Isolation, culture, and transplantation of human hepatocytes.

The in situ two-step collagenase perfusion technique used for the isolation of hepatocytes from rat liver was adapted into a procedure applicable to pieces of human liver obtainable from surgical procedures. Human hepatocytes obtained by this method were maintained in primary culture for 10 days. The cellular changes observed at the light microscopic and electron microscopic levels are described. The changes in microsomal enzymes as a function of the age of the cultures were also measured. Exposure of the human hepatocytes to procarcinogens known to be metabolized by rodent liver resulted in unscheduled DNA synthesis. The isolated hepatocytes were also transplanted into two-thirds partially hepatectomized athymic nude mice. The transplanted cells formed nodules with characteristic hepatic architecture. These studies demonstrate that hepatocytes obtained from human liver by the described modified collagenase technique can be used for in vitro studies in chemical carcinogenesis.

Inhibition of DNA synthesis by chemical carcinogens in cultures of initiated and normal proliferating rat hepatocytes.

Rat hepatocytes in primary culture can be stimulated to replicate under the influence of rat serum and sparse plating conditions. Higher replication rates are induced by serum from two-thirds partially hepatectomized rats (Michalopoulos, G., Cianciulli, H. D., Novotny, A. R., Kligerman, A. D., Strom, S. C., and Jirtle, R. L. Cancer Res., 42: 4673-4682, 1982). The effects of carcinogens and noncarcinogens on the ability of hepatocytes to synthesize DNA were examined by measuring the incorporation of [3H]thymidine by liquid scintillation counting and autoradiography. Hepatocyte DNA synthesis was not decreased by ethanol or dimethyl sulfoxide at concentrations less than 0.5%. No effect was observed when 0.1 mM ketamine, Nembutal, hypoxanthine, sucrose, ascorbic acid, or benzo(e)pyrene was added to cultures of replicating hepatocytes. Estrogen, testosterone, tryptophan, and vitamin E inhibited DNA synthesis by approximately 50% at 0.1 mM, a concentration at which toxicity was noticeable. Several carcinogens requiring metabolic activation as well as the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine interfered with DNA synthesis. Aflatoxin B1 inhibited DNA synthesis by 50% (ID50) at concentrations between 1 X 10(-8) and 1 X 10(-7) M. The ID50 for 2-acetylaminofluorene was between 1 X 10(-7) and 1 X 10(-6) M. Benzo(a)pyrene and 3'-methyl-4-dimethylaminoazobenzene inhibited DNA synthesis 50% between 1 X 10(-5) and 1 X 10(-4) M. Diethylnitrosamine and dimethylnitrosamine (ID50 between 1 X 10(-4) and 5 X 10(-4) M) and 1- and 2-naphthylamine (ID50 between 1 X 10(-5) and 5 X 10(-4) M) caused inhibition of DNA synthesis at concentrations which overlapped with concentrations that caused measurable toxicity. The ability of hepatocytes to activate 2-acetylaminofluorene to reactive intermediates capable of binding to DNA and inhibiting new DNA synthesis decreased as a function of time in culture. gamma-Glutamyl-transferase-positive hepatocytes from diethylnitrosamine-treated rats were observed to be relatively insensitive to carcinogen inhibition of DNA synthesis.

Promoting effect of nicotinamide on the development of renal tubular cell tumors in rats initiated with diethylnitrosamine.

Nicotinamide administered in the drinking water of male Fischer 344 rats increased the number of renal tubular cell tumors of rats treated with an i.p. injection of diethylnitrosamine (DEN) (25-mg/kg body weight). The incidence of kidneys with tumors in rats treated with DEN alone was 5%. In rats which received DEN and then were promoted with either 30 or 6.7 mM nicotinamide in their drinking water, the incidence of kidneys with tumors rose to 59 and 28%, respectively. Rats which were on 30 mM nicotinamide but did not receive DEN had no kidney tumors present. These results show that nicotinamide promoted DEN-induced renal tubular cell tumorigenesis.

