Hepatocytes cultured in alginate microspheres: an optimized technique to study enzyme induction.

An important application of hepatocyte cultures is identification of drugs acting as inducers of biotransformation enzymes that alter metabolic clearance of other therapeutic agents. In the present study we optimized an in vitro system with hepatocytes cultured in alginate microspheres that allow studies of enzyme induction with excellent sensitivity. Induction factors obtained with standard inducers, such as 3-methylcholanthrene or phenobarbital, were higher compared to those with conventional hepatocyte co-cultures on collagen coated dishes. This is illustrated by activities of 7-ethoxyresorufin-O-deethylase (EROD) after incubation with 5 microM 3-methylcholanthrene (3-MC), a standard inducer for cytochrome P4501A1 and 1A2. Mean activities for solvent controls and 3-MC exposed cells were 2.99 and 449 pmol/min/mg protein (induction factor: 150) for hepatocytes cultured in microspheres compared to 2.72 and 80.6 pmol/min/mg (induction factor: 29.6) for hepatocytes on collagen coated dishes. To compare these in vitro data to the in vivo situation male Sprague Dawley rats, the same strain that was used also for the in vitro studies, were exposed to 3-MC in vivo using a protocol that guarantees maximal induction. Activities were 29.2 and 1656 pmol/min/mg in liver homogenate of solvent and 3-MC treated animals (induction factor: 56.7). Thus, the absolute activities of 3-MC exposed hepatocytes in microspheres are lower compared to the in vivo situation. However, the induction factor in vitro was even higher compared to the in vivo situation (150-fold versus 56.7-fold). A similar scenario was observed using phenobarbital (0.75 mM) for induction of CYP2B and 3A isoenzymes: induction factors for testosterone hydroxylation in position 16beta were 127.5- and 50.4-fold for hepatocytes in microspheres and conventionally cultured hepatocytes, respectively. The new in vitro system with hepatocytes embedded in solid alginate microspheres offers several technical advantages: (i) the solid alginate microspheres can be liquefied within 60s, allowing a fast and complete harvest of hepatocytes; (ii) alginate capsules are stable allowing transport and mechanical stress; (iii) high numbers of hepatocytes can be encapsulated in short periods; (iv) defined cell numbers between 600 hepatocytes, the approximate number of cells in one capsule, and 18 x 10(6) hepatocytes, the number of hepatocytes in 6 ml alginate, can be transferred to a culture dish or flask. Thus, encapsulated hepatocytes allow a flexible organization of experiments with respect to cell number. In conclusion, we optimized a technique for encapsulation of hepatocytes in alginate microspheres that allows identification of enzyme induction with an improved sensitivity compared to existing systems.

4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models.

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.

Information processing in healthcare at the start of the third Millennium: potential and limitations.

The 21st century is said to be a century of the information society. We should be aware that continuing progress in information processing methodology (IPM) and information and communication technology (ICT) is changing our societies, including medicine and health care. At the start of the third Millennium we should ask ourselves, what progress can we expect from modern IPM/ICT for healthcare in the coming decade, what concerns does the information society have to face, and what steps have to be taken. These questions were addressed by clinicians, researchers and industrial representatives in a panel discussion at the joint conference ISCB-GMDS-99 of the International Society of Clinical Biostatistics and the German Society for Medical Informatics, Biometry and Epidemiology. Important aspects raised by the panelists and in the subsequent discussion were: (1) the main goal of expanding IPM/ICT should be to further improve quality of care, while maintaining reasonable costs; (2) with the support of modern IPM and ICT the boundaries between inpatient and outpatient care will fade away enabling a more efficient, patient-centered health care; (3) cooperation between health-care professionals will increase; there will be different ways of communication between them and with the patient, including modern ICT and the Internet; (4) society must be concerned with achieving equal opportunities in being informed about and in using new ICT; (5) misuse of data will remain a serious problem and can become an obstacle to progress.

[Between symbol and symptom: pain and its meanings in classical antiquity]. / Zwischen Symbol und Symptom: Der Schmerz und seine Bedeutung in der Antike Medizinhistorischer Beitrag zur Eröffnung des 1. Deutschen Schmerzkongresses 1995 in der Alten Aula der Universität Heidelberg am 4. Oktober 1995.

According to semiotics, which may be defined as the doctrine of the essential nature and fundamental varieties of signs, objects, and interpretants, pain is considered to be a sign (significant) with very different meanings (significance) either as a naturalistic symptom (of disease) or as a symbol used in a metaphorical context. When following this methodological perspective it is possible to interpret medical as well as poetic writings on equal terms. In Graeco-Roman medical texts pain was mostly understood as a result and an indicator of disease, but nonetheless as a symptom which seemed to be actively produced by the affected body. Especially in the Corpus Hippocraticum dating from the 5th and 4th century B. C. this materialistic and at the same time psychosomatic attitude can be noticed. Aristotle (4th century B. C.), Celsus (1st century A. D.), and the famous experimental physiologist Galen (2nd century A. D.) agreed that pain was a sign of evil which should be fought without exception. It was Galen who added the disturbance of function (functio laesa) as the fifth cardinal sign of inflammation to the four well-known cardinal signs of Celsus (rubor, calor, tumor, dolor). He also coined the term [see text] to characterize an attack of migraine. In algotherapy, Galen used a complex pharmacological system which was based upon the four cardinal qualities of humoral pathology. On the other hand, pain was designed as a multi-dimensional symbol by the famous Graeco-Roman epic poets. In Homer's Odyssey (8th century B. C.), pain appears transformed into the shape of a scar which is visible and palpable on the hero's leg like an identification tag, whereas in Virgil's Aeneids (1st century B. C.) pain symbolizes weakness and defencelessness which can only be alleviated by the goddess Venus.

Intrapelvic calculi demonstrated in a patient after laparoscopic laser cholecystectomy: a case report.

Laparoscopic laser cholecystectomy is becoming increasingly popular in the surgical community for the treatment of gallbladder disease. Physicians will need to familiarize themselves with the imaging consequences of this new therapy. Described below is a case report of a woman in whom calculi were incidentally found within the pelvis on a plain radiograph of the abdomen after she presented to the hospital with pancreatitis. Initial confusion regarding the etiology of these calculi was solved after it was discovered that the patient had proven gallstones and a recent laparoscopic procedure. In the appropriate clinical setting, gallstones should be added to the differential consideration of intrapelvic calcifications.