Justification for conducting neurological clinical trials as part of patient care within private practice.

The aim of this review was to assess the benefits and drawbacks of conducting neurological clinical trials and research in private practice for the patients, clinician, Practice Manager, sponsors/Clinical Research Organisations (CROs) and Clinical Trial Coordinator (CTC) to determine if this is justified for all involved. A combination of literature reviews, original research articles and books were selected from 2005 to 2015. Provided that the practice has sufficient number of active trials to prevent financial loss, support staff, adequate facilities and equipment and time, the benefits outweigh the drawbacks. Clinical trials provide patients with more thorough monitoring, re-imbursement of trial-related expenses and the opportunity to try an innovative treatment at no charge when other options have failed. For the clinician, clinical trials provide more information to ensure better care for their patients and improved treatment methods, technical experience and global recognition. Trials collect detailed and up-to-date information on the benefits and risks of drugs, improving society's confidence in clinical research and pharmaceuticals, allow trial sponsors to explore new scientific questions and accelerate innovation. For the CTC, industry-sponsored clinical trials allow potential entry for a career in clinical research giving CTCs the opportunity to become Clinical Research Associates (CRAs), Study Start-Up Managers or Drug Safety Associates.

Delaying gratification for food and tokens in capuchin monkeys (Cebus apella) and chimpanzees (Pan troglodytes): when quantity is salient, symbolic stimuli do not improve performance.

Capuchin monkeys have been tested for the capacity to delay gratification for accumulating rewards in recent studies and have exhibited variable results. Meanwhile, chimpanzees have consistently excelled at this task. However, neither species have ever been tested at accumulating symbolic tokens instead of food items, even though previous reports indicate that tokens sometimes facilitate performance in other self-control tasks. Thus, in the present study, we tested capuchin monkeys and chimpanzees for their capacity to delay gratification in a delay maintenance task, in which an experimenter presented items, one at a time, to within reach of an animal for as long as the animal refrained from taking them. In Experiment 1, we assessed how long capuchin monkeys could accumulate items in the delay maintenance task when items were food rewards or tokens exchangeable for food rewards. Monkeys accumulated more food rewards than they did tokens. In Experiment 2, we tested capuchin monkeys and chimpanzees in a similar accumulation test. Whereas capuchins again accumulated more food than tokens, all chimpanzees but one showed no difference in performance in the two conditions. These findings provide additional evidence that chimpanzees exhibit greater self-control capacity in this task than do capuchin monkeys and indicate that symbolic stimuli fail to facilitate delay maintenance when they do not abstract away from the quantitative dimension of the task. This is consistent with previous findings on the effects of symbols on self-control and illuminates what makes accumulation a particularly challenging task.

Immunohistochemistry and serum values of S-100B, glial fibrillary acidic protein, and hyperphosphorylated neurofilaments in brain injuries.

INTRODUCTION: Traumatic brain injury (TBI) triggers a series of reactions resulting in cytoskeletal-related changes varying between focal and diffuse injuries. METHODS: The patients (n=38) were divided into group of diffuse axonal injuries (DAI, n=10) and focal (n=28) injuries. Serum hyperphosphorylated neurofilaments (NF-H) and glial fibrillary acidic protein (GFAP) were measured by Biovendor immunoassay, and serum S-100B protein was measured by Cobas e411 (Roche) by immunoassay. Immunohistochemistry was performed with monoclonal antibodies (Chemicon, USA). RESULTS: The median serum S-100B concentration was higher in patients with focal mass lesions (1.72±0.4 µg/l vs. 0.37±0.1 µg/l, p<0,05) compared to patients with DAI during 10 days of hospitalisation. With respect to all patients, the highest peak of serum S-100B values (4.21±1.1 µg/l) and GFAP (8.58±2.4 µg/l) were found in expansive lesions. The median serum NF-H was higher in DAI compared to focal TBI (0.625±0.14 vs 0.139±0.02 ng/l, p<0.05) during all 10 days after admission. Further, immunohistochemical investigation, in deceased patients with DAI , using NF-H antibody proved positive varicose and waving axons, and retraction balls. Time-dependent profile of serum NF-H demonstrated the increase of values within 4th up to 10th day in both groups. Values ranged from 0.263 up to 1.325 ng/l in DAI, and from 0.103 up to 1.108 ng/l in focal injuries. Patients with expansive contusions had similar levels of serum NF-H as patients without expansive lesions. Immunohistochemistry of cytoskeletal proteins presented strong positive staining of vinculin, vimentin in vessels, GFAP, and S-100B in DAI compared to weak staining in expansive lesions. CONCLUSION: The time-profile kinetics of all markers may reflect different types of pathophysiological changes of the BBB or axonal damage in focal and diffuse injuries. KEYWORDS: brain contusions - diffuse axonal injury - S-100B protein - GFAP - hyperphosphorylated neurofilaments.

