Increased insulin-like growth factor-1 protein in human left ventricular hypertrophy.

Animal models of cardiac hypertrophy demonstrated increased expression of insulin-like-growth factor-1 (IGF-1) in the heart. To study protein expression of insulin-like-growth factor 1 in left ventricular hypertrophy (LVH) in humans 11 hearts of autopsy cases with LVH were compared to 11 controls using immunohistochemical staining with anti-human IGF-1. LVH was defined as thickening of the left ventricular wall which ranged from 1.6 to 2.5 cm with hearts weights from 400 to 900 g. Immunohistochemical staining for IGF-1 was increased in the presence of LVH. In cases of LVH 37.9 +/- 3.5% of the cross-sectional myocardial area stained positively for IGF-1 compared to 6.8 +/- 2.9% in controls (P < 0.001). The findings support the hypothesis that IGF-1 has a role in the pathogenesis of LVH in humans. The increase of IGF-1 protein with LVH suggests reactivation of the cardiac IGF-1 genes in the hypertrophied adult cardiomyocyte.

Six month follow-up: the crucial test of multidisciplinary developmental assessment.

Participation of parents in the developmental assessment process is thought to be beneficial in promoting understanding of their child's disability, and improving consensus between parents and professionals about appropriate intervention programmes. If costly multidisciplinary assessments are to be justified, it is necessary to establish long-term benefits for the child. This highlights a need for research identifying how families use services after diagnostic assessment and what they understand to be important for their child. Poor parent-professional agreement about diagnosis may be a factor contributing to low compliance with recommendations. The major purpose of the current study was to follow-up families 6 months after developmental assessment, in order to investigate use of recommended intervention services. In addition, mothers' opinions about diagnostic findings, recommendations and early intervention services were examined. Subjects were 40 pre-school children who presented for developmental assessment, and their mothers. The majority were diagnosed with developmental problems in multiple domains. Results indicated that most mothers recalled and agreed with their child's diagnosis, but underestimated the severity of developmental delay. Families had not accessed the range of multidisciplinary intervention programmes recommended, given the complexity of their children's disabilities. Speech therapy was considered the service of highest priority by mothers, and was the treatment most frequently received. Mothers recognized a need for more therapeutic interventions for their child. An unexpected finding was the high prevalence of families who organized nonprescribed therapies. Possible explanations of the findings and implications for service delivery are discussed.

Interferon-inducible T cell alpha chemoattractant (I-TAC): a novel non-ELR CXC chemokine with potent activity on activated T cells through selective high affinity binding to CXCR3.

Chemokines are essential mediators of normal leukocyte trafficking as well as of leukocyte recruitment during inflammation. We describe here a novel non-ELR CXC chemokine identified through sequence analysis of cDNAs derived from cytokine-activated primary human astrocytes. This novel chemokine, referred to as I-TAC (interferon-inducible T cell alpha chemoattractant), is regulated by interferon (IFN) and has potent chemoattractant activity for interleukin (IL)-2-activated T cells, but not for freshly isolated unstimulated T cells, neutrophils, or monocytes. I-TAC interacts selectively with CXCR3, which is the receptor for two other IFN-inducible chemokines, the IFN-gamma-inducible 10-kD protein (IP-10) and IFN-gamma- induced human monokine (HuMig), but with a significantly higher affinity. In addition, higher potency and efficacy of I-TAC over IP-10 and HuMig is demonstrated by transient mobilization of intracellular calcium as well as chemotactic migration in both activated T cells and transfected cell lines expressing CXCR3. Stimulation of astrocytes with IFN-gamma and IL-1 together results in an approximately 400,000-fold increase in I-TAC mRNA expression, whereas stimulating monocytes with either of the cytokines alone or in combination results in only a 100-fold increase in the level of I-TAC transcript. Moderate expression is also observed in pancreas, lung, thymus, and spleen. The high level of expression in IFN- and IL-1-stimulated astrocytes suggests that I-TAC could be a major chemoattractant for effector T cells involved in the pathophysiology of neuroinflammatory disorders, although I-TAC may also play a role in the migration of activated T cells during IFN-dominated immune responses.

IP6 in treatment of liver cancer. I. IP6 inhibits growth and reverses transformed phenotype in HepG2 human liver cancer cell line.

