In 1986, the World Health Organization convened a Study Group to review the safety issues related to products derived from CCLs. The Study Group made a clear recommendation to pursue CCLs in product development because of the demonstrated capability of modern manufacturing processes to cope with contaminants. Issues such as acceptable levels of cellular DNA in products, the relationship of purity to safety, and the relevance of the genetic stability of recombinant cells to product consistency are current examples of areas in need of discussion and agreement. A system in which regulatory authorities, industry, and the general biomedical community cooperate in finding solutions is ultimately in everyone's best interest.


Experience in cell line testing.

Experience in cell line testing. For more than ten years Microbiological Associates, Inc. has provided testing services for cell lines, products and processes associated with the safety of recombinant biologics. The results of numerous cell line characterizations performed under the guidance of "Points to Consider" documents shed light on the incidence of contamination of production cell banks. The most common sources of compromise of production substrates are also identified. Findings from this short review, though necessarily general, permit attention to be focused on areas of cell line characterization where concern is warranted. Conversely, where an alternative technique of equal sensitivity to existing tests is available for specific cell substrates, these may be given consideration. The observations presented here support the overall safety of biopharmaceuticals produced from genetically engineered cell lines and the wisdom of a multifaceted approach to assure product safety.


Integration and stability of CHO amplicons containing plasmid sequences. Fluorescence in situ hybridization (FISH, 15) and high density, non-selective long term perfusion culture were used to study aspects of genetic stability and productivity in recombinant CHO cells. We analysed the distribution and structure of recombinant amplicons in chromosomes of CHO cells used for the production of proteins. In the presence, but not in the absence, of methotrexate (MTX) we found a high proportion of cells (40-60%) with multiple and/or unusually structured and extended chromosome regions containing amplified sequences. Removal of MTX from culture media resulted in the rapid decline in the frequency of cells containing amplified sequences exhibiting multiple and heterogeneous integrations. In cloned lines cultivated in the absence of MTX, a well defined signal motif on a specific chromosome, interpreted as the "master integration" unit, became increasingly abundant over time until almost all cells contained that signal motif. We used fluorescence in situ hybridization (FISH) with chemically modified DNA probes complementary to the integrated sequences to verify the stability of master integrations in cells cultured for extended periods in medium lacking MTX. In a second approach, we studied long-term stability of recombinant sequences during non-selective perfusion culture of CHO populations with non-cloned, MTX resistant heterogeneous populations of cells producing CD4IgG chimeric molecules. Due to the mode of transfection and primary selection the resulting cell lines consisted of subpopulations of cells derived from various independent integration events. The amplification and MTX selection procedure used consecutively generated multiple, structurally different amplicon types in the cell population, thus increasing the degree of heterogeneity. High density perfusion culture of these cells in the absence of MTX was used to maximize growth rates and was thought to select against cells with reduced growth rates, due to the highly amplified state of their introduced DNA, with concomitantly high productivity. However we found no evidence for such a selection; cells showed no reduction in copy number or total loss of amplified sequences at the end of the culture. More significantly, specific productivity of these cell lines grown under non-selective conditions did not decrease over the 100 days observation period.


Physiological relevance of protein glycosylation. The glycosylation of proteins is a complex biological pathway which is ordered and non-random. It is also a deterministic pathway dependent upon protein sequence, cellular phenotype, and the physiological environment. Two principal physiological roles have emerged within the past decade for protein-linked glycans: as recognition determinants and as modulators of various protein attributes such as bioactivity, pharmacokinetics, folding, and immunogenicity. All these attributes are crucial to development and application of protein-based pharmaceuticals. However, protein glycosylation represents a difficult structure/function problem since most glycoproteins exhibit microheterogeneity and oligosaccharides frequently contribute to this heterogeneity. Nevertheless, recent data suggest that different members of the heterogeneous ensemble exhibit distinguishable intrinsic properties, suggesting that the microheterogeneity of protein glycosylation represents a sophisticated mechanism of biological control.


Bioprocess factors affecting glycoprotein oligosaccharide structure. The oligosaccharide structures of glycoproteins can have a profound effect on properties critical to the development of glycoprotein products for human therapeutic or diagnostic use, including clearance rate, antigenicity, specific activity, solubility, resistance to thermal inactivation, and resistance to protease attack. Therefore, it is important to understand how bioprocess factors influence oligosaccharide structure. In this presentation, I will summarize literature data concerning potential effects of bioprocess factors on glycoprotein oligosaccharide biosynthesis. These data are drawn from two recent detailed reviews published by our laboratory describing the effect of cell culture conditions on N-linked glycosylation (10) and the effects of other bioprocess factors on both N-linked and O-linked glycosylation (11).


The effect of the calcium antagonist nifedipine on peripheral nerve function in streptozotocin-diabetic rats. Recent data suggests that reduced nerve blood flow is implicated in the aetiology of experimental diabetic neuropathy, which may be prevented by manipulations that reduce receptor-mediated vasoconstrictor activity. This investigation examines the effects of nifedipine, a voltage-sensitive calcium channel antagonist which has a direct vasodilatory effect on vessels, on nerve conduction, hypoxic resistance and capillary density in streptozotocin-induced diabetic rats. Treated and non-treated non-diabetic and diabetic groups were employed. Diabetes duration was 2 months. Treatment was preventive, groups received a nifedipine dietary supplement (40 mg.kg-1.day-1) for 2 months from the start of the study. Conduction was measured in sciatic motor branches supplying tibialis anterior and gastrocnemius muscles, and sensory saphenous nerve. Diabetes resulted in a 23-28% reduction in motor conduction velocity (p < 0.001), and a 15% deficit for sensory saphenous nerve (p < 0.001). In the nifedipine-treated diabetic group, motor and sensory conduction deficits were minimal compared with non-treated diabetes (p < 0.001). Nifedipine treatment had no significant effect on conduction velocity in non-diabetic rats. In vitro measurement of sciatic nerve hypoxic resistance revealed a 60% increase in the time taken for compound action potential amplitude to reach half its initial value with diabetes (p < 0.001). This was not significantly affected by nifedipine treatment. Experimental diabetes or nifedipine treatment did not significantly alter sciatic nerve endoneurial capillary density. We conclude that nifedipine, a vasodilator which acts directly on vascular smooth muscle, prevents nerve conduction deficits in experimental diabetes.


Increased vascular permeability in pancreas of diabetic rats: detection with high resolution protein A-gold cytochemistry. The role of the pancreatic microcirculation in the pathogenesis of Type 1 (insulin-dependent) diabetes mellitus remains poorly understood. Herein, a method is described for the ultrastructural investigation of the integrity of the pancreatic microvasculature. The method consists of histochemical detection and isolation of the islets followed by albumin and protein A-gold immunocytochemistry, whereby the distribution of endogenous albumin is used as a marker of endothelial integrity. This technique, applied to the study of spontaneously diabetic rats, reveals a selective increase in permeability of islet capillaries and post-capillary venules at the onset of diabetes, while acinar capillaries and arterioles remain intact. At 50 days of age, before the onset of diabetes, the microvasculature of diabetes-prone rats shows no alterations in permeability to albumin. When used in conjunction with morphometric analyses, this methodological approach may be useful for further studies in pathologic or experimental conditions involving the pancreatic microvasculature.


