Atomic radiations in the decay of medical radioisotopes: a physics perspective.

Auger electrons emitted in nuclear decay offer a unique tool to treat cancer cells at the scale of a DNA molecule. Over the last forty years many aspects of this promising research goal have been explored, however it is still not in the phase of serious clinical trials. In this paper, we review the physical processes of Auger emission in nuclear decay and present a new model being developed to evaluate the energy spectrum of Auger electrons, and hence overcome the limitations of existing computations.

Long-term disease-free survival after nonmyeloablative cyclophosphamide/fludarabine conditioning and related/unrelated allotransplantation for acute myeloid leukemia/myelodysplasia.

A total of 50 consecutive patients (median age, 57.5 years) with AML (n=30) or myelodysplasia (MDS, n=20) underwent HLA matched related donor (MRD, n=27) or unrelated donor (MUD, n=23) peripheral blood hematopoietic cell transplantation after nonmyeloablative CY/fludarabine (Flu) conditioning. GVHD prophylaxis included CsA (n=19)+/-mycophenolate mofetil (n=31). At a median follow-up of 59 months, 21 patients (42%) were alive without evidence of disease. By Kaplan-Meier analysis, year 1-4 disease-free survival (DFS) and OS estimates were 0.50/0.58, 0.40/0.46, 0.37/0.43 and 0.37/0.41. MUD recipients were engrafted quickly (13.5 days) compared to MRD recipients (16 days) and relapsed/progressed less frequently (P=0.005). Overall grade 3/4 acute GVHD (aGVHD) occurred in 26% in the absence of antecedent mucositis and was associated with chronic GVHD (cGVHD) and poor OS. Extensive cGVHD developed in 51.2% of 100 day survivors. Rates of aGVHD, cGVHD and survival were similar between MRD and MUD recipients. Of 14 survivors with cGVHD, 5 (35.7%) experienced resolution off immunosuppression, suggesting that tolerance with HLA matched grafts is possible at an advanced age by this method. This study provides further evidence for prolonged DFS after CY/Flu MRD allotransplantation for AML/MDS, and extends the findings to older patients and those with unrelated donors.

Assessment of iron status and the role for iron-replacement therapy in anaemic cancer patients under the care of a specialist palliative care unit.

Anaemia is common in advanced cancer, may develop for several reasons, and is not always symptomatic. Our observations of the seemingly indiscriminate prescription of iron-replacement therapy (IRT) for anaemic palliative care patients, and our practice of discontinuing IRT in patients with normal red-cell indices, prompted a study to determine (1) the prevalence of anaemia in our patients, (2) what proportion had iron deficiency, (3) the prevalence and benefits of IRT and (4) the prevalence of side effects attributable to IRT. The prevalence of anaemia was 65%. We found a 9% prevalence of iron deficiency, and suggestive but inconclusive evidence of iron deficiency in a further 41%, but only three (27%) of these patients had typical iron deficiency red-cell indices. Only two patients within the study population were taking IRT. Haemoglobin increased significantly in one, but fell in the other, and both experienced side effects attributable to iron. IRT should neither be indiscriminately prescribed nor withheld for anaemic palliative care patients, and the decision should not be based on red-cell indices alone. When symptomatic anaemia is found in patients whose general condition indicates that IRT would be acceptable, iron status should be fully assessed. A therapeutic trial of IRT may be justified where ferritin is elevated, but other parameters suggest iron deficiency.

Studies of self-incompatibility in wild tomatoes: I. S-allele diversity in Solanum chilense (Dun.) Reiche [corrected] (Solanaceae).

We characterized the molecular allelic variation of RNases at the self-incompatibility (SI) locus of Solanum chilense Dun. We recovered 30 S-RNase allele sequences from 34 plants representing a broad geographic sample. This yielded a species-wide estimate of 35 (95% likelihood interval 31-40) S-alleles. We performed crosses to confirm the association with SI function of 10 of the putative S-RNase allele sequences. Results in all cases were consistent with the expectation that these sequences represent functional alleles under single-locus gametophytic SI. We used the allele sequences to conduct an analysis of selection, as measured by the excess of nonsynonymous changes per site, and found evidence for adaptive changes both within the traditionally defined hypervariable regions and downstream, near the 3'-end of the molecule.

Notification of rheumatic fever in South Africa -- evidence for underreporting by health care professionals and administrators.