Anterior cruciate ligament tear patterns in young patients: An arthroscopic multicenter study.

INTRODUCTION: In the last decade, there has been a renewed interest in anterior cruciate ligament (ACL) preservation surgeries in the younger patients. Several ACL preservation techniques such as primary repair, augmented repair, and scaffold repair have been described based on the particular tear type and pattern. The purpose of this study was to determine the distribution of tear patterns in young patients presenting with an acute ACL injury. METHODS: A prospective observational study was performed at two tertiary children's hospitals. Patients under 18 years undergoing ACL reconstruction within 8 weeks of initial injury were included from 2017 to 2019. Tear patterns were classified by two orthopedic surgeons from each of the two centers during arthroscopic ACL reconstruction into 4 types: I. Avulsion off the femur, II. <10% of total ACL length tear from femoral end, III. Mid-substance tear and IV. Single bundle tear. For reliability, the four surgeons classified ACL injury (2 rounds each) based on de-identified intraoperative videos of 33 randomly selected surgical ACL cases. Inter and intra-rater reliability studies were calculated using Kappa statistics. RESULTS: 224 patients (123 males, 101 females) with mean age of 16 (range: 9-18) years were enrolled in this study. Fifty-seven (25%) patients reported contact injury while 167 (75%) reported non-contact. Isolated ACL injury was recorded in 70 (31%) patients, while concomitant injuries were recorded in 154 patients (69%). The most common associated injury was lateral meniscus tear (35%), followed by lateral and medial meniscus tears (20%). According to our classification, 31 (14%) patients were Type I, 30 (13%) were Type II, 139 (62%) were Type III, 18 (8%) were Type IV. The intra-rater reliability was excellent for 2 reviewers, good for 1 and marginal for another. The overall inter-rater reliability for all 4 reviewers was marginal for both readings. There was no statistical difference in the occurrence of type of tear based on the mechanism of injury (contact vs non-contact) or age of the patients. CONCLUSIONS: This is the first multicenter study using an arthroscopic assessment to classify the location of ACL tear in the young population. It gives us further insight on the possible application for surgeries to preserve the ACL in this group. Larger studies incorporating these findings with MRI evaluation and ACL repair techniques are needed to confirm the utility of this information to decide the eligibility for repair in pediatric patients.

Subcellular localization of Bcr, Abl, and Bcr-Abl proteins in normal and leukemic cells and correlation of expression with myeloid differentiation.

We used specific antisera and immunohistochemical methods to investigate the subcellular localization and expression of Bcr, Abl, and Bcr-Abl proteins in leukemic cell lines and in fresh human leukemic and normal samples at various stages of myeloid differentiation. Earlier studies of the subcellular localization of transfected murine type IV c-Abl protein in fibroblasts have shown that this molecule resides largely in the nucleus, whereas transforming deletion variants are localized exclusively in the cytoplasm. Here, we demonstrate that the murine type IV c-Abl protein is also found in the nucleus when overexpressed in a mouse hematopoietic cell line. However, in both normal and leukemic human hematopoietic cells, c-Abl is discerned predominantly in the cytoplasm, with nuclear staining present, albeit at a lower level. In contrast, normal endogenous Bcr protein, as well as the aberrant p210BCR-ABL and p190BCR-ABL proteins consistently localize to the cytoplasm in both cell lines and fresh cells. The results with p210BCR-ABL were confirmed in a unique Ph1-positive chronic myelogenous leukemia (CML) cell line, KBM5, which lacks the normal chromosome 9 and hence the normal c-Abl product. Because the p210BCR-ABL protein appears cytoplasmic in both chronic phase and blast crisis CML cells, as does the p190BCR-ABL in Ph1-positive acute leukemia, a change in subcellular location of Bcr-Abl proteins between cytoplasm and nucleus cannot explain the different spectrum of leukemias associated with p210 and p190, nor the transition from the chronic to the acute leukemia phenotype seen in CML. Further analysis of fresh CML and normal hematopoietic bone marrow cells reveals that p210BCR-ABL, as well as the normal Bcr and Abl proteins, are expressed primarily in the early stages of myeloid maturation, and that levels of expression are reduced significantly as the cells mature to polymorphonuclear leukocytes. Similarly, a decrease in Bcr and Abl levels occurs in HL-60 cells induced by DMSO to undergo granulocytic differentiation. The action of p210BCR-ABL and its normal counterparts may, therefore, take place during the earlier stages of myeloid development.