Contribution of neural intrapancreatic non-cholinergic non-adrenergic mechanisms to glucose-induced insulin release in the isolated rat pancreas. In the isolated rat pancreas the effect of intrapancreatic non-adrenergic non-cholinergic nerves was examined upon insulin, glucagon and somatostatin release during perturbations of perfusate glucose. Elevation of glucose from 1.6 to 8.3 mmol/l increased insulin and somatostatin secretion and inhibited glucagon release. The first phase of insulin secretion was significantly reduced by the neurotoxin tetrodotoxin to 55% of the controls (p < 0.05). The somatostatin response was attenuated by tetrodotoxin while the change of glucagon remained unaffected. In contrast the combined adrenergic and cholinergic blockade with atropine, phentolamine and propranolol (10(-5) mol/l) did not modify the insulin, glucagon and somatostatin response. When glucose was changed from 8.3 to 1.6 mmol/l, the reduction of insulin and somatostatin release was not modified by tetrodotoxin, but stimulation of glucagon was significantly attenuated by 60-70% (p < 0.03), which was similar to the effect of combined adrenergic and cholinergic blockade. Subsequently, the effect of neural blockade was examined during more physiological perturbations of perfusate glucose levels. When glucose was changed from 3.9 to 7.2 mmol/l, tetrodotoxin also attenuated first phase insulin response by 40% while cholinergic and adrenergic blockade had no effect. The nitric oxide synthase inhibitor NG-Nitro-L-arginine-methyl-ester (L-NAME) did not alter the glucose-induced insulin response indicating that nitric oxide is not involved in this mechanism. It is concluded that neural non-adrenergic non-cholinergic mechanisms contribute to the first, but not second phase of glucose-induced insulin release. Non-adrenergic non-cholinergic effects do not participate in regulation of glucagon and somatostatin secretion under the conditions employed.(ABSTRACT TRUNCATED AT 250 WORDS)


Insulin resistance in type 2 (non-insulin-dependent) diabetic patients with hypertriglyceridaemia. Hypertriglyceridaemia, which is frequently seen in Type 2 (non-insulin-dependent) diabetes mellitus, is associated with insulin resistance. The connection between hypertriglyceridaemia and insulin resistance is not clear, but could be due to substrate competition between glucose and lipids. To address this question we measured glucose and lipid metabolism in 39 Type 2 diabetic patients with hypertriglyceridaemia, i.e. mean fasting serum triglyceride level equal to or above 2 mmol/l (age 59 +/- 1 years, BMI 27.4 +/- 0.5 kg/m2, HbA1c 8.0 +/- 0.2%, serum triglycerides 3.2 +/- 0.2 mmol/l) and 41 Type 2 diabetic patients with normotriglyceridaemia, i.e. mean fasting serum triglyceride level below 2 mmol/l (age 58 +/- 1 years, BMI 27.0 +/- 0.7 kg/m2, HbA1c 7.8 +/- 0.2%, serum triglycerides 1.4 +/- 0.1 mmol/l). Insulin sensitivity was assessed using a 340 pmol.(m2)-1 x min-1 euglycaemic insulin clamp. Substrate oxidation rates were measured with indirect calorimetry and hepatic glucose production was estimated using a primed (25 microCi)-constant (0.25 microCi/min) infusion of [3-3H]-glucose. Suppression of lipid oxidation by insulin was impaired in patients with hypertriglyceridaemia vs patients with normal triglyceride levels (3.5 +/- 0.2 vs 3.0 +/- 0.2 mumol.kg-1 x min-1; p < 0.05). Stimulation of glucose disposal by insulin was reduced in hypertriglyceridaemic vs normotriglyceridaemic patients (27.0 +/- 1.3 vs 31.9 +/- 1.6 mumol.kg-1 x min-1; p < 0.05) primarily due to impaired glucose storage (9.8 +/- 1.0 vs 14.6 +/- 1.4 mumol.kg-1 x min-1; p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)


Correlations between nerve function and tissue oxygenation in diabetic patients: further clues to the aetiology of diabetic neuropathy? Transcutaneous oxygen, laser Doppler flowmetry, peroneal nerve motor conduction velocity and skin temperature were assessed in both legs of 34 diabetic patients, who had a mean age of 41 (range 29-77) years, and diabetes duration of 21 (3-34) years. Transcutaneous oxygen significantly correlated with peroneal nerve motor conduction velocity (r = 0.59 p < 0.001) and laser Doppler flowmetry (r = 0.7 p < 0.001). Laser Doppler flowmetry correlated weakly with peroneal motor conduction velocity, (r = 0.34 p < 0.05). In each patient the leg with the higher transcutaneous oxygen (mean 70.2 +/- 9.3 (SD) mmHg) had a significantly higher peroneal motor conduction velocity (45.3 +/- 7.1 vs 41.5 +/- 6.3 m/s, p < 0.01), than the leg with the lower transcutaneous oxygen (61.0 +/- 11.9 mmHg), though no difference in skin temperature was observed, 31.4 +/- 0.4 vs 31.1 +/- 0.5 degrees C. We then assessed the potential for reversibility of conduction velocity deficits in ten non-diabetic patients, aged 59 (52-77) years, undergoing unilateral femoro-popliteal bypass, measuring transcutaneous oxygen, peroneal nerve motor conduction velocity and skin temperature pre- and 6 weeks post-surgery. In the control leg (unoperated) there was no significant change in transcutaneous oxygen (63.2 +/- 8.8 vs 63.0 +/- 4.6 mm Hg), peroneal nerve motor conduction velocity (45.1 +/- 7.8 vs 43.4 +/- 7.2 m/s) or skin temperature (30.8 +/- 1.3 vs 30.2 +/- 1.2 degrees C) after surgery (all NS).(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of C-peptide on blood flow, capillary diffusion capacity and glucose utilization in the exercising forearm of type 1 (insulin-dependent) diabetic patients. Microvascular dysfunction is frequently seen in patients with Type 1 (insulin-dependent) diabetes. The present study was undertaken to examine whether skeletal muscle microcirculation in Type 1 diabetic patients is influenced by C-peptide. Forearm blood flow, capillary diffusion capacity and substrate exchange were studied during strenuous rhythmic forearm exercise on a hand ergometer. Measurements were made before and during i.v. infusion for 60 min of C-peptide or 0.9% NaCl in Type 1 diabetic patients and healthy subjects. During infusion the C-peptide levels in the diabetic patients increased from less than 0.05 nmol/l to 1.32 +/- 0.08 nmol/l. Prior to infusion forearm blood flow and capillary diffusion capacity during exercise were lower in the diabetic patients than the control subjects. During C-peptide infusion both variables increased in the diabetic patients (blood flow +27 +/- 4%, capillary diffusion capacity +52 +/- 9%) to levels similar to those in the healthy subjects, while no significant change was seen in the healthy control subjects or the diabetic patients given NaCl. Forearm uptake of oxygen and glucose in the diabetic patients increased markedly after C-peptide administration but were unchanged after NaCl infusion. Significant uptake of C-peptide to the deep forearm tissues was observed in the resting state; approximately 7 +/- 2% of the arterial C-peptide concentration was extracted by forearm tissues in diabetic patients as well as in healthy control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)