OBJECTIVE: To determine whether under-reporting of rheumatic fever occurs at hospital, municipal, provincial and national levels of the South African health system. BACKGROUND: Information on the incidence of rheumatic fever (RF) and the prevalence of rheumatic heart disease (RHD) is required for the prevention of valvular heart disease in developing countries. In South Africa, RF was made a notifiable condition in 1989. It has recently been suggested that the reporting of RF cases may be incomplete, possibly because of underreporting by health care professionals and deficient administration of the disease notification system in South Africa. METHOD AND RESULTS: We assessed whether underreporting of RF cases occurs by comparing the numbers of RF cases reported per year at hospital, municipal, provincial and national levels from 1990 to 2004. There was a fall in the number of RF cases reported per year at national and provincial level over the 15 years of observation. A detailed analysis of the number of RF cases reported at hospital, municipal and provincial level for a 5-year period showed that more cases were diagnosed in one hospital (serving a smaller population) than were captured at municipal and provincial level (serving a larger population), suggesting underreporting by health care professionals. There were discrepancies in the number of cases reported at municipal, provincial and national level, suggesting poor administration of the notification system. CONCLUSION: There appears to be underreporting of RF cases by health care professionals, and poor administration of the RF notification system. Health care professionals need to be educated about the statutory requirement to notify all RF cases in South Africa. An effective national disease notification system is required.

Altered expression of Ape1/ref-1 in germ cell tumors and overexpression in NT2 cells confers resistance to bleomycin and radiation.

The human AP endonuclease (Ape1 or ref-1) DNA base excision repair (BER) enzyme is a multifunctional protein that has an impact on a wide variety of important cellular functions including oxidative signaling, transcription factor regulation, and cell cycle control. It acts on mutagenic AP (baseless) sites in DNA as a critical member of the DNA BER repair pathway. Moreover, Ape1/ref-1 stimulates the DNA-binding activity of transcription factors (Fos-Jun, nuclear factor-kappaB, Myb, ATF/cyclic AMP-responsive element binding protein family, HIF-1alpha, HLF, PAX, and p53) through a redox mechanism and thus represents a novel component of signal transduction processes that regulate eukaryotic gene expression. Ape1/ref-1 has also been shown to be closely linked to apoptosis associated with thioredoxin, and altered levels of Ape1/ref-1 have been found in some cancers. In a pilot study, we have examined Ape1/ref-1 expression by immunohistochemistry in sections of germ cell tumors (GCTs) from 10 patients with testicular cancer of various histologies including seminomas, yolk sac tumors, and malignant teratomas. Ape1/ref-1 was expressed at relatively high levels in the tumor cells of nearly all sections. We hypothesized that elevated expression of Ape1/ref-1 is responsible in part for the resistance to therapeutic agents. To answer this hypothesis, we overexpressed the Ape1/ref-1 cDNA in the GCT cell line NT2/D1 using retroviral gene transduction with the vector LAPESN. Using an oligonucleotide cleavage assay and immunohistochemistry to assess Ape1/ref-1 repair activity and expression, respectively, we found that the repair activity and relative Ape1/ref-1 expression in GCT cell lines are directly related. NT2/D1 cells transduced with Ape1/ref-1 exhibited 2-fold higher AP endonuclease activity in the oligonucleotide cleavage assay, and this was reflected in a 2-3-fold increase in protection against bleomycin. Lesser protection was observed with gamma-irradiation. We conclude that: (a) Ape1/ref-1 is expressed at relatively high levels in some GCTs; (b) elevated expression of Ape1/ref-1 in testicular cancer cell lines results in resistance to certain therapeutic agents; and (c) Ape1/ref-1 expression in GCT cell lines determined by immunohistochemistry and repair activity assays parallels the level of protection from bleomycin. We further hypothesize that elevated Ape1/ref-1 levels observed in human testicular cancer may be related to their relative resistance to therapy and may serve as a diagnostic marker for refractory disease. To our knowledge, this is the first example of overexpressing Ape1/ref-1 in a mammalian system resulting in enhanced protection to DNA-damaging agents.

Regression of a murine gammaherpesvirus 68-positive b-cell lymphoma mediated by CD4 T lymphocytes.