Opinions, attitudes and practices of Australian neurologists with regard to epilepsy and driving.

INTRODUCTION: Austroads Guidelines for fitness to drive were promulgated in 2003. Epilepsy was one of the conditions included and this paper reports results of a survey of Australian neurologists regarding opinions and practices relevant to the guidelines. METHODS: The survey was developed, piloted and Human Research Ethics Committee approved. Members of the Australian Association of Neurologists received three mailings and results were analysed. RESULTS: Almost 70% of 236 surveyed indicated assessment of epilepsy and driving with <9% not doing so--establishing approximately 77% response for eligible neurologists. Most questions achieved 90% response. Almost 90% respondents assessed epilepsy and 70% found the guidelines helpful. Seventy-seven per cent endorsed doctor assessors although half discounted General Practitioners as insufficiently knowledgeable and half advocated that only neurologists evaluate potential drivers with epilepsy. Most respondents supported reporting recalcitrant patients; yet only <30% did so. Three-quarters favoured licences carrying a warning to self-report and two-thirds felt that product information should identify driving implications. Although many questions attracted expected responses, the surprise was the large undecided numbers, which were greater than expected. Neurologists were more lenient than prescribed by the guidelines with neither consensus for controlled epilepsy nor mandatory driving restrictions. CONCLUSION: Respondents supplied predictable answers regarding ideal circumstances; yet most did not report recalcitrant patients. Most claimed to adhere to the guidelines and yet advocated more lenient driving restrictions that may allow preventable accidents. There was agreement between neurologists and guidelines for more rigorous restrictions for commercial drivers although again neurologists were more lenient. There is need for prospective research on epilepsy and driving.

Human adherent cell contact-mediated modulation of normal myeloid colony formation.

The effects of coincubation of normal nonadherent bone marrow cells on adherent monolayers created from human peripheral blood mononuclear cells or marrow cells were investigated. Nonadherent marrow cells were coincubated for 4 hours with peripheral blood adherent cells at ratios of adherent cells to marrow cells of 2:1 to 5:1. This coincubation suppressed subsequent neutrophilic agar colony growth but not eosinophilic growth. Further studies suggested that this suppression was a cell-cell-mediated process and not secondary to soluble factors. However, coincubation on marrow adherent cells caused increased neutrophilic colony recovery. The possible in vivo relevance is discussed.

Development and characterization of a human myelogenous leukemia cell line resistant to 4'-(9-acridinylamino)-3-methanesulfon-m-anisidide.

The human myelogenous leukemia cell line HL-60 was made resistant to amsacrine (m-AMSA) by repeated exposure in vitro to increasingly large doses of the drug. Resistance to m-AMSA developed in a triphasic process and was accompanied by a slightly slower growth rate and cloning efficiency and a more differentiated morphological phenotype. Extensive chromosomal rearrangement also took place. Among other chromosomal aberrations, one of the No. 6 homologues showed an added segment on the long arm in the form of an homogeneously staining region. One of the homologues of chromosome 14 in every cell showed a deletion of the distal end of the long arm that was replaced by an unidentified homogeneously staining segment. Membrane-associated 170 kd glycoprotein was not overexpressed in the resistant cells, which together with an absence of cross-resistance to Vinca alkaloids and anthracyclines points toward a mechanism of resistance different from multidrug resistance. The ability of resistant cells to respond to differentiation-inducing agents was not significantly changed as compared with that of the parental line. Growth of resistant cells in the absence of m-AMSA for over 200 population doublings within a period of more than 1.5 years did not result in reversion of the resistance, suggesting a stable genomic change. Resistance was not due to a decrease in the bioavailability of the drug. Uptake of [14C]m-AMSA by either whole cells or isolated nuclei of resistant cells exceeded that of the parental cell line, and outward transport of the drug was not more active; thus there were higher levels of intracellularly bound drug. The cell line represents an excellent model for studies of the mechanisms of resistance to m-AMSA and its modulation in human myelogenous leukemia.