Neuroleptic malignant syndrome. Review of response to therapy.

The purpose of this study was to evaluate options available for the treatment of neuroleptic malignant syndrome. We conducted a computer search of English-language literature using a Medline database and review of bibliographies of identified articles. Sixty-four case reports containing sufficient data about patients' demographics and clinical response to therapy to allow evaluation were combined with three previously unreported cases. The mean time to clinical response was 6.8 days when supportive measures alone were used in the treatment of neuroleptic malignant syndrome. The addition of dantrolene (response time, 1.15 days) or bromocriptine (response time, 1.03 days) significantly shortened the time to clinical response. We conclude that the addition of either dantrolene or bromocriptine to conservative measures decreases the time to clinical response. A randomized trial would address potential case selection biases.

Physicians as patients. Choices regarding their own resuscitation.

BACKGROUND: Attitudes toward cardiopulmonary resuscitation have changed considerably during the last 30 years. Although physicians are routinely involved in the decision making about cardiopulmonary resuscitation for their patients, little is known about their collective preferences regarding it for themselves. METHODS: A questionnaire was distributed at an internal medicine primary care review course at an urban community hospital. Of the 111 physicians registered at the meeting, 72 (65%) completed the questionnaire and serve as the basis for the results. Physicians were asked if they would want cardiopulmonary resuscitation for themselves in the presence of an acute myocardial infarction, Alzheimer's disease, and nine other advanced chronic diseases at the projected ages of 40, 60, and 80 years. RESULTS: At all projected ages, physicians' desire for cardiopulmonary resuscitation with any advanced chronic disease was significantly less than with an acute myocardial infarction (P < or = .000001 except for rheumatoid arthritis). Fewer physicians wanted cardiopulmonary resuscitation at age 80 years than at 40 years for any disease (P < or = .002). The results did not differ when analyzed by respondents' age, gender, or primary care specialty, or the size of the community in which they practiced. CONCLUSIONS: The results of this initial survey indicate that most physicians would not want cardiopulmonary resuscitation with a variety of underlying chronic diseases and corresponding functional impairments--particularly with advancing age. Conversely, with an acute myocardial infarction, all physicians surveyed would desire cardiopulmonary resuscitation at age 40 years, and many would continue to desire it with advancing age.

Internal medicine training in ambulatory gynecology. A house of staff survey.

OBJECTIVES: Practicing internists commonly provide gynecologic care, yet internists receive little formal training in ambulatory gynecology. To evaluate current residency training, we surveyed house staff perceptions of their training in gynecology. METHODS: Data were collected from house staff at five Portland, Ore, internal medicine training programs by questionnaire using a Likert scale about training in 24 gynecologic and five nongynecologic internal medicine problems. Responses were compared by level of training, institution, and gender. RESULTS: Responses were received from 195 (89%) of 221 residents. House staff reported less than adequate training in gynecology, particularly in the areas of family planning and medical complications of pregnancy, while reporting much superior training in the traditional medicine problems. CONCLUSIONS: House staff in internal medicine report little training in the diagnosis and treatment of gynecologic problems. Training in family planning may be particularly deficient. Residency programs must seek effective teaching strategies to address this deficiency.

Teaching behavioral medicine in a primary care setting. An integrated approach.

The identification and management of psychosocial complaints is increasingly being recognized as an important function of primary care physicians. Residency programs are slowly incorporating training in behavioral medicine into their curricula. A wide variety of methods for teaching behavioral medicine have been used. This article describes a model in which behavioral medicine education is fully integrated into a primary care setting.

Kidney proximal tubular cells isolated by collagenase perfusion grow in defined media in the absence of growth factors.