Murine gammaherpesvirus 68-infected S11 cells were injected subcutaneously into nude mice. Adoptively transferred restimulated lymphocytes consistently elicited the regression of S11 tumors. CD4 T lymphocytes were most effective in preventing tumor formation, and immunohistochemistry highlighted populations of CD4 T cells in regressing tumors.

Cost analysis of an intensive home follow-up program for first-time post-myocardial infarction patients and their families.

OBJECTIVE: The general goal of this research was to determine the effectiveness of a home follow-up program in order to acquire guidance in how to plan the future structure and contents of post-myocardial infarction (MI) patients' care and rehabilitation. The specific aim of this study was to evaluate the cost-effectiveness of the program in reducing the rate of rehospitalization of first-time post-MI patients when measured at six weeks and six months post-discharge. HYPOTHESIS: The supportive-educative home follow-up program will prove to be cost-effective by indicating an inverse correlation with the cost of post-MI patients being rehospitalized for unplanned and preventable diagnoses. DESIGN AND SETTING: Cost analysis, using data from a one year randomized control clinical trial conducted in a small urban hospital in eastern Canada. An experimental post test only control group design, including the process of randomization, was used in this study. SUBJECTS: 62 people admitted with a diagnosis of a first-time acute MI during a one-year period with no co-morbidity likely to affect rehabilitation. MAIN OUTCOME MEASURES: Health care costs. RESULTS: Early supportive home follow-up reduced inpatient rehospitalization by more than half (three rehospitalizations vs seven rehospitalizations) and reduced the average length of stay (five days vs seven days). Cost analysis demonstrated that intense home follow-up in the time immediately following patient discharge could still produce cost savings to the health care system. CONCLUSION: Intensive home follow-up provided a cost-effective alternative to traditional cardiac rehabilitation programs; however, a larger study is required to assess the generalizability of the results and long-term cost effectiveness.

Clinical major option: a model for implementing critical care nursing into baccalaureate preparation.

What was initiated as a directive from a provincial government in an attempt to increase the number of critical care nurses has evolved into an exciting educational opportunity for many nurses and student nurses in the year 2000. Between 1993 and 1997 there has been significant downsizing of acute care beds across Canada (Code Blue: Critical Care Nursing in Nova Scotia, 1998). At the same time patient acuity has increased, due to shorter hospital stays, and the number of nurses working full-time has decreased with the increased use of casual nurses. Several studies at both the provincial and national levels report current and future shortages of specialized nurses (emergency, critical care and perioperative). It is expected that this shortage will continue into the future, a shortage that is driven by technological advances, as well as an aging general and nursing population. Continued shortages of these acute care nurses will result in fierce competition for skilled nurses as well as aggressive recruitment and retention strategies (Code Blue: Critical Care Nursing in Nova Scotia, 1998). It is generally agreed within the nursing community that specialty nurses in critical care require a unique body of knowledge that is not acquired in a basic undergraduate nursing program (Fitzsimmons, Hadley, & Shively, 1999). This specialized knowledge can be gained informally through experience; however, it is largely developed in additional formal education programs. The purpose of this article is to outline a strategy for the delivery of specialty education at three educational levels in acute care nursing with three streams: emergency, critical care and perioperative nursing. This clinical major option is to be delivered in partnership among the Queen Elizabeth Hospital II, the Health Science Centre and Dalhousie University School of Nursing, Halifax, Nova Scotia, Canada. This model of offering specialty education in university preparation could be a template for preparing nurses in the new millennium.

Electro-oculography, electroretinography, visual evoked potentials, and multifocal electroretinography in patients with vigabatrin-attributed visual field constriction.

PURPOSE: Symptomatic visual field constriction thought to be associated with vigabatrin has been reported. The current study investigated the visual fields and visual electrophysiology of eight patients with known vigabatrin-attributed visual field loss, three of whom were reported previously. Six of the patients were no longer receiving vigabatrin. METHODS: The central and peripheral fields were examined with the Humphrey Visual Field Analyzer. Full visual electrophysiology, including flash electroretinography (ERG), pattern electroretinography, multifocal ERG using the VERIS system, electro-oculography, and flash and pattern visual evoked potentials, was undertaken. RESULTS: Seven patients showed marked visual field constriction with some sparing of the temporal visual field. The eighth exhibited concentric constriction. Most electrophysiological responses were usually just within normal limits; two patients had subnormal Arden electro-oculography indices; and one patient showed an abnormally delayed photopic b wave. However, five patients showed delayed 30-Hz flicker b waves, and seven patients showed delayed oscillatory potentials. Multifocal ERG showed abnormalities that sometimes correlated with the visual field appearance and confirmed that the deficit occurs at the retinal level. CONCLUSION: Marked visual field constriction appears to be associated with vigabatrin therapy. The field defects and some electrophysiological abnormalities persist when vigabatrin therapy is withdrawn.