Significance of cellular pharmacokinetics for the cytotoxic effects of daunorubicin.

The effect of free and DNA-linked daunorubicin on the colony-forming ability of granulocyte-macrophage committed stem cells and spleen colony-forming cells (i.e., multipotent stem cells) from normal mice has been studied in vitro and in vivo. After incubation of bone marrow cells in short-term suspension cultures, both committed and multipotent stem cells were more sensitive to the free drug than to the DNA complex, whereas the reverse was found in vivo after i.v. injection. However, when the in vitro cell-killing effect was related to the cellular retention of daunorubicin, no difference in activity was found between free and DNA-linked drug. Incubation of the bone marrow cells with a higher drug concentration for a shorter time resulted in a considerably lower cell survival than incubation with a lower concentration for a longer time, the intracellular exposure dose being the same. When the in vivo cell survival was related to the cellular retention of daunorubicin, the DNA complex was slightly more toxic than free drug, which can be explained by the higher peak concentration obtained. The results obtained with committed granulocytic stem cells and multipotent stem cells were comparable. Thus, the observed discrepancy between the in vitro and in vivo toxicity of free and DNA-linked daunorubicin can be explained by the differences in cellular retention of daunorubicin under these two conditions; i.e., the DNA complex probably acts as a slow-release preparation of daunorubicin. The results also demonstrated for the first time the importance of the peak concentration of daunorubicin in the target cells and indicate an important role of dose scheduling for the cytostatic effect of the drug.

The effects of acetaldophosphamide, a novel stable aldophosphamide analogue, on normal human and leukemic progenitor cells in vitro: implications for use in bone marrow purging.

Acetaldophosphamide (A-ALD), a novel in vitro active and stable derivative of aldophosphamide, kills human bone marrow-derived granulocyte-macrophage colony-forming cells (GM-CFC) independent of the cell cycle. The surviving fraction of GM-CFC is an exponential function of the drug concentration and time of exposure. Variation of marrow light-density cell concentration between 2 x 10(6) and 10 x 10(6)/ml does not significantly influence its GM-CFC toxicity. Marrow depleted of GM-CFC by A-ALD subsequently generates GM-CFC when grown in suspension cultures. During the early period after treatment with A-ALD the number of surviving GM-CFC (size of surviving GM-CFC compartment) does influence the speed of the GM-CFC repopulation in suspension cultures. The importance of the number of surviving GM-CFCs for the growth and maintenance of GM-CFC population in such suspension cultures diminishes with time. No significant differences are observed after 2 wk, indicating that the ancestor stem cell population and its regenerative potential responsible for in vitro hematopoiesis have not been significantly affected by the drug treatment. A-ALD-treated progenitor cells retain their ability to integrate with the previously established marrow stromal cell layer and generate GM-CFC within this layer to an extent comparable to that of untreated marrow cells. The effect of A-ALD on human hematopoiesis is comparable to that of 4-hydroperoxycyclophosphamide. Its advantage over 4-hydroperoxycyclophosphamide is a greater stability in vitro. It has sparing effect on GM-CFC ancestor cells. Its toxicity to myeloid leukemia cell line (KBM-3)-derived clonogeneic cells is higher than to the GM-CFC. It is similar in doxorubicin-sensitive (KBM-3) and -resistant (KBM-3/DOX) leukemic cells. Thus, A-ALD appears to be a promising drug for in vitro purging of bone marrow cells.

Drug sensitivity and cross-resistance of the 4'-(9-acridinylamino)methanesulfon-m-anisidide-resistant subline of HL-60 human leukemia.