Cellular and humoural autoimmunity markers in type 2 (non-insulin-dependent) diabetic patients with secondary drug failure. In some cases patients with Type 2 (non-insulin-dependent) diabetes mellitus fail to respond to treatment with oral hypoglycaemic agents. These patients may respond in the same way as Type 1 (insulin-dependent) diabetic patients. Cellular immune aggression (defined as the capacity of peripheral mononuclear cells to inhibit stimulated insulin secretion by dispersed rat islet cells), insulin autoantibodies, C-peptide response and HLA antigens were determined in 31 Type 2 diabetic patients with secondary failure to oral hypoglycaemic agents and in 22 control subjects. Nine (29.03%) of the 31 Type 2 diabetic patients showed positive cellular immune aggression (2 SD below control group) and 22 (70.97%) presented no cellular immune aggression. There was a relationship between positive cellular immune aggression and each of the following parameters: age, body mass index and microangiopathy. No correlation was found between positive cellular immune aggression and glycaemia, HbA1, blood lipids or atherosclerosis. Patients with positive cellular immune aggression showed a significantly lower glucagon-stimulated C-peptide response vs those with no cellular immune aggression. Within a sub-group of patients who had never been treated with insulin, insulin autoantibodies were present in four of six patients with positive cellular immune aggression. DR2 antigen was found with decreased frequency in patients whereas no DR3/DR4 heterozygotes were observed. Our data support the hypothesis that a group of Type 2 diabetic patients with secondary failure to oral hypoglycaemic agents presented autoimmunity towards pancreatic Beta cells.


Glomerular charge selectivity and the influence of improved blood glucose control in type 1 (insulin-dependent) diabetic patients with microalbuminuria. We first compared glomerular charge selectivity index in two matched groups of Type 1 (insulin-dependent) diabetic patients with micro- and normoalbuminuria respectively, and secondly, investigated prospectively in a randomized clinical trial, the influence of improved metabolic control on selectivity index in diabetic patients with microalbuminuria. In Study 1, 27 patients with microalbuminuria (albumin excretion > or = 15 micrograms/min in at least two out of three overnight urine samples) were matched (age, diabetes duration, mean 1-year HbA1c, gender) with normoalbuminuria patients (n = 24), and in Study 2, 23 microalbuminuric patients were randomly allocated to either intensive (continuous subcutaneous insulin infusion) or conventional treatment. Glomerular charge selectivity index was measured as IgG/IgG4 selectivity index, i.e. total IgG/IgG4 clearance ratio in timed overnight urine samples. The microalbuminuric patients had a significantly reduced selectivity index compared to the normoalbuminuric patients: 1.20 (0.92-1.40) vs 1.68 (1.22-2.21), median and 95% confidence interval (p < 0.01). In Study 2, the HbA1c improved in the intensive-treatment group compared to the conventional-treatment group: at 2, 6 and 12 months the difference in mean percentage HbA1c between the groups was 1.1, 1.2 and 1.4, respectively (p < 0.01). A sharp 50% increment in IgG/IgG4 selectivity index was seen in the intensive-treatment group during the first 6 months (p < 0.05 compared to the conventional group). We conclude that adolescents and young adults in an early stage of diabetic nephropathy have reduced glomerular charge selectivity, which may be improved by reducing the mean blood glucose level.


Hyperproinsulinaemia in patients with myotonic dystrophy. Hyperinsulinaemia is a reported feature of the inherited multisystem disorder myotonic dystrophy. This phenomenon has been attributed to a compensatory beta cell response to tissue insulin resistance. In this study, circulating concentrations of insulin, proinsulin, and split proinsulin molecules were determined after an overnight fast in ten patients with myotonic dystrophy using two-site monoclonal antibody-based immunoradiometric assays. Results were compared with ten healthy control subjects matched for age, gender, and body mass index. Oral glucose tolerance (75 g), as defined by World Health Organization criteria, was normal in all subjects. Fasting plasma immunoreactive insulin concentration, as determined using a conventional radioimmunoassay, was almost three times higher (p < 0.005) in the myotonic dystrophy patients than the healthy control subjects. By contrast, fasting concentrations (mean +/- SEM) of C-peptide (0.75 +/- 0.09 vs 0.52 +/- 0.03 nmol/l, p = 0.07) and immunoradiometrically-determined insulin (60 +/- 12 vs 38 +/- 4 pmol/l, p = 0.09) were not significantly different between the groups. Fasting concentrations of proinsulin (10.3 +/- 2.9 vs 1.6 +/- 0.3 pmol/l, p < 0.01), and 32-33 split proinsulin (7.8 +/- 2.5 vs 2.9 +/- 0.4 pmol/l, p < 0.05) were significantly elevated in the patients with myotonic dystrophy. Accordingly, the mean fasting proinsulin:insulin ratio, expressed as a percentage, was significantly increased in the myotonic patients (20 +/- 5 vs 4 +/- 1%, p < 0.01). The overall C-peptide response to the oral glucose challenge was significantly greater in the myotonic patients compared with the healthy control subjects (p < 0.001). These results provide corroborative evidence of increased beta-cell secretion in myotonic dystrophy.(ABSTRACT TRUNCATED AT 250 WORDS)