Analysis of engraftment, graft-versus-host disease, and immune recovery following unrelated donor cord blood transplantation.

Unrelated cord blood (UCB) is being used as a source of alternative hematopoietic stem cells for transplantation with increasing frequency. From November 1994 to February 1999, 30 UCB transplant procedures were performed for both malignant and nonmalignant diseases in 27 children, aged 0.4 to 17.1 years. Patients received either HLA-matched (n = 3) or 1- or 2-antigen-mismatched (n = 27) UCB following 1 of 2 standardized preparative and graft-versus-host disease regimens (hyperfractionated total body irradiation, cyclophosphamide, and antithymocyte globulin [ATG] with cyclosporine A and methotrexate; or busulfan, melphalan, and ATG with cyclosporine A and prednisone). The median time to neutrophil and platelet engraftment was 27 days (12-60 days) and 75 days (33-158 days) posttransplantation, respectively. No correlation was noted between neutrophil and platelet engraftment and nucleated cells per kilogram, CD34(+) cells per kilogram infused, or cytomegalovirus status of recipient. The cumulative probability of acute grade 2 or greater graft-versus-host disease (GVHD) was 37.2%, and of grade 3 or greater GVHD was 8.8%. No patients developed chronic GVHD. CD4, CD19, and natural killer cell recovery was achieved at a median of 12, 6, and 2 months, respectively. CD8 recovery was delayed at a median of 9 months. Normal mitogen response was achieved at 6 to 9 months. The probability of survival, disease-free survival, and event-free survival at 1 year was 52.3% (34.1%-70.5%), 54.7% (34.5%-74.9 %) and 49.6% (29.9%-69.4%), respectively. This series of 30 UCB transplants suggests that although CD8 cell recovery is delayed, the pattern of immune reconstitution with UCB is similar to that reported for other stem cell sources. (Blood. 2000;96:2703-2711)

Separating the retinal electrophysiologic effects of vigabatrin: treatment versus field loss.

OBJECTIVE: To separate the retinal electrophysiologic markers associated with vigabatrin-attributed visual field loss (VGB-VFL) from those associated with current vigabatrin therapy. METHODS: A nonrandomly selected cohort of 8 previous and 18 current vigabatrin users and a reference cohort of 8 never vigabatrin-treated patients with epilepsy receiving other antiepilepsy drugs (AED) underwent electro-oculography (EOG), electroretinography (ERG), and automated static threshold perimetry. A cohort of 22 normal subjects underwent ERG. The validity of the retinal electrophysiologic variables to detect the presence and severity of VGB-VFL was assessed using receiver operator characteristic curves. RESULTS: Of 26 patients exposed to vigabatrin, 18 exhibited VGB-VFL. No patients receiving alternative AED showed this type of visual field abnormality. The presence and severity of VGB-VFL was significantly associated with the latency (implicit time) and amplitude of the ERG cone function. The amplitude of the cone flicker response was the strongest predictor of VGB-VFL and revealed a sensitivity of 100% at a specificity of 75%. The EOG, the photopic and scotopic ERG, and the latency of the ERG second oscillatory potential (OP2) were not significantly related to the presence of VGB-VFL. Vigabatrin therapy was significantly associated with the photopic amplitude, the scotopic a-wave latency, and the latency of OP2. CONCLUSION: In patients who cannot perform reliable perimetry, the cone-specific ERG flicker amplitude provides the best screening method for detecting VGB-VFL.

Lung infections: role of apoptosis in host defense and pathogenesis of disease.

Apoptosis is a form of cell death that has gained enormous attention during the past few years, and its mechanisms, important to biology and medicine, are being unraveled at an accelerating pace. Apoptosis of lung cells occurs during lung infections and may be either a host defense mechanism or reflect the pathogenesis of the infection. In the first part of this review, the biochemistry and physiology of apoptotic pathways and its regulators are discussed. This is followed by an overview of apoptotic mechanisms in selected lung infections. The implications of apoptosis in host immunity, pathogenesis, and treatment of pulmonary infections will be discussed in this context.