Cells from kidney proximal tubules have been successfully isolated, characterized, and cultured from male Fischer 344 rats between 150-400 g using a two-step collagenase perfusion. The cells undergo high levels of DNA synthesis and mitosis in both serum free media (with an without hormone supplementation) and media containing 10% fetal bovine serum. Confluent monolayers were observed between 5 to 7 days after seeding 2 X 10(5) cell/35 mm collagen-coated plate. Approximately 50% of the total kidney and 70% of the cortex was isolated using this technique. The viability of the isolated tubules was 75 +/- 8% and the estimated number of viable cells was 12 +/- 3 X 10(6) cells. At the time of isolation greater than 90% of the isolated tubules and cells were positive for gamma glutamyltransferase (GGT), periodic acid-schiff (PAS), and glucose-6-phosphatase (G-6-Pase). Both GGT and G-6-Pase decreased rapidly during the first 3 days in primary culture as assessed by histochemistry. Ultrastructurally the isolates consisted of cells with numerous microvilli and mitochondria. The size and number of microvilli decrease rapidly in primary culture. The morphologic and biochemical evidence suggests that the primary isolates and cultures are proximal tubular in origin.

Effect of hydrocortisone and nicotinamide on gamma glutamyltransferase in primary cultures of rat hepatocytes.

Isolated rat hepatocytes cultured on collagen coated plates exhibit a gradual fetal phenotypic change during time in culture. The fetal liver marker gamma glutamyltransferase (GGT) was used to follow this change. Inasmuch as a significant overgrowth of nonparenchymal liver derived cells is seen frequently in primary cultures of hepatocytes, a technique was utilized that corrects for the presence of nonparenchymal cells. In media supplemented with either hydrocortisone (10(-5) M) or nicotinamide (25 mM) the original epithelial morphology of hepatocytes was preserved for a longer period of time than in unsupplemented media. Hepatocytes in unsupplemented media exhibited an increase in GGT specific activity over time. Hydrocortisone (10(-5) M) induced an increase in GGT activity compared to controls. Nicotinamide (25 mM) inhibited the increase in GGT activity compared to the unsupplemented hepatocytes. Our results indicate that GGT is regulated by hydrocortisone and nicotinamide.

Induction of rat kidney ornithine decarboxylase by nicotinamide without a concomitant increase in DNA synthesis.

Four hours after an i.p. injection of 500 mg/kg nicotinamide, there was a decrease in kidney ODC activity, followed by a substantial increase by 24 h. Exposing rats to 0, 0.67, 6.7 and 30 mM nicotinamide in their drinking water for 7 and 28 days also resulted in a statistically significant increase in kidney ODC activity, but the rates of DNA synthesis were unaffected, as measured by incorporation of [3H]thymidine into DNA and % labeled proximal tubule nuclei; the total amount of DNA/kidney was also unaffected. The results of this investigation demonstrate that nicotinamide, a known renal tumor promoter, was able to induce a significant increase in ODC activity in the rat kidney without a concomitant increase in DNA synthesis, suggesting that early stimulation of ODC is associated with tumor promotion even in the absence of effects on DNA replication.

Binding of alpha 2-macroglobulin to hepatocytes: mechanism of in vivo clearance.

The binding of 125I-labeled human alpha 2-macroglobulin-methylamine to adult rat hepatocytes in primary culture was studied at 4 degrees C. Cells which had been in culture for 4 hours exhibited steady state ligand binding after 1 hour, a receptor number of 22,400 receptors per cell, and a dissociation constant of 0.6 nM. Adult rat hepatocytes exhibited a significant decrease in receptor number with increased time in primary culture with less than 10% of the initial number of receptors remaining after 2 days (p less than 0.01). In autopsy studies of mice injected intravenously with 125I-labeled alpha 2-macroglobulin-methylamine, greater than 90% of the cleared ligand was found in the liver. Autoradiography of the liver demonstrated that 80% of the ligand was cleared by hepatocytes. It is concluded that the hepatocytes are the primary pathway for clearance from the circulation of receptor recognized alpha 2-macroglobulin.