A subline of the HL-60 leukemia resistant to 4'-(9-acridinylamino)methanesulfon-m-anisidide (HL-60/AMSA) was developed by intermittent long-term in vitro treatment. Resistance to 4'-(9-acridinylamino)methanesulfon-m-anisidide remained unchanged after 180 doublings in the absence of the drug, suggesting a stable phenotypic alteration. The pattern of cross-resistance of HL-60/AMSA was evaluated for a spectrum of antileukemic agents using the clonogenic assay. Modest cross-resistance to doxorubicin (Adriamycin) was observed in the resistant subline on continuous exposure to the drug for 8 to 9 days; however, HL-60/AMSA cells retained their sensitivity to doxorubicin following short-term exposure for 60 min. HL-60/AMSA was also sensitive to the anthracycline aclacinomycin, Vinca alkaloids, and alkylating agents. Furthermore, enhanced sensitivity to 1-beta-D-arabinofuranosylcytosine was observed. The subline was cross-resistant to etoposide.

Antisense RNA inhibition of phosphoprotein p18 expression abrogates the transformed phenotype of leukemic cells.

Phosphoprotein p18 was identified originally on the basis of its very high level of expression in leukemic cells of different lineages. Changes in the level of p18 accumulation and phosphorylation associated with induction of differentiation of leukemic cells suggested a potential role for this phosphoprotein in cellular proliferation and differentiation and possibly in malignant transformation. Recent studies have demonstrated that p18 plays an important role in cell cycle progression by serving as a substrate for p34(cdc2) kinase. These studies showed that inhibition of p18 expression in leukemic cells results in growth retardation and accumulation of cells in G(2)-M. In this study, we explore the potential role of p18 in cellular transformation by investigating the effects of inhibition of p18 expression on the malignant phenotype of K562 erythroleukemia cells. These studies show that antisense inhibition of p18 expression in leukemic cells results in growth arrest at a lower saturation density, loss of serum independence, and loss of anchorage-independent growth in vitro. In addition, inhibition of p18 expression results in a marked inhibition of tumorigenicity of leukemic cells in vivo in the severe combined immune deficiency mouse model. These studies demonstrate that the high level of p18 expression in leukemic cells is necessary for the maintenance of the transformed phenotype and suggest p18 as a potential target for antileukemic interventions.

Phase I clinical investigation of benzisoquinolinedione (amonafide) in adults with refractory or relapsed acute leukemia.

After Phase I studies of benzisoquinolinedione (amonafide) in solid tumors identified myelosuppression as the dose-limiting toxicity, we conducted a Phase I study in patients with relapsed or refractory acute leukemia to define the optimal dose. Amonafide was given i.v. over 2-4 h daily for 5 days. The starting dose was 600 mg/m2/day with subsequent escalation to 750, 900, 1100, 1400, and 1800 mg/m2/day. Thirty-eight courses were administered to 24 patients, of whom 12 participated in concomitant pharmacological studies. Nausea and vomiting, transient orange discoloration of the skin, and tinnitus occurred at all dose levels. The latter symptom, along with lightheadedness and flushing, was related to infusion duration; this was increased to 4 h with doses greater than or equal to 900 mg/m2. The dose-limiting toxicities were mucositis and painful skin erythema which occurred in all 4 patients treated with 1800 mg/m2. No remissions occurred. Clearing of peripheral blood blasts occurred in 67% of patients treated with 1100 mg/m2 and in all patients treated with greater than or equal to 1100 mg/m2/day. A decrease in marrow leukemic infiltrate (% blasts x % cellularity) to less than 10% occurred in 15 and 50% of patients treated at these levels, respectively. There were 10 deaths (42%), which were unrelated to dosage. The harmonic mean terminal plasma half-life was 4.6 h (range, 2.5-35.5 h). Three patients had long drug half-lives of 9.7, 16.4, and 35.5 h and each had initial bilirubin levels greater than 1.0 mg/dl. The average urinary excretion of amonafide over 5 days was 3.5% of the total dose. This establishes 1100-1400 mg/m2/day for 5 days as the maximally tolerated dose of amonafide for studies in acute leukemia.

CI-973, a new platinum derivative with potential antileukemic activity.