Patient compliance and satisfaction with mechanical devices for preventing deep venous thrombosis after joint replacement.

A consecutive series of patients having total joint arthroplasty at a single university hospital were sequentially treated with two mechanical devices for prevention of deep venous thrombosis (DVT). The first 104 patients (group 1) wore thigh-high sequential compression device (SCD). The next 120 patients (group 2) wore a foot pump. Daily documentation of hourly compliance with each respective device was recorded until discharge. A patient satisfaction questionnaire was also obtained. Patient understanding about the devices' function aided compliance (73% compliance in group 1, and 77% in group 2). The satisfaction questionnaire revealed significantly greater satisfaction in group 2 (73%) versus group 1 (55%). Of a subgroup of 35 patients who had used both devices, 24 preferred the foot pump, 7 the SCD, and 4 had no preference. This study showed a higher degree of compliance and satisfaction for foot pumps as prophylaxis against DVT.

Bcl-2 protects against beta-lapachone-mediated caspase 3 activation and apoptosis in human myeloid leukemia (HL-60) cells.

We previously demonstrated that beta-lapachone (beta-lap) killed cancer cells solely by apoptosis. Beta-Lap induced apoptosis in HL-60 cells in a dose-dependent manner as measured by flow cytometry and DNA ladder formation. Cell cycle changes, such as accumulations in S and G2-phases, were not observed. Apoptosis was accompanied by activation of caspase 3 and concomitant cleavage of poly(ADP-ribose) polymerase (PARP) to an 89 kDa polypeptide. PARP cleavage was blocked by zDEVD-fmk or zVAD-fmk, caspase-specific cleavage site inhibitors. Retrovirally introduced bcl-2 prevented beta-lap-mediated caspase 3 activation and PARP cleavage and increased the viability of Bcl-2-expressing HL-60 cells compared to cells with vector alone. Various beta-lap-related analogs (e.g., dunnione and naphthoquinone derivatives) induced equivalent apoptosis in HL-60 cells, but no compound was more effective than beta-lap. These data provide further evidence that the primary mode of cell killing by beta-lap is by the initiation and execution of apoptosis in human cancer cells.

High dose chemotherapy with autologous peripheral blood progenitor cell transplantation in an anephric child with multiply recurrent Wilms tumor.

PURPOSE: An autologous peripheral blood progenitor cell (APBPC) transplant in an anephric child with multiply recurrent Wilms tumor using a conditioning regimen of high dose chemotherapy in conjunction with hemodialysis (HD) and peritoneal dialysis is described. PATIENT AND METHODS: The child had a left nephrectomy at 9 months of age for a stage II Wilms tumor. At 6 years of age, she required a right nephrectomy because of progressive, recurrent disease unresponsive to treatment with doxorubicin, actinomycin, and vincristine. She was maintained on peritoneal dialysis. Salvage chemotherapy consisted of 5 cycles of carboplatin and cyclophosphamide after APBPCs were collected after granulocyte colony-stimulating factor mobilization. After a preparative regimen of carboplatin, cyclophosphamide, and etoposide with closely timed HD, peripheral blood progenitor cells were infused and peritoneal dialysis was resumed. RESULTS: No nonhematopoietic toxicity occurred. Pharmacokinetic studies demonstrated that HD effectively eliminated carboplatin and provided safe, effective plasma concentrations in this anephric patient. Trilineage engraftment occurred by day +10 and the child was discharged from the hospital on day +14. She had a local recurrence on day +194 and died of progressive disease on day +660. CONCLUSIONS: With dialysis support and dose modification, high-dose chemotherapy followed by APBPC transplantation can be successfully performed in the anephric child. Given the lack of organ toxicity in this patient, increased doses of the drugs used in this preparative regimen may be possible for anephric children.

Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation.

Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell-depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/microg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had >/=40,000 copies of EBV DNA/microg DNA, all of whom were recipients of T-cell-depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with >/=40,000 copies EBV/microg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.