Pulmonary complications of type 1 (insulin-dependent) diabetic patients. We have investigated the influence of diabetes mellitus including the presence of late complications of the pulmonary system. To check this relationship 31 Type 1 (insulin-dependent) diabetic patients (mean age 30.6 +/- 5.32 years, mean duration of diabetes 12.9 +/- 5.05 years) were admitted into the trial and compared with 18 control subjects. Pulmonary function tests were measured including spirometric parameters, diffusing capacity, specific diffusing capacity and dynamic compliance measured at 20 and 60 breaths per min. No disturbance of the spirometric parameters was observed in the diabetic patients. Diffusing capacity in the diabetic patients with complications was significantly lower than in both the diabetic patients without complications and the control group (81.2 +/- 16.2%, 104 +/- 13.7%, 99.3 +/- 2.8%; p < 0.001, p < 0.005 respectively). Specific diffusing capacity was significantly lower in the diabetic patients than in the control subjects (80.3 +/- 13.1% vs 89.4 +/- 12.9%; p < 0.05). In the group with late complications specific diffusing capacity was lower than in the group without complications (69.7 +/- 9.17%; 87.2 +/- 10.7%, respectively; p < 0.001). Dynamic compliance at 20 breaths per min in diabetic patients was 84.06 +/- 17.08% vs 95.2 +/- 11.59% in the control subjects (p < 0.05). It was particularly low in the group with late complications 80.6 +/- 13.2% and patients with metabolic poor control, 80.3 +/- 12.02% (both p < 0.005 vs the control group). Dynamic compliance at 60 breaths per min was 60.1 +/- 15.0% as compared to 83.2 +/- 13.3% in the control group (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)


Microdialysis measurement of the absolute glucose concentration in subcutaneous adipose tissue allowing glucose monitoring in diabetic patients. The possibility of continuously monitoring the absolute glucose concentration in subcutaneous adipose tissue, using microdialysis of the extracellular water space, was investigated in six Type 1 (insulin-dependent) diabetic patients. By using a large microdialysis probe (30 x 0.62 mm), and by perfusing with a low flow rate (0.5 microliters/min), complete recovery of glucose was attained in vitro. In the patients the dialysis probe was implanted subcutaneously, perfused by a wearable microinfusion pump, and dialysate samples were collected in 60-min fractions over 10 h. The absolute glucose concentration in the tissue dialysate was the same or almost the same as the blood glucose concentration (range 87-101% of the blood glucose value). The changes in blood glucose were closely paralleled by the variations in adipose tissue glucose (r = 0.93, p < 0.01), and the recovery of glucose in the microdialysate remained constant during the 10-h study period. In conclusion, it is possible, using microdialysis, to directly determine the absolute glucose concentration in subcutaneous adipose tissue. Hence, this technique may be used for continuous glucose monitoring in diabetic patients.


Genetic control of diabetes mellitus.

Genetic control of diabetes mellitus. Genetic inheritance predisposing individuals to diabetes mellitus was discussed in this work group. The two forms of the disease, Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) were discussed separately since the pattern of inheritance and genes involved appear to be distinctly different. Within these subtypes there is considerable genetic heterogeneity, and superimposed environmental factors confound the analysis. New technologies that will allow finer molecular analysis, as well as new candidates genes, were presented.


The process of atherogenesis--cellular and molecular interaction: from experimental animal models to humans. Atherogenesis is a disorder of the artery wall that involves: adhesion of monocytes and lymphocytes to the endothelial cell surface; migration of monocytes into the sub-endothelial space and differentiation into macrophages; ingestion of low density lipoproteins and modified or oxidised low density lipoproteins by macrophages by several pathways, including a scavenger pathway, leading to accumulation of cholesterol esters and formation of "foam cells". These foam cells together with T lymphocytes form the fatty streak. Vascular smooth muscle cells migrate from the media into the intima and proliferate with the formation of atherosclerotic plaques. These processes which involve cell adhesion, migration, differentiation, proliferation and cell interaction with the extracellular matrix are regulated by a complex network/cascade of cytokines and growth regulatory peptides. Thus, atherosclerosis may be the result of a specialised chronic inflammatory fibroproliferative process which has become excessive and in its excess this protective response has become the disease state.


Cellular and molecular biology of the beta cell. Considerable progress has been made in our understanding of islet-cell function and its relationship to regulation of whole body glucose metabolism. At the genetic level, the regulatory regions in islet-specific genes are being characterised. Transcription factors that interact with these regions have been cloned and these will be instructive in elucidating how islet-specific genes are regulated during development and regeneration. Identification of the enzymes responsible for proteolytic conversion of proinsulin to insulin represents a major advance in understanding prohormone processing. Cleavage of proinsulin is mediated by at least two prohormone convertases (PC3/PC1 and PC2). Their activity is regulated by an acidic gradient between the Golgi and secretory granules and by calcium ions. It is not yet clear how insulin or the PC's are specifically diverted into the regulated secretory pathway. Regulation at this step may be defective in some diabetic patients resulting in relatively elevated circulating proinsulin levels. Specific features of GLUT 2 and glucokinase (GK), proteins that regulate Beta-cell glucose transport and phosphorylation, indicate that these may be key components of the glucose sensor. GLUT 2 is necessary to reconstitute glucose-sensitive insulin secretion in pituitary tumour cells expressing a proinsulin cDNA. Furthermore, the expression of GLUT 2 in Beta cells, but not in hepatocytes, is decreased in diabetes mellitus. However, under normal circumstances GK is probably rate limiting for Beta-cell glucose utilisation. Thus, it is likely that both GLUT 2 and GK determine the set point for glucose-stimulated insulin secretion. Elucidation of distal effectors that regulate insulin secretion is also crucial to our understanding of Beta-cell function.(ABSTRACT TRUNCATED AT 250 WORDS)


Autoimmune tolerance and type 1 (insulin-dependent) diabetes mellitus. The autoimmune process that results in Type 1 (insulin-dependent) diabetes mellitus may be viewed as a failure to develop or maintain tolerance to self-antigens expressed in the islets of Langerhans. During T-cell development in the thymus, cells that are reactive with self antigens encountered there may undergo clonal deletion or, as more recently described, clonal anergy which effectively removes these cells from the pool of mature antigen reactive T cells. For antigens not found in the thymus, tolerance to self antigens is more complex and may depend on site of antigen expression, ambient concentrations of lymphokines, and availability of antigen-presenting cells that can deliver co-stimulatory signals. Transgenic mice in which the majority of T cells express T-cell receptors against "self" antigens or in which expression of antigens is targeted to peripheral tissues have proven useful for studies of tolerance in both T- and B-cell compartments. In general, T-cell reactivity against foreign antigen expressed on Beta cells does not occur because of the failure to activate T cells reactive with the antigen, termed clonal ignorance. This may be broken with, for example, viral infection or cytokines. In one transgenic model, dendritic cells that surround the islets of Langerhans have been shown to be responsible for presentation of islet antigens to the immune system. B-cell tolerance can also involve mechanisms of clonal deletion or clonal anergy similar to that occurring with T cells. In addition, a mechanism for changing the affinity of the B-cell antigen receptor termed "receptor editing" has been described, which may play an important role in diversifying the B-cell repertoire while removing self-reactive cells. Tolerance to antigens may also be inducible. For example, monoclonal antibodies against T-cell epitopes may induce antigen-specific tolerance that is transferable to other animals, and MHC blocking peptides which can inhibit T-cell responses that are restricted by disease associated MHC molecules. In conclusion, although several possible triggers and mechanisms of autoimmune diabetes can be envisioned, none can be excluded by existing data. However, advances in understanding mechanisms of tolerance to islet and other self antigens suggest potentially useful therapeutic approaches to arresting the autoimmune response.