Hepatocellular carcinoma (HCC) is a common tumor world-wide with extremely poor prognosis. Recent studies have shown that inositol hexaphosphate (IP6), a naturally occurring carbohydrate, has novel anti-cancer function in various in vitro and in vivo models. The aim of this study was to assess whether IP6 could inhibit the growth of human hepatocellular carcinoma. We treated HepG2, a human liver cancer cell line in vitro with IP6 and evaluated its effect on growth and differentiation. IP6 treatment of HepG2 cells caused a dose-dependent growth inhibition. Compared to other cancer cell lines, HepG2 cells were quite sensitive to IP6, IC50 (50% inhibition of cell growth) of IP6 being < 1.0 mM (0.338 mM). Treatment with IP6 decreased the ability of HepG2 cells to form colonies, as assessed in the plating efficiency assay. Morphological changes induced by IP6 were consistent with differentiation of HepG2 cells. Exposure of HepG2 cells to IP6 drastically decreased the rate of production of alpha-fetoprotein (AFP), a tumor marker of HCC, indicating also that IP6 treatment leads to differentiation of malignant liver cells. Further, IP6 treatment caused a decreased expression of mutant p53 protein in HepG2 cells, with no significant change in the expression of wild-type p53. The expression of p21WAF1 protein was increased by 1.5 fold, as determined by immunocytochemical staining and ELISA assay. These data demonstrate that IP6 inhibits the growth, and induces differentiation, and a less aggressive phenotype of HepG2 cells, suggesting a role of IP6 in the treatment of HCC.

IP6: a novel anti-cancer agent.

Inositol hexaphosphate (InsP6 or IP6) is ubiquitous. At 10 microM to 1 mM concentrations, IP6 and its lower phosphorylated forms (IP(1-5)) as well as inositol (Ins) are contained in most mammalian cells, wherein they are important in regulating vital cellular functions such as signal transduction, cell proliferation and differentiation. A striking anti-cancer action of IP6 has been demonstrated both in vivo and in vitro, which is based on the hypotheses that exogenously administered IP6 may be internalized, dephosphorylated to IP(1-5), and inhibit cell growth. There is additional evidence that Ins alone may further enhance the anti-cancer effect of IP6. Besides decreasing cellular proliferation, IP6 also causes differentiation of malignant cells often resulting in a reversion to normal phenotype. These data strongly point towards the involvement of signal transduction pathways, cell cycle regulatory genes, differentiation genes, oncogenes and perhaps, tumor suppressor genes in bringing about the observed anti-neoplastic action of IP6.

National survey of state trauma registries--1992.

Registries, such as those for oncology, have demonstrated usefulness in collating information. Trauma care can be improved through the accumulation of local, regional, and state trauma statistics. The efforts to develop a National Trauma Registry in the United States are still in their infancy. A four-page survey questionnaire was returned by each of the 50 State Emergency Medical Services (EMS) Directors, as well as the EMS Directors of the District of Columbia and five American possessions, to evaluate the status of state trauma registries in the United States. In 1992, 24 (48%) states had a registry. Development costs average $101,107 and annual maintenance costs averaged $72,105. An average of 1.7 full-time equivalents (FTE) was necessary to maintain the registry. Fourteen (58%) states have effected legislation through the registry. Trauma prevention has been promoted in nine (38%) states and a decrease in mortality recognized through the registry in five (21%) states. Trauma registries are labor intensive and expensive but are effective in decreasing morbidity and mortality. The need for a National Trauma Registry incorporating and comparing data from health care facilities around the United States and its possessions has the potential of improving trauma health care.

Simian virus 40 large tumor antigen-immortalized normal human liver epithelial cells express hepatocyte characteristics and metabolize chemical carcinogens.

Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a limited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was extended by introduction of the simian virus 40 large T antigen gene. Two cell lines, THLE-2 and -3, established with a recombinant simian virus 40 large T antigen virus have undergone > 100 population doublings, are nontumorigenic when injected into athymic nude mice, have near-diploid karyotypes, and do not express alpha-fetoprotein. The cells express cytokeratin 18 and albumin in early passage, whereas higher-passage cells in logarithmic-phase growth also express cytokeratin 19. THLE-2 and -3 cells metabolize benzo[a]pyrene, N-nitrosodimethylamine, and aflatoxin B1 to their ultimate carcinogenic metabolites that adduct DNA, which indicates functional cytochrome P450 pathways. Other enzymes involved in metabolism of chemical carcinogens, such as epoxide hydrolase, NADPH cytochrome P450 reductase, superoxide dismutase, catalase, glutathione S-transferases, and glutathione peroxidase are also retained by THLE cells. Thus, these immortalized human liver cells constitute an in vitro model for pharmacotoxicological studies and for the investigation of etiology and pathogenesis of human hepatocellular carcinoma.