A positive effect of p21cip1/waf1 in the colony formation from murine myeloid progenitor cells as assessed by retroviral-mediated gene transfer.

p21cip1/waf1 is a cyclin dependent kinase inhibitor. We have previously reported stimulation of p21cip1/waf1 by steel factor and GM-CSF in a factor dependent cell line and of p21cip1/waf1 involvement in hematopoiesis in vivo in p21cip1/waf1 gene knockout (-/-) mice. To further assess a role for increased p21cip1/waf1 in hematopoietic progenitor cells, we developed the retroviral vector L(p21cip1)SN to transcriptionally regulate p21cip1/waf1 from the Mo-MLV LTR. L(p21cip1)SN and the control vector LXSN were used to transduce murine bone marrow progenitor cells from p21cip1/waf1 (-/-) and littermate control (+/+) mice, as well as from other mouse strains. Hematopoietic colony formation by transduced cells was assessed in semi-solid culture medium with multiple growth factors. Myeloid colony formation by bone marrow cells from p21cip1/waf1 (-/-) mice was significantly lower than that by (+/+) mouse cells. Transduction of cells with LXSN had no effect on colony formation: however, (-/-) cells transduced with L(p21cip1)SN formed significantly greater numbers of colonies than either LXSN-transduced (-/-) or (+/-) cells. Moreover, L(p21cip1)SN-transduced (+/+) cells formed significantly more colonies than LXSN-transduced (+/+) cells. Increased cloning efficiency of progenitors from normal strains of mice induced by L(p21cip1)SN compared to LXSN-transduced cells was seen whether unseparated or highly purified populations of Sca1+ Lin- marrow cells were used. Gene transfer of L(p21cip1)SN increased the size and number of cells per colony, as well as the number of colonies compared to LXSN gene transfer. No colonies grew from non-transduced, LXSN-, or L(p21cip1)SN-transduced cells when no growth factors were added to the cultures. These results document the positive effect of p21cip1/waf1 in the proliferation and/or differentiation of the murine myeloid progenitor cells that lead to colony formation.

Differentiation of LA-N-5 neuroblastoma cells into cholinergic neurons: methods for differentiation, immunohistochemistry and reporter gene introduction.

The use of model systems derived from cell lines has been a valuable tool in understanding the molecules and cellular processes that govern differentiation processes (T.R. Breitman, S.E. Selonick, S.J. Collins, Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid, Proc. Natl. Acad. Sci. USA 77 (1980) 2936-2940 [2]; N. Gomez, S. Traverse, P. Cohen, Identification of a MAP kinase in phaeochromocytoma (PC12) cells, FEBS Lett. 314 (1992) 461-465 [4]). The use of such systems provides an inexpensive, quick and simple way to identify and test molecules that can be further studied in more complex in vivo experiments. Some cell lines such as embryonic stem cells can be induced to differentiate in vitro, however, the differentiation is difficult to control and most often leads to the generation of a wide variety of cell types. Cell lines derived from sources committed to a restricted cell fate provide an opportunity to examine cell growth and differentiation within a specific cell type (G.M. Keller, In vitro differentiation of embryonic stem cells, Curr. Opin. Cell Biol. 7 (1995) 862-869 [10]). In this article we describe a simple system for the differentiation of the human neuroblastoma cell line LA-N-5 into cholinergic neurons using all-trans retinoic acid (G. Han, B. Chang, M.J. Connor, N. Sidell, Enhanced potency of 9-cis versus all-trans retinoic acid to induce the differentiation of human neuroblastoma cells, Differentiation, 59 (1995) 61-69 [5]; D.P. Hill, K.R. Robertson, Characterization of the cholinergic neuronal differentiation of the human neuroblastoma cell line LA-N-5 after treatment with retinoic acid, Dev. Brain Res. 102 (1997) 53-67 [6]; J.A. Robson, N. Sidell, Ultrastructural features of a human neuroblastoma cell line treated with retinoic acid, Neuroscience 14 (1985) 1149-1162 [12]; N. Sidell, C.A. Lucas, G.W. Kreutzberg, Regulation of acetylcholinesterase activity by retinoic acid in a human neuroblastoma cell line, Exp. Cell Res. 155 (1984) 305-309 [14]). These cells provide a setting for the study of cholinergic neuronal differentiation and of the factors that influence that process. We also discuss procedures that can be used to study gene expression in LA-N-5 cells by immunohistochemistry and reporter gene analysis.