Treatment of diabetes mellitus.

Treatment of diabetes mellitus. New treatment strategies for subjects with Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetes mellitus are being developed. Pilot studies utilising insulin itself have been reported to prevent Type 1 diabetes in subjects likely, by immunogenetic and physiologic criteria, to develop clinically overt disease, while the results of nicoti-namide trials in these subjects remain preliminary. Immunotherapy with cyclosporin A and azathioprine can slow disease progression and may produce long-term remissions when given within two months of onset of clinically overt disease. In subjects with established disease, familial clustering of diabetic nephropathy may be related to concomitant susceptibility to hypertension and elevated rates of Na/H countertransport. Treatment of hypertension associated with the nephropathy appears to slow renal deterioration. Whether reversal of the metabolic consequences of insulin deficiency or resistance also prevents chronic diabetic complications has not been firmly established. In the presence of a reduced Beta-cell mass, moderate hyperglycaemia may itself contribute to decreased muscle glucose uptake but not glycogen synthesis in Type 1 and Type 2 diabetes. Reversal of chronic hyperglycaemia by pancreas and islet cell transplantation, vanadate, and sulphonylureas are discussed as alternate strategies to insulin treatment in establishing normoglycaemia and furthering our understanding of insulin action and secretion at the cellular level. There remains a need to develop more sensitive biochemical and genetic markers to identify subjects at increased risk for developing chronic diabetic complications.


[Left atrial electric isolation in the treatment of atrial fibrillation secondary to rheumatic valvular disease]. Surgical isolation of the left atrium was performed for the treatment of chronic atrial fibrillation secondary to valvular disease in 100 patients who underwent valve surgery. From May 1989 to September 1991, 62 patients underwent mitral valve surgery (Group I), 19 underwent mitral valve surgery and DeVega tricuspid annuloplasty (Group II), 15 underwent mitral and aortic surgery (Group III), and 4 patients underwent mitral and aortic surgery and DeVega tricuspid annuloplasty (Group IV). Left atrial isolation was performed prolonging the usual left paraseptal atriotomy towards the left fibrous trigone anteriorly, and the postero-medial commissure posteriorly. The incision was conducted a few millimeters apart from the mitral valve annulus, and cryolesion were placed at the edges to ensure complete electrophysiological isolation of the left atrium. Operative mortality accounted for 3 cases (3%). In 79 patients (81.4%) sinus rhythm recovered and persisted until discharge from the hospital. No differences were found between the groups (Group I: 80.7%; Group II: 68.5%; Group III 86.7%, Group IV 75% - p = N.S.). Three cases of late mortality (3.1%) were registered. long-term results showed persistence of SR in 71% of Group I, 61.2% of Group II, 85.8% of Group III, and 100% of Group IV. The unique risk factor for late recurrency of atrial fibrillation was found to be a duration of preoperative AF longer than 6 months. Due to the high success rate in recovering the sinus rhythm, we suggest left atrial isolation in patients with chronic atrial fibrillation undergoing valvular surgery.


[Pordenone study: HDL-cholesterol and obesity indexes in the young]. In 1987, ten years after the first observation, we performed the follow-up of the subjects included in the "Pordenone Study on the precursors of atherosclerosis in childhood". The anthropometric, biologic and anamnestic indicators of coronary risk were evaluated. The W.H.O. protocol was always used. 439 (90%) subjects underwent reexamination, (234 males and 205 females between 18 and 26 years). HDL cholesterol, tricipital and subscapular skinfold thickness, weight and height were evaluated. The aim of our study was to find possible correlations between obesity indexes and HDL cholesterol values. We found that HDL cholesterol levels are lower in males compared to females and that differences exist below and over the 80 degrees percentile of BMI. Obese subjects have lower HDL cholesterol levels in both sexes. Females with android obesity (subjects with subscapular skinfold thickness values over 80 degrees percentile) had low HDL cholesterol values. Because of this inverse correlation between HDL cholesterol and coronary risk, and because young obese generally have low HDL cholesterol levels, we believe that the study of coronary risk factors is also useful starting from this age in overweight subjects. This will be useful for preventive purposes. Particular attention must be given to young girls with android obesity.


[Arrhythmogenicity in left ventricular hypertrophy in mild to moderate arterial hypertension]. Several studies have evidenced that hypertensive patients (pts) with left ventricular hypertrophy (LVH) have an increased incidence of malignant ventricular arrhythmias and sudden death. The purpose of our study was to investigate the prevalence of risky ventricular arrhythmias in uncomplicated hypertensive pts (untreated during last 10 days) in comparison with normotensive ones. In this context, not only the value of left ventricular mass index (LVMI) was taken into account, but also the type of LVH and the related functional behaviour. 59 untreated mild to moderate essential hypertensives (EH), without symptoms or signs of coronary artery disease, were classified in 3 groups: normal (i.e. without hypertrophy) EH (NEH: 12 pts, 6 M and 6 F, mean age +/- SD 52 +/- 10 yrs), concentric hypertrophic EH (CEH: 30 pts, 15 M and 15 F, mean age +/- SD 59 +/- 10 yrs), and eccentric hypertrophic EH (EEH: 17 pts, 7 M and 10F, mean age +/- SD 60 +/- 10 yrs), according to echocardiographic measurements. Values and duration of arterial hypertension were comparable among the groups. A normotensive, age-matched group was studied as control (C: 21 pts, 11 M and 10 F, mean age +/- SD, 57 +/- 10 yrs). 24-hour Holter electrocardiographic monitoring (ECG-H) and Signal-Averaged electrocardiography (SAECG) were performed seeking to identify the arrhythmogenic risk. Echocardiographic analysis was accomplished by means of a computerized system: LVMI, ratio of LV wall thickness to LV internal radius (relative wall thickness = RWTh), systolic velocity of circumferential fractioning (VCFs), peak of LV relaxation rate (pLVRr) and peak-systolic stress (pSS) were evaluated. Normal LV systolic function was generally found, but both NEH and EEH groups showed a significant reduction in pLVRr in comparison with C and CEH groups (mean values +/- SD: 3.52 +/- 1,3 and 3.40 +/- 0.9 vs 4.92 +/- 0.4 and 4.27 +/- 1.4 sec-1, respectively, p < .05 for both). pSS was significantly higher in EEH and NEH than in CEH and C (mean values +/- SD: 149 +/- 42 and 157 +/- 66 vs 116 +/- 28 and 122 +/- 15 10(3) dynes/cm2, respectively; p < .05 for both). At ECG-H, EEH had a prevalence of potentially malignant ventricular arrhythmias (PMVA: ventricular extrasystoles > or = 30/h; ventricular couplets, > or = 2 episodes/24h, or triplets, > or = 1 episode/24h; R on T), significantly larger than in C (35.3% vs 4.8%, p < .05) and almost significantly larger than in NEH and CEH (8.3% and 10%, respectively). No differences in LVMI were found between EEH with or without PMVA. In respect of functional LV behaviour, the former group showed lower values of VCFs (2.33 +/- 0.6 vs 3.71 +/- 1.32 sec-1, (p < .005) than the latter group. At SAECG, the EEH exhibited again a greater prevalence of abnormal findings than C (35.3% vs 0%, p < 0.5). No correlations were found between ECG-H and SAECG abnormalities, nor between the latter group and LVMI or LV functional indexes. Among pts showing a more pronounced impairment of diastolic function (pLVRr < 4 sec-1), EEH exhibited the highest prevalence of both PMVA (50%) and late potentials (41%). Our data suggest that uncomplicated mild to moderate essential hypertension may be associated with higher risk of ventricular arrhythmias, particularly when cardiac involvement is characterized by eccentric LVH. On the contrary, in this stage of hypertensive disease, LVMI as well as LV function do not seem to influence the ventricular arrhythmogenesis. The clinical importance of these findings is uncertain, and further studies are needed.