We examined the effects of CI-973 (supplied by Parke-Davis) on several human leukemia cell lines and a Chinese hamster ovary (CHO) line and their drug-resistant counterparts. The cell lines used were HL-60, HL-60/mAMSA, HL-60/DOX, KBM3, KBM3/mAMSA 6, KBM3/mAMSA 6(85), CHO, and CHO/AC-7. DOX, mAMSA, and AC-7 indicate resistance to doxorubicin, amsacrine, or 1-beta-D-arabinofuranosylcytosine, respectively. Cells were incubated with CI-973, and the effect was evaluated by two methods: growth inhibition assay and inhibition of colony formation. All cell lines examined were inhibited by CI-973; two of three amsacrine-resistant lines and the one cytarabine-resistant line demonstrated collateral sensitivity. At equivalent dosages, a 4-day exposure provided much greater cell kill than a 1-h exposure. Clonogenic assay showed exponential killing over 3 log units. Maximum CI-973 levels required to kill 50% of cells were 10-fold lower than the peak plasma levels achieved in a phase I solid tumor study. A continuous infusion phase I study in acute leukemia has been initiated.

In vitro toxicity and DNA cleaving capacity of benzisoquinolinedione (nafidimide; NSC 308847) in human leukemia.

Benzisoquinolinedione (nafidimide; NSC 308847) is an investigational drug currently in phase I clinical testing. We have studied the antileukemic activity in vitro, the cellular drug transport, and the molecular mechanism of action with DNA of this new compound. By agarose gel electrophoresis, we verified that nafidimide is an intercalating agent, through its alteration of the electrophoretic migration of DNA products produced by the relaxing action of DNA topoisomerase I. Concentrations of up to 100 microM of nafidimide did not produce topoisomerase I-mediated DNA cleavage. Nafidimide produced DNA single-strand breaks (SSB), double-strand breaks, and DNA-protein cross-links in human myeloid leukemia cells (measured with filter elution). The ratio of SSB/DNA-protein cross-links was 1.32 +/- 0.36, a value similar to that produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), suggesting that nafidimide, like m-AMSA, produced protein-associated DNA-strand breaks through a topoisomerase II-mediated reaction. The production of double-strand breaks by nafidimide also suggests the involvement of topoisomerase II in the drug-induced DNA cleavage. The cytotoxic activity of nafidimide was quantified in human myeloid leukemia cell lines differing by a factor of 70 in their cytotoxic sensitivity to m-AMSA. The m-AMSA-resistant line was less than 2-fold resistant to nafidimide. Cellular drug uptake was rapid and reached a steady state level in 30 min at 37 degrees C. At the end of exposure, drug egress was rapid, as was the disappearance of the DNA SSB. Rapid cellular uptake of nafidimide, with low retention at the end of exposure and rapid rejoining of DNA SSB suggest that prolonged cellular exposure may be necessary for optimal antitumor effect. In vitro cloning data suggest that nafidimide may be a therapeutic option for patients with leukemia resistant to m-AMSA.

Possible mechanisms of action of lithium on augmentation of in vitro spontaneous myeloid colony formation.

To understand the possible mechanisms of lithium carbonate-induced neutrophilia, the in vitro effect on human myeloid progenitor cells was examined. A significant increase in spontaneous colony formation (15 of 24 experiments) was observed with the addition of lithium. Increased colony formation seldom occurred when human placental conditioned media as a source of colony-stimulating activity (CSA) was simultaneously added to the cultures. Further data suggest that lithium requires an adherent marrow cell population for this action and that increases in CSA-containing cultures may be due to suboptimal CSA concentrations. Lithium was shown to release CSA from marrow cells and adherent cell population prepared from human bone marrow. Lithium possibly increases spontaneous human myeloid colony development indirectly through CSA release by adherent cells.

Biological properties and growth in SCID mice of a new myelogenous leukemia cell line (KBM-5) derived from chronic myelogenous leukemia cells in the blastic phase.

The establishment and the biological properties of a new leukemic cell line (KBM-5) derived from a patient in the blastic phase of chronic myelogenous leukemia are described. The cells exhibited multiple copies of the Philadelphia chromosome, and a high level of p210Bcr-Abl kinase activity was detected with rabbit anti-Abl and anti-Bcr (exon 3) peptide antisera. Use of specific primers and polymerase chain reaction followed by Southern blotting revealed that KBM-5 cells carried a bcr3-ABLII splice junction. While a normal BCR message was detected, no normal ABL message was found. The cells were phenotypically myeloid with monocytic differentiation. The high cloning efficiency in semisolid media was independent of the presence of exogenous colony-stimulating factors. In vitro exposure to induces of differentiation, such as retinoic acid, dimethyl sulfoxide, or hemin, failed to influence the growth rate of the cells and their level of differentiation. KBM-5 cells are highly resistant to the antiproliferative action of recombinant alpha- and gamma-interferons. Although sensitive to recombinant tumor necrosis factor alpha, they were completely resistant to natural killer cell action. KBM-5 cells constitutively expressed mRNA for tumor necrosis factor alpha but not for gamma-interferon, other interleukins, or hematopoietic growth factors. The KBM-5 cells that were transplanted into SCID mice manifested metastatic potential and tissue invasiveness similar to the way leukemic cells in humans do. This new KBM-5 cell line represents a helpful model for examining in vitro and in vivo modulation of the growth and properties of leukemic cells by using biological and chemotherapeutic agents.

Clinical implications of decreased retinoblastoma protein expression in acute myelogenous leukemia.

The retinoblastoma (RB) protein levels in blast-enriched mononuclear fractions from the peripheral blood of 33 newly diagnosed patients with acute myelogenous leukemia were studied. Ten patients who had previously been treated were also analyzed, nine of whom had achieved prior complete remission. Low RB protein expression was found in 13 of 43 (30%) of the acute myelogenous leukemia patients as determined by Western blotting and immunochemical analysis. Of particular interest among the 20 newly diagnosed patients treated with the same therapeutic regimen, the median survival was 39 days for those with low RB protein expression compared to 333 days for those with high levels of RB protein expression in their leukemic cells (P less than or equal to 0.02). This preliminary study suggests that decreases of RB protein expression in peripheral blood of myeloid leukemic cells occur frequently and may be associated with shortened survival of acute myelogenous leukemia patients.

Ethics in clinical drug trial research in private practice.

INTRODUCTION: Private clinics and clinicians have been involved in clinical drug trials for approximately two decades. This paper reviews the ethical consideration inherent in this process. METHODS: Involvement of a single community based, private, Australian neurological clinic in the conduct of trials was audited. Changes in ethical considerations were analysed. RESULTS: The clinic previously audited its clinical trial involvement, starting with pharmaceutical company orchestrated trials. These were vetted by hospital based ethics committees (ECs) which then refused to review private research. A private EC accommodating NH & MRC standards was formed to assess private research. Indemnity concerns forced return to institutional ECs with government guaranteed indemnification. Trials evolved to investigator initiated, company sponsored studies thence a company asking the clinic to devise, sponsor and manage a trial. The latter relegated trial co-ordination to the clinic which would control publication thereby creating new ethical standards. DISCUSSION: Private practice trial involvement evolved from reluctant inclusion to a pivotal role in privately sponsored studies. Access to ECs is government endorsed and publication is independent for investigator-sponsored trials. There has been modification of standard operating procedures and enhanced ethical standards.

Alternative 5' end of the bcr-abl transcript in chronic myelogenous leukemia.

Philadelphia chromosome positive acute lymphocytic leukemia and chronic myelogenous leukemia are strongly associated with two distinct forms of bcr-abl chimeric protein, known as P190 and P210, respectively. By studying cDNA clones obtained from the cell line KBM-5, we identified two new bcr-abl transcripts. These are formed by alternative splicing of at least two exons to the known bcr exon 2. One novel transcript can encode a protein kinase of approximately 190 kd, while the other can direct the synthesis of a larger protein whose amino terminus remains to be defined. The alternative exons can be spliced also to the two normal bcr transcripts, reflecting the activation of a cryptic promoter. These messages were present at low abundance in two cases of blastic crisis but were not detected in the chronic phase. It is conceivable that the proteins encoded by the new bcr-abl mRNAs are involved in the transformation to the acute phase in some cases of chronic myelogenous leukemia.