Clinical development of Taxol.

Taxol is the first of a novel class of anticancer drugs, the taxanes. Taxol's unique effects include its ability to polymerize tubulin into stable microtubules in the absence of cofactors and to induce the formation of stable microtubule bundles. During its development, formidable challenges were overcome: a suitable formulation was developed, an adequate supply was ensured, severe hypersensitivity reactions were diminished in incidence and severity, and clinical efficacy was demonstrated. Phase II evaluation is still underway; to date, clinical efficacy has been demonstrated in ovarian, breast, non-small-cell lung, and head and neck cancer. Response rates were low in early studies in melanoma, prostate, colon, cervix, and renal cancer, but for these tumors, additional evaluation is ongoing with a higher Taxol dose or different schedule. In December 1992, Food and Drug Administration approval was granted for use of Taxol as second-line therapy in ovarian cancer patients. Nevertheless, important questions regarding optimal use of this important new drug remain. These include determination of optimal dose and schedule and development of suitable combination chemotherapy regimens. The clinical development of Taxol and current status of phase I, II, and III clinical trials are reviewed.

16,16-dimethyl prostaglandin E2 modulates aflatoxin B1-induced injury of rat hepatocytes in primary culture: possible role of cAMP.

The hepatocellular cytoprotective effects of 16,16-dimethyl prostaglandin E2 (dmPGE2), an analogue of PGE2, were investigated using primary cultures of rat hepatocytes and aflatoxin B1 as the hepatotoxin. Lactic dehydrogenase (LDH) release by hepatocytes was used as an index of hepatotoxicity. When aflatoxin-treated hepatocytes were co-cultured with 16,16-dmPGE2 (0.01-0.5 micrograms/mL) LDH release was significantly reduced and ultrastructural changes of hepatocellular injury were markedly diminished. The magnitude of the cytoprotective effect was not dependent on the concentration of the prostaglandin over the range tested. A significant cytoprotective effect was also induced when hepatocellular cyclic AMP (cAMP) levels were increased by the addition of dibutyl-cAMP. In contrast to 16,16-dmPGE2, PGF2 alpha Tromethamine, an analogue of PGF2 alpha, which does not stimulate cAMP, induced insignificant changes in cytoprotection. These findings indicate that only a low concentration of 16,16-dmPGE2 (> or = 0.01 micrograms/mL) is necessary to induce a maximal hepatocellular cytoprotective effect and suggest that this effect may be dependent on activation of cAMP.

Comparative effects of three carcinogens on human, rat and mouse hepatocytes.

Ultrastructural changes commonly observed in liver cells of rodents exposed to carcinogens in vivo can be induced in hepatocytes exposed to carcinogens in vitro. Human, rat and mouse hepatocytes in primary culture were treated with actinomycin D, aflatoxin B1 (AFB1) and dimethylnitrosamine (DMN). These cultured hepatocytes were examined for ultrastructural alterations following carcinogen exposure for 24 h. Similar to the effects on liver cells in vivo, the most prominent change was a segregation of the nucleolar components. Human, rat and mouse hepatocytes, dosed with 7.9 X 10(-8) M actinomycin D, developed nucleolar segregation in 86%, 98% and 55% of cells, respectively. When incubated with 3.2 X 10(-6) M AFB1, 60% of human and 84% of rat hepatocytes developed nucleolar segregation. However, exposures of mouse hepatocytes less than or equal to 3.2 X 10(-5) M of AFB1 failed to induce segregation of the nucleolus. DMN administered at a dose of 2.0 X 10(-2) M caused segregation in 11% of the rat hepatocytes and in 60% of the mouse hepatocytes. Distinct nucleolar segregation did not occur in human hepatocytes until they were exposed to a concentration of 5.0 X 10(-2) M DMN (31%). Actinomycin D, AFB1, DMN, as well as other compounds that bind to DNA and interfere with template activity cause nucleolar segregation. Morphologic changes observed in cultured rat and mouse hepatocytes correlate well with in vivo experiments with regard to the relative sensitivity of rats and mice to toxicological effects of these carcinogens. Thus, hepatocyte cultures may provide a realistic system to determine the sensitivity of human liver cells to carcinogens.

Replicative cultures of adult human and rhesus monkey liver epithelial cells.