[Effects of aerobic training in patients with moderate chronic heart failure]. To evaluate the effects of a program of moderate intensity in patients (pts) with clinically stable chronic heart failure (CHF), we studied 20 pts (18M, 2F, mean age 61 years) with dilated cardiomyopathy, Weber Class B, ejection fraction (EF) < 40% and aerobic capacity of 16 +/- 2 ml/kg/min on cardiopulmonary exercise testing. We randomly assigned pts to 2 groups, a training group (T, 10 pts) and a control group (C, 10 pts), similar for anatomical and clinical characteristics, group T underwent a thrice weekly, 8 week-long ambulatory program of aerobic activity, beginning at 40% of maximal oxygen uptake. At the end, in Group T we observed a significant increase of exercise tolerance (+45%; p < 0.005), peak oxygen uptake (VO2 max) (+20%; p < 0.001), anaerobic ventilatory threshold (AT) (+37%; p < 0.005), lactate threshold (+36%; p < 0.005), peak heart rate (< 10%; p < 0.01) and of peak systolic pressure (+12%; p < 0.007); and a significant reduction in resting heart rate (-17%; p < 0.005), resting diastolic pressure (-11%; p < 0.005), plasma lactate (LA) at rest (-26%; p < 0.01), at peak (-21%; p < 0.005) and at recovery (-22%; p < 0.005), plasma norepinephrine (NE) both at rest (-38%; p < 0.005) and at peak (-13%; p < 0.005) and of plasma epinephrine (E) (-38%; p < 0.005; -32%, p < 0.001, respectively). We observed no change in EF at the end in both groups nor any untoward cardiac effects during training. We didn't note any correlation between AT and venous oxygen saturation (r = 0.15; p = 0.65) changes at the end. The increase in peak VO2 after training was not correlated to any AT increase (r = 0.12; p = 0.72). We observed, however, a significant correlation between lactate threshold and AT changes after training (r = 0.81; p = 0.005) and between LA and resting, submaximal and peak NE (r = 0.89; p = 0.005) and E (r = 0.78; p = 0.007) changes at the end of training. a) in pts with clinically stable CHF a program of aerobic activity well tolerated in terms of frequency, intensity and duration may determine a significant increase in exercise tolerance, aerobic capacity, AT and LA threshold, and a significant decrease in plasma LA, NE and E at rest, submaximal and peak levels; b) in our opinion, such modifications are partly determined by a delay in lactate accumulation and partly by sympathetic tone lowering, and permit us to underline the concept that physical inactivity may provoke important peripheral changes that, in turn, may reduce exercise tolerance and aerobic capacity, by creating a vicious circle difficult to break by medical therapy alone.


[Perception threshold of angina and transient myocardial ischemia in ambulatory electrocardiography]. We studied the predictive value of prolonged angina perception threshold in identifying patients with stable coronary artery disease at risk of silent myocardial ischemia during daily life. 71 patients with documented coronary artery disease (previous myocardial infarction or stenotic lesion > 60% at angiography) underwent a symptom-limited exercise test and out-of-hospital Holter monitoring after drug withdrawal. A second exercise test was performed before disconnecting the dynamic EKG in order to validate the ST-depression recorded during ambulatory monitoring. 23 patients (32.4%) (Group A) had angina perception threshold > 60 sec after onset of ischemia (ST > 1 mm), while in 48 (67.7%) the delay in the perception of angina was shorter than 60 sec (Group B). The demographic, clinical and angiographic variables did not influence the angina perception threshold; however, this parameter was the most powerful predictor of ambulatory ischemia among the two groups (4.8 vs 2.8 p < 0.02), and in particular of the painless episodes (3.8 vs 1.8 p < 0.002). Moreover, the silent ischemic time was longer in patients of group A (4362 vs 1774 sec p < 0.017). Finally, the event-free survival was similar in the two groups of patients during the 2 years of follow-up (cardiac death 1 vs 3, nonfatal myocardial infarction 1 vs 1, aorto-coronary bypass 2 vs 7, PTCA 2 vs 2, unstable angina 0 vs 2), total events 6 vs 15 p = ns. These results demonstrate that the patients at risk for silent ischemia during ambulatory monitoring may be identified simply by evaluating their angina perception threshold during exercise test; however, silent ischemia does not have an adverse prognostic value.


[Aortic dissection after aortic valve replacement: clinical and anatomic features]. Cardiac surgery has become a possible cause of ascending aortic dissection, particularly in patients who have undergone aortic valve replacement. From January 1979 to December 1989, 134 patients with angiographic evidence of aortic dissection, were studied in the Haemodinamic Laboratory of the University of Turin. Twelve of these patients (7 men and 5 women), mean age 51.6 years, had been previously operated for aortic valve replacement. We evaluated incidence, clinical and anatomic features of ascending aortic dissection in this group. A dilated ascending aorta with a mean diameter of 4.92 cm. was noted in all these patients. The interval between valve replacement and diagnosis of aortic dissection was 57 months (range 1 day to 9 years). Reoperation was performed in 11 patients. Four intraoperative deaths occurred and 2 other patients died in the early post-operative period. Survival rate at 12 months was 50%. Our incidence of ascending aortic dissection after aortic valve replacement was 0.66% (10 patients out of 1499 operated for aortic valve replacement) and it is in agreement with reports from other Authors in literature. Replacement of the ascending aorta must be considered if an important dilatation of aortic root is found at time of aortic valve surgery. New efforts must be undertaken to identify patients at high risk for this complication because an elevated mortality rate is observed in reoperation for aortic dissection.


[Increased incidence of spontaneous conversion to sinus rhythm in patients with paroxysmal atrial fibrillation]. The aim of our study was to evaluate spontaneous conversion rate to sinus rhythm in patients with paroxysmal atrial fibrillation (AF) not submitted to any treatment (pharmacological and/or electrical). From January 1985 to September 1990, 123 consecutive patients with paroxysmal AF were hospitalized in our department. In 11 patients arrhythmia was due to arrhythmogenic conditions; 34 patients were submitted to emergency treatment with drugs (23 cases) or electrical cardioversion (11 cases); 78 patients (41 males; mean age 65.1 years; 37 females: mean age 68.6 years), without emergency problems were enrolled in our study and were submitted to a four-day observation period without any therapy, except in case of worsening. 35 patients were free from heart disease; in the other 43, 28 had chronic coronary disease, 11 hypertensive cardiovascular disease, 2 rheumatic valvular disease, 1 hypertrophic cardiomyopathy and 1 chronic cor pulmonale. In all 78 patients sinus rhythm was restored spontaneously - in about 90% of them within 24 hours. Mean time to conversion was 21 hours (range 1-96 hours). Cardioversion occurred in similar percentage and at the same time in both subgroups of patients (with and without heart disease). Therefore, given the risks and cost of every treatment, a 24-hour observation period without therapy could be useful in those patients presenting with paroxysmal atrial fibrillation without emergency problems.


[Comparison of results of echo-dobutamine and ECG-dobutamine tests in the diagnosis of coronary disease]. The aim of the study was to compare the results of 2D-Echocardiographic (ECHO) vs 12-lead Electrocardiographic (ECG) monitoring during Dobutamine (DOB) infusion performed as a stress test in patients referred for the evaluation of chest pain of suspected ischemic origin. Fourty-seven consecutive patients, 40 m and 7 f, mean age 52 +/- 9 years, were studied after interruption of any antianginal therapy. DOB was infused in 5-minute stages with incremental doses of 5 mcg/kg/min up to a maximal dose of 40 mcg/kg/min. 2D-ECHO monitoring could not be performed in 3 out of 47 patients because of a poor acoustic window. Thus, the overall feasibility was 94% for 2D-ECHO DOB test vs 100% for ECG DOB test (p = ns). The ECG and 2D-ECHO findings were compared in the remaining 44 patients. Criteria for positivity were: transient regional dyssynergy absent or of lesser degree in the baseline examination for 2D-ECHO; ST-segment shift > 0.1 mV from baseline for ECG. The test was stopped when a regional wall motion abnormality developed even in the absence of significant ECG changes. Angiographically assessed coronary artery disease (CAD) was considered significant when a > 50% reduction of the luminal diameter in at least 1 major coronary vessel occurred. There were 10/44 patients with no significant CAD and 34/44 patients with CAD; 16 had single, 18 double or triple and/or left main vessel disease.(ABSTRACT TRUNCATED AT 250 WORDS)


[Endomyocardial biopsy in childhood].

[Endomyocardial biopsy in childhood]. The experience with endomyocardial biopsy in pediatric age is still limited. From February 1986 to August 1990, 144 right ventricle endomyocardial biopsies were performed in 84 patients (age range 33 days--14 years, median age 31 months, weight range 3--57 kgs). Clinical diagnosis was: dilated cardiomyopathy in 50 patients; graft reject in 19; hypertrophic cardiomyopathy in 4; restrictive cardiomyopathy in 5; heart tumor in 3; ventricular arrhythmia in 3. The bioptome was introduced directly, without the use of a long sheath. There were no major complications; 2 patients experienced complete transient atrioventricular block and in 1 case right ventricular perforation occurred. In 11/45 patients (27%) with the clinical diagnosis of dilated cardiomyopathy and available myocardial specimens, acute myocarditis was diagnosed. In 47/65 procedures in the transplanted patients, a moderate to severe rejection was diagnosed. In the remaining patients, endomyocardial biopsy did not help the clinical diagnosis. We conclude that the right ventricular endomyocardial biopsy is a safe procedure in pediatric age; its utility is mostly limited to the diagnosis of acute myocarditis and graft reject after cardiac transplantation.


[Evaluation of the effects of molsidomine and propranolol-molsidomine combination on portal hemodynamics by pulsed Doppler ultrasound]. Molsidomine, a long acting vasodilator with antianginal properties, has been shown to decrease porto-hepatic pressure gradient in patients with cirrhosis. The present study aimed at assessing the effects of molsidomine, propranolol and of the association of these two drugs on portal vein blood flow as measured using Doppler and B-mode sonography. In 10 patients without liver disease (group 1), portal flow time average mean velocity (TAV) and portal vein blood flow (PVBF) were measured under basal conditions, 1 hour then 2 hours after ingestion of 4 mg of molsidomine. The same measurements were performed in 15 patients with cirrhosis (group 2) under basal conditions, 1 then 2 hours after double-blind administration of either molsidomine (10 patients) or placebo (5 patients). Fifteen further patients with cirrhosis (group 3) were studied after the double blind administration of 80 mg of propranolol and two hours later of 4 mg of molsidomine (10 patients) or placebo (5 patients); TAV and PVBF were measured under basal conditions, two hours after propranolol ingestion or placebo, then one and two hours after molsidomine or placebo ingestion. TAV and PVBF remained unchanged in patients treated with placebo. Molsidomine reduced TAV by 23.8 +/- 19.5% in group 1 (P < 0.01) and by 25.6 +/- 21.4% in group 2 (P < 0.01). In group 3, a 10% decrease was observed after propranolol (NS). When molsidomine was added, TAV was further decreased (-17.6 +/- 13.3% vs baseline, P < 0.01). PVBF remained unchanged in the three groups of patients.(ABSTRACT TRUNCATED AT 250 WORDS)


[Treatment of ascites in patients with liver cirrhosis without neither hyponatremia nor renal insufficiency. Results of a randomized study comparing diuretics and punctures compensated by albumin]. Previous studies have suggested that treatment of ascites in cirrhotic patients by repeated paracenteses and albumin infusion is fast, effective and safe. In one of these studies including patients with hyponatremia or renal impairment, this treatment was associated with a reduction of duration of hospital stay in comparison with large dose diuretics. The aim of this randomized study was to compare paracentesis with albumin perfusion and low dose diuretics in cirrhotic patients with ascites, but without hyponatremia or renal impairment. Twenty-six patients (group 1) were treated with paracentesis (4 L/day) and 27 patients (group 2) were treated with spironolactone (225 to 300 mg/day), associated with furosemide (40 to 80 mg/day), when inefficient alone. Ascites and peripheric edema disappeared more rapidly in group 1 than in group 2, 8.6 +/- 9.6 vs 13.5 +/- 6.7 days (P = 0.001) and 4.1 +/- 2.6 vs 10.5 +/- 6.5 days (P = 0.001) respectively. During hospitalisation, the incidence of complications was higher in group 2 than in group 1: 56 vs 26% (P = 0.03). Hyponatremia occurred in 30% of patients in group 2 and 4% of patients in group 1 (P = 0.04). The duration of hospital stay was shorter in group 1 (15.0 +/- 10.4 days) than in group 2 (21.0 +/- 11.7 days) (P = 0.007). During follow-up, ascites reappeared in 32% of patients in group 1 and 57% of patients in group 2 (P = 0.09). At 3 months, one patient in group 1 and 2 patients in group 2 developed spontaneous peritonitis whereas survival was similar in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)


[Incidence of duodenal ulcer in the city of Reims 1988]. No epidemiological data on the incidence of duodenal ulcer are available for a well-defined population in France. We therefore conducted a prospective study in the urban population of Reims. All radiologists, gastroenterologists and surgeons of this town were asked to report all new cases of duodenal ulcer seen during one year. During 1988, 114 new cases were identified. Age standardized incidence using world population standards were 83.6/100,000 for males and 29.5/100,000 for females. The male/female ratio was 2.8. Mean age at the time of diagnosis was 51.9 years, higher for females (63.4 years) than for males (48.0 years). Nearly one third (32.5%) of duodenal ulcers presented with complications (haemorrhage or perforation) at the time of initial diagnosis.


[Metabolism of plasma cholesterol and lipoproteins after total resection of the small intestine in patients with parenteral nutrition. Effects of the amount of phospholipids infused]. The aim of this study was to investigate the plasma lipoprotein profile in 2 patients treated by parenteral nutrition for total small bowel resection over a 15 month period. According to the amount of infused phospholipids (6 g/d vs 3 g/d), infused during 4 non consecutive 6 month or 6 week periods, HDL-cholesterol, apolipoproteins AI and B plasma levels were 30 to 50% below normal values. During the higher phospholipid supply, cholesterolemia seemed normal; each phospholipid supply decrease was followed by a reduction of cholesterol, phospholipids and apolipoprotein B plasma levels of 40, 50 and 25%, respectively, while HDL-cholesterol and apolipoprotein AI plasma levels remained unchanged. Density gradient ultracentrifugation showed that plasma cholesterol changes were mainly due to cholesterol changes (as free cholesterol associated with phospholipids) located in the density range of 1.019-1.040, reflecting the presence of lipoprotein X-like particles, whose levels remained unchanged during each period. An apolipoprotein E, CII and CIII enrichment of plasma was observed and was more pronounced when patients received higher phospholipid infusion. These results show that, in patients without a small bowel, minor changes in phospholipids supply are responsible for serious alterations of the lipoprotein profile; formation of lipoprotein X-like particles could be favored by the low HDL levels in these patients.


[Bacterial translocation in Crohn disease]. Bacterial translocation is the passage of viable endogenous bacteria from the gastrointestinal tract to mesenteric lymph nodes and other internal organs. The aim of this work was to study bacterial translocation in patients operated on for Crohn's disease. Twenty-eight patients, mean age 29 years, not having received any antibiotics since at least 8 days, presenting with ileal (n = 12), ileo-colonic (n = 14) or colonic (n = 2) Crohn's disease were studied. In 25 out of 28 cases (89%) indication for surgery was strictures inducing an upper small bowel distension in 9 out of 25 patients. Mesenteric lymph nodes and liver biopsies, portal blood samples and peritoneum swabs were harvested after laparotomy and before gut opening. Bacterial translocation, defined as the presence of intestinal bacteria in at least one of the specimens, was present in 8 out of 28 patients. This was found in lymph nodes draining surgical territories in 7 out of 8 cases. Bacterial strains involved in translocation included E. coli (n = 5), Enterococcus (n = 3), Clostridium perfringens (n = 2), Proteus (n = 2), and Bacteroides fragilis (n = 1). The rate of translocation differed neither according to Crohn's disease site nor with perforating or non perforating type of the disease. Five out of 9 patients operated on for strictures with proximal distension had a translocation. In conclusion, bacterial translocation was identified in 29% of patients operated on for Crohn's disease in this series. Distension of the intestine proximal to a digestive stricture could favor the occurrence of bacterial translocation in Crohn's disease.


[Efficacy of a daily application of mesalazine (Pentasa) suppository with progressive release, in the treatment of ulcerative proctitis. A double-blind versus placebo randomized trial]. Therapeutic efficacy of mesalazine controlled-release suppository 1 g once daily was compared with that of a placebo during 2 weeks in 50 patients (26 in the mesalazine group, 24 in the placebo group) with ulcerative proctitis, in a double-blind randomized trial. Endoscopic and clinical remission was seen in 69 and 65% of mesalazine-treated patients and in 33 and 25% of placebo-treated patients respectively (P < or = 0.01). No side effects were seen. It is concluded that a once-a-day administration of 1 g mesalazine controlled-release suppository is effective for topical treatment of patients with ulcerative proctitis.


[Role of rectal endoscopic ultrasonography in the pre-therapeutical study of villous tumors]. The staging of rectosigmoid villous adenomas is difficult when based only on clinical or endoscopic findings, even when superficial biopsies are taken. Echoendoscopy (EE) with a 7.5 and 12 MHz transducer heads provides good visualization of these lesions. The role of EE in the staging of villous adenomas presenting as benign lesions was prospectively studied. Among 47 patients who had an echoendoscopic investigation for villous adenoma during a 18 month period, 25 had lesions believed to be benign based on clinical and endoscopic findings. In 23 patients (group I, superficial biopsies showed no malignancy; in 2 patients (group II), with 3 lesions, biopsies were positive for in situ carcinoma. Comparisons were made between echographic images and operative specimens. A complete EE investigation was possible in all cases. In group I, there was one tumor T3N+ that was diagnosed by EE only. Twelve other patients had neoplastic lesions limited to the mucosa or submucosa only, and 10 had benign lesions. The integrity of the muscularis propria was imaged in all cases but EE did not differentiate benign lesions from neoplastic lesions invading the mucosa. In group II, parietal staging was correct in 2 of 3 cases. In addition to clinical examination and endoscopic investigations, EE seems useful in the staging of large or high located villous adenomas as well as for the best therapeutic choice.


[Liver damage induced by the ingestion of a product of phytotherapy containing wild germander. Four cases]. We report 4 cases of patients who developed hepatic injury during administration of herbal medicines for loosing weight with Wild Germander. Three developed jaundice and one had fatigue. Aminotransaminase activities were increased in all patients. Outcome was favorable after drug withdrawal in all patients. Liver biopsy showed centrolobular necrosis and portal lymphoplasmocytic infiltration. Another drug than Wild Germander might have been implicated in hepatic injury in two patients. The similarity of these cases suggests, however, that Wild Germander, like other herbal medicines, may be responsible for hepatic injury.