Exploratory experiments indicate that media containing lipids, phosphoethanolamine, epidermal growth factor, insulin, cholera toxin, bovine pituitary extract, chemically denatured serum, and triiodothyronine will support replicative cultures of normal liver epithelial cells obtained from adult Rhesus monkey and human donors. In addition, we have extended the culture population doubling potential of the human liver epithelial cells by their transfection with a plasmid containing the SV40 virus T-antigen gene. The T-antigen gene-containing cells continued to express albumin through 40 population doublings. Finally, results of preliminary experiments suggest that it may be possible to induce human liver epithelial cells to undergo differentiation to hepatocyte-like cells either by injecting them into the spleen of an athymic nude mouse or by incorporating them into a collagen "tissue equivalent" matrix.

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity.

DNA binding levels were determined and compared in cultured hepatocytes from male and female rats as well as other animal species following exposure to aflatoxin B1 (AFB1) or 2-acetylaminofluorene (2-AAF). When human, rat (both male and female) and mouse hepatocytes in primary culture were exposed to 2.0 X 10(-7) M [3H]AFB1 (sp. act. 2.63 microCi/nmol) for 24 h, male rat hepatocytes had the highest degree of [3H]AFB1-DNA binding (203 pmol/mg DNA) and human hepatocytes contained the next highest binding level (42 pmol/mg DNA). Hepatocytes from female rats contained 38 pmol/mg DNA while cultured mouse hepatocytes contained only 1.4 pmol/mg DNA. When the same dose of [3H]AFB1 was administered to the cultured male rat hepatocytes at 24 h, 48 h, 72 h and 1 week after seeding, and incubated for 24 h, the DNA binding levels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallel experiments to the cultured male rat hepatocytes above, the AFB1-DNA binding levels in the cultured female hepatocytes were 42, 41, 37 and 34 pmol/mg DNA respectively. Human, male and female rat hepatocytes in primary culture were exposed to 5.2 X 10(-5) M 2-acetylamino[9-14C]fluorene (sp. act. 0.0094 microCi/nmol) for 24 h. It was determined that male rat hepatocytes contained the highest amount of radioactively labeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), female rat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytes contained 0.29 nmol/mg DNA. Results from our in vitro hepatocyte culture system correlate well with in vivo animal studies dealing with species and sex differences in DNA binding and carcinogenic susceptibility. This indicates that hepatocytes in vitro maintain many of the biological properties necessary for carcinogen response similar to liver cells in vivo. In addition, comparison of genotoxic effect in cultured hepatocytes from animals as well as humans may be useful in evaluating carcinogenic potential of xenobiotics in human liver.

Comparative ultrastructural effects of aflatoxin B1 on mouse, rat, and human hepatocytes in primary culture.

Human, rat, and mouse hepatocytes in primary culture were treated with aflatoxin B1 (AFB1) and examined for ultrastructural alterations. As early as 1 h following in vitro exposure to AFB1, there were ultrastructural changes in the nuclei of rat and human hepatocytes. The most prominent change in the nuclei was a segregation of nucleolar components that resembled the segregation in liver cells of rats exposed to AFB1 in vivo. The nucleolar segregations were developed by incubating rat hepatocytes for 24 h in a medium containing as little as 0.01 micrograms of AFB1 per ml. The minimum concentration to induce the same change in human hepatocytes was 0.1 micrograms/ml. No distinct nucleolar alteration was observed in mouse hepatocytes incubated in a medium containing 10 micrograms of AFB1 per ml. Irregular nuclear chromatin condensation also developed in the cells exposed to a higher concentration of AFB1, whereas little damage was observed in mitochondria and lysosomes. The similarity in morphological changes between our in vitro model and in vivo models previously investigated indicates that the hepatocytes in primary culture maintain the biological properties necessary for carcinogen responses similar to liver cells in vivo. In addition, the morphological changes in cultured rat and mouse hepatocytes induced by AFB1 correlate with in vivo experiments insofar as mice are relatively resistant, whereas rats are sensitive to AFB1 carcinogenesis. Thus, cultured hepatocyte systems may be a valuable tool to study genetic damage which may lead to hepatocellular carcinomas in human and animal livers.

Isolation and culture of hepatocytes from human liver of immediate autopsy.

Human livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function. Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intact lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 X 10(6) cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes, 632 pmol/mg protein of cytochrome p450 and 536 pmol/mg protein cytochrome b5 were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture.