Role of cysteine 288 in nucleophosmin cytoplasmic mutations: sensitization to toxicity induced by arsenic trioxide and bortezomib.

Mutations in exon 12 of the nucleophosmin (NPM1) gene (NPMc+ (NPM1 COOH terminal mutations)) define a distinct subset of acute myelogenous leukemias (AMLs), in which the NPMc+ protein localizes aberrantly to the leukemic cell cytoplasm. We have found that introduction of the most common NPMc+ variant into K562 and 32D cells sensitizes these cells to apoptosis induced by drugs such as bortezomib and arsenic trioxide (ATO) that induce reactive oxygen species (ROS) formation, and that cytotoxicity is prevented in the presence of N-acetyl-L-cysteine (NAC), an ROS scavenger. The substitution of tryptophan 288 (W288) by cysteine occurs in the great majority of NPM1c+ mutations. Mutagenesis of cysteine 288 to alanine re-localizes NPMc+ from the cytoplasm to the nucleolus and attenuates the sensitivity of cells expressing this mutation to bortezomib and ATO. Primary AML cells expressing NPMc+ are also significantly more sensitive than other AML cells to apoptosis induced by both drugs at pharmacologically achievable doses. We conclude that the presence of a cysteine moiety at position 288 results in the cytoplasmic localization of NPM1c+ and the increased sensitivity to bortezomib and ATO. These data suggest that bortezomib and ATO may have increased therapeutic efficacy in NPM1c+ leukemias.

Understanding disparities in leukemia: a national study.

PURPOSE: Disparities in solid tumors have been well studied. However, disparities in hematologic malignancies have been relatively unexplored on population-based levels. The purpose of this study is to examine the relationship between race/ethnicity and acute leukemia mortality. METHODS: All patients with acute leukemia [acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML)] were identified in the Surveillance Epidemiology and End Results database, 1999-2008. Kaplan-Meier curves were generated to reflect survival probabilities by race/ethnicity. Multivariable Cox proportional hazard models estimated hazard of mortality by race with adjustment for individual (age, gender, year of diagnosis) and select genetic factors. RESULTS: A total of 39,002 patients with acute leukemia were included in the study. Overall, there was a mortality disparity in acute leukemia for blacks (HR 1.17, p < 0.0001) and Hispanics (HR 1.13, p < 0.0001) compared with non-Hispanic whites. In stratified analysis, disparities in ALL were greater than AML; blacks (HR[ALL]1.45, p < 0.0001; HR[AML]1.12, p < 0.0011); Hispanics (HR[ALL]1.46, p < 0.0001; HR[AML]1.06, p < 0.0001). Adjustment for individual patient and select genetic factors did not explain disparities. CONCLUSIONS: Blacks and Hispanics suffer decreased survival in acute leukemia as compared to others. Further investigation is needed to understand the drivers of poor cancer outcomes in these populations.

Safety, efficacy and biological predictors of response to sequential azacitidine and lenalidomide for elderly patients with acute myeloid leukemia.

Acute myeloid leukemia (AML) is a disease of the elderly. Poor outcomes with standard therapies necessitate novel approaches. Outpatient regimens sufficiently potent and well tolerated to induce remissions and enable continuation therapy may be beneficial. In this phase-1 study, we determined the maximum tolerated dose (MTD) and the efficacy for sequential azacitidine and lenalidomide as remission induction and continuation therapy in elderly, previously untreated patients. We investigated the impact on global DNA methylation and bone marrow cytokines, and sought biological predictors of response. Eighteen patients were enrolled. The MTD was not reached. Median follow-up was 8.2 months (10.3 months for survivors). Common adverse events included fatigue, injection site reactions, constipation, nausea, pruritus and febrile neutropenia. Ten patients responded (56%), and the rate of complete remissions (CRs) or CRs with incomplete recovery of blood counts for evaluable patients was 44% (7/16). The median response duration was 6.2 months. DNA demethylation and changes in bone marrow cytokines were observed; responders had a unique cytokine profile and a trend towards lower methylation levels. Sequential azacitidine and lenalidomide was well tolerated with encouraging clinical and biological activity in previously untreated elderly AML patients. This trial is registered at ClinicalTrials.gov (NCT00890929).

Relationship between bone ingrowth, mineral apposition rate, and osteoblast activity.

To better understand skeletal attachment of porous coated total hip and knee implants over time, this study investigated the dynamics of osteoblast populations at the interface of porous coated implants in a weight-bearing ovine model. The relationship between cancellous bone ingrowth, mineral apposition rate (MAR), and osteoblast activity indicators such as osteoblast area, relative osteoblast number, osteoid width, and osteoid area (O.Ar.) were investigated. The data demonstrated that the percent O.Ar. was a marginally significant predictor of bone ingrowth and MAR over time, suggesting that the amount of osteoid present influenced bone ingrowth and MAR in the porous coated implants. The data also demonstrated that all osteoblast activity indicators were significantly greater in the porous coated region compared to the host bone region, while controlling for in situ time (p < 0.05). This may have been due to the trauma of implantation or the influence of the implant load on the bone tissue promoting a regional acceleratory phenomenon. The localized response suggests that specific therapies may be developed to affect the physiology of osteoblasts at the interface of implants, which may allow for improve skeletal attachment of biomaterials and clinical outcomes of cementless joint replacements.

Looking 'back to the future': alumni perceptions of a UK undergraduate medical programme.

The five year Bachelor of Medicine (BM5) programme of the University of Southampton commenced in 1971. In keeping with other medical schools, the Southampton BM5 programme has been involved in a number of incremental curriculum reforms over the years. Complementing the School's annual pre-registration house officer (PRHO) questionnaire, this study of alumni cohorts (2000-2003) sought to investigate further how past graduates view their medical education and whether there are emerging priorities in medical practice. Findings confirm that alumni rate the BM5 highly and generally value the BM5 aims. Considering the impact of the social context on individual well-being and patient care, increased emphasis may need to be placed on preventive medicine, including greater alignment of several curriculum areas with clinical practice.

Phase I study of 506U78 administered on a consecutive 5-day schedule in children and adults with refractory hematologic malignancies.

PURPOSE: A phase I study was conducted to determine the maximum-tolerated dose (MTD), toxicity profile, and pharmacokinetics of a novel purine nucleoside, nelarabine, a soluble prodrug of 9-beta-D-arabinosylguanine (araG; Nelarabine), in pediatric and adult patients with refractory hematologic malignancies. PATIENTS AND METHODS: Between April 1994 and April 1997, 93 patients with refractory hematologic malignancies were treated with one to 16 cycles of study drug. Nelarabine was administered daily, as a 1-hour intravenous infusion for 5 consecutive days, every 21 to 28 days. First-cycle pharmacokinetic data, including plasma nelarabine and araG levels, were obtained on all patients treated. Intracellular phosphorylation of araG was studied in samples of leukemic blasts from selected patients. RESULTS: The MTDs were defined at 60 mg/kg/dose and 40 mg/kg/dose daily x 5 days in children and adults, respectively. Dose-limiting toxicity (DLT) was neurologic in both children and adults. Myelosuppression and other significant organ toxicities did not occur. Pharmacokinetic parameters were similar in children and adults. Accumulation of araGTP in leukemic blasts was correlated with cytotoxic activity. The overall response rate was 31%. Major responses were seen in patients with T-cell malignancies, with 54% of patients with T-lineage acute lymphoblastic leukemia achieving a complete or partial response after one to two courses of drug. CONCLUSION: Nelarabine is a novel nucleoside with significant cytotoxic activity against malignant T cells. DLT is neurologic. Phase II and III trials in patients with T-cell malignancies are encouraged.

Student attitudes towards anatomy teaching and learning in a multiprofessional context.

CONTEXT: We carried out a survey of attitudes to learning anatomy amongst students from a range of health care disciplines in a multiprofessional context. SETTING: A joint course called the Common Foundation Programme (CFP) presented by a hospital medical school and a joint university faculty of health and social care sciences in the UK in the first term of the students' courses. PARTICIPANTS: Students following degree courses in biomedical science, medicine, nursing, physiotherapy, diagnostic radiography and therapeutic radiography. OBJECTIVES: To assess student attitudes to cadaveric work, learning anatomy and multiprofessional learning, and to compare student performance between degree courses in an anatomy assessment. DESIGN: A questionnaire was designed that requested demographic information and the students' attitudes to cadaveric work, anatomy learning and multiprofessional learning on a Likert scale. All students sat the same anatomy assessment at the end of the first term. RESULTS: The biomedical science and medical students were the most apprehensive about entering the dissecting room. The biomedical science students enjoyed working in a multidisciplinary group the most. Assessment results varied widely and the physiotherapy and medical students scored more highly than students in other disciplines, although all students had participated in the same course. CONCLUSIONS: It was possible to teach anatomy in the context of the shared learning experience of the CFP, although performance varied widely. Reasons for the differences are discussed and suggestions for the design of multiprofessional courses involving anatomy are made.

Trends in radiological anatomy teaching in the U.K. and Ireland.

AIM: To determine the prevalence of radiological anatomy teaching in modern medical curricula. MATERIALS AND METHODS: The present paper details a survey carried out amongst 21 medical schools in the U.K. and Ireland to determine the variability of the inclusion of radiological anatomy in their different curricula. RESULTS: In all the institutions surveyed teaching of anatomy as revealed by radiological techniques was delivered by anatomy departments. There was considerable variation, however, in the way in which it was delivered, by whom and in the range of radiological material available. Most anatomy departments had links with radiology departments, though the precise arrangements of these also varied. CONCLUSION: This study highlights the need for standardization between anatomy departments with regard to the content and level of radiological anatomy taught to ensure that an appropriate basis for clinical undergraduate training is provided.

Alemtuzumab in previously treated chronic lymphocytic leukemia patients who also had received fludarabine.

PURPOSE: This phase II pilot study determined the efficacy and safety of alemtuzumab (Campath-1H; Burroughs Wellcome, United Kingdom) in patients with chronic lymphocytic leukemia (CLL), all of whom had previously received fludarabine and other chemotherapy regimens. PATIENTS AND METHODS: Twenty-four patients were treated with intravenous alemtuzumab at six centers in the United States. The target dose of 30 mg over 2 hours, three times weekly, was administered for up to 16 weeks. Responses were evaluated by an independent panel of experts using 1996 National Cancer Institute-sponsored Working Group criteria. Safety assessments included analysis of lymphocyte subpopulations. Antimicrobial prophylaxis was not mandatory. RESULTS: Eight patients (33%) achieved a major response (all partial remissions), with a median time to response of 3.9 months (range, 1.6 to 5.3 months). The median duration of response was 15.4 months (range, 4.6 to >or= 38.0 months), the median time to disease progression was 19.6 months (range, 7.7 to >or= 42.0 months), and the median survival time was 35.8 months (range, 8.8 to >or= 47.1 months). Acute infusion-related events, mainly grades 1 and 2, were most common and most severe in the first week. Ten patients (eight nonresponders and two responders) experienced major infections on-study. Pneumocystis carinii pneumonia was reported in two patients on-study; neither had received prophylaxis. Median CD4+ and CD8+ counts decreased and then began to increase by the end of the study, with further recovery by 1-month follow-up. One of 53 samples obtained from 10 patients had a low titer of alemtuzumab antibodies. CONCLUSION: Alemtuzumab has significant activity in poor-prognosis, fludarabine-treated CLL patients. However, because of a relatively high incidence of opportunistic infections accompanying profound lymphopenia, future protocols should include mandatory prophylaxis.

Anatomic relation between the nuchal ligament (ligamentum nuchae) and the spinal dura mater in the craniocervical region.

There are inconsistencies between the descriptions of the physical connections between the spinal cervical dura and the surrounding tissues. This study was undertaken to clarify the relationship between the spinal dura, the nuchal ligament and the suboccipital muscles. Dissections were performed on embalmed cadavers: in nine the relevant structures were removed en bloc, whereas in one a sagittal section was prepared. In all specimens it was possible to demonstrate continuity in the midline between the nuchal ligament and the posterior spinal dura at the atlanto-occipital and atlanto-axial intervals. No such attachments were found caudal to the arch of the axis. In addition, there was a connective tissue bridge between the deep aspect of the rectus capitis posterior minor muscle to the transverse fibers of the posterior atlanto-occipital membrane that extended laterally to blend with the perivascular tissue of the vertebral arteries. The present study is, we believe, the first to describe continuity between the nuchal ligament and the dura at the atlanto-occipital interspace, and confirms previous descriptions of similar connections at the atlanto-axial level. Knowledge of the exact attachments of the dura may contribute to understanding the biomechanics of the cervical spine and of the possible etiology of some types of cervicogenic headaches.

Nutritional support of the pediatric trauma patient: a practical approach.

It is mandatory to evaluate and develop a plan for nutritional support for all injured children who are hospitalized. Although most childre" will rapidly resume normal oral intake, more severely injured children should be started on parenteral or enteral nutrition as soon as possible after admission. The mode of delivery and composition of nutritional support differ depending on the clinical setting and can change during the recovery period. Development of an initial plan and modification of the plan depending on the child's response will most effectively meet the metabolic demands after injury.

Tumor uptake and elimination of 2',2'-difluoro-2'-deoxycytidine (gemcitabine) after deoxycytidine kinase gene transfer: correlation with in vivo tumor response.

PURPOSE: We hypothesized that tumor uptake and elimination of 2',2'-difluoro-2'-deoxycytidine/2',2'-difluoro-2'-deoxycytidine 5'-triphosphate (dFdCyd/dFdCTP) would be altered after dCK gene transfer and that this change would result in an enhanced cytotoxic effect. To test this hypothesis, we examined dFdCyd/dFdCTP uptake and clearance in HT-29 human colon carcinoma xenografts in nude mice by high-performance liquid chromatography (HPLC) and fluorine-19 magnetic resonance spectroscopy (F-19 MRS). EXPERIMENTAL DESIGN: HT-29 tumors were grown from cells infected with either the retroviral vector alone (LNPO-LacZ) or vector containing the dCK gene (LNPO-dCK). HPLC and F-19 MRS analyses were performed after a single 160 mg/kg i.p. injection of dFdCyd. Tumor response was determined in animals receiving a similar dosing schedule of dFdCyd. RESULTS: HPLC experiments revealed an increased tumor accumulation of dFdCTP in xenografts overexpressing dCK compared with wild-type controls (P < or = 0.05). dFdCTP in the dCK-infected tumors was easily identified at 24 h postinjection. Conversely, no dFdCTP could be detected in the control xenografts 14 h postinjection. Subsequent F-19 MRS experiments confirmed an altered uptake, revealing a 2.5-fold greater accumulation of dFdCyd/dFdCTP in the dCK xenografts. Whereas a modest tumor growth delay was observed in the wild-type tumors receiving dFdCyd, dCK xenografts demonstrated a marked tumor growth delay following treatment (P < or = 0.05). CONCLUSIONS: These data support the hypothesis that increased expression of dCK cDNA in HT-29 xenografts results in an enhanced dFdCTP accumulation and prolonged elimination kinetics, and ultimately a potentiated in vivo tumor response to dFdCyd. Related to these effects, changes in the overall tumor metabolism of dFdCyd/dFdCTP was detectable by noninvasive F-19 MRS. These data are relevant to future preclinical and clinical studies evaluating dCK gene transfer and dFdCyd therapy.

Innovative approaches to comprehensive cardiovascular disease risk reduction in clinical and community-based settings.

We have developed, tested, and successfully implemented an affordable, evidence-based, comprehensive cardiovascular disease risk-reduction program for use in primary and secondary prevention settings. The program is administered at hospitals, physician practices, cardiac rehabilitation programs, work sites, shopping malls, and health clubs. The program is also delivered from a call center using the telephone and the Internet. Program staff are guided by a computerized participant management and tracking system. Lifestyle management interventions are based on several behavior change models, primarily social learning theory, the stages of change model, and single concept learning theory. Typically, the program is administered entirely by non-physician healthcare professionals whose services are integrated with the care provided by the participants' physicians. Outcome data have documented the clinical effectiveness of this innovative approach.

Human cytosolic 5'-nucleotidase I: characterization and role in nucleoside analog resistance.

Nucleoside analogs are important in the treatment of hematologic malignancies, solid tumors, and viral infections. Their metabolism to the triphosphate form is central to their chemotherapeutic efficacy. Although the nucleoside kinases responsible for the phosphorylation of these compounds have been well described, the nucleotidases that may mediate drug resistance through dephosphorylation remain obscure. We have cloned and characterized a novel human cytosolic 5'-nucleotidase (cN-I) that potentially may have an important role in nucleoside analog metabolism. It is expressed at a high level in skeletal and heart muscle, at an intermediate level in pancreas and brain, and at a low level in kidney, testis, and uterus. The recombinant cN-I showed high affinity toward dCMP and lower affinity toward AMP and IMP. ADP was necessary for maximal catalytic activity. Expression of cN-I in Jurkat and HEK 293 cells conferred resistance to 2-chloro-2'-deoxyadenosine, with a 49-fold increase in the IC(50) in HEK 293 and a greater than 400-fold increase in the IC(50) in Jurkat cells. Expression of cN-I also conferred a 22-fold increase in the IC(50) to 2',3'-difluorodeoxycytidine in HEK 293 cells and an 82-fold increase in the IC(50) to 2',3'-dideoxycytidine in Jurkat cells. These data indicate that cN-I may play an important role in the regulation of physiological pyrimidine nucleotide pools and may also alter the therapeutic efficacy of certain nucleoside analogs.

Inhibition of T lymphocyte activation in mice heterozygous for loss of the IMPDH II gene.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides, which are also synthesized from guanine by a salvage reaction catalyzed by the X chromosome-linked enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). Since inhibitors of IMPDH are in clinical use as immunosuppressive agents, we have examined the consequences of knocking out the IMPDH type II enzyme by gene targeting in a mouse model. Loss of both alleles of the gene encoding this enzyme results in very early embryonic lethality despite the presence of IMPDH type I and HPRT activities. Lymphocytes from IMPDH II(+/-) heterozygous mice are normal with respect to subpopulation distribution and respond normally to a variety of mitogenic stimuli. However, mice with an IMPDH II(+/-), HPRT(-/o) genotype demonstrate significantly decreased lymphocyte responsiveness to stimulation with anti-CD3 and anti-CD28 antibodies and show a 30% mean reduction in GTP levels in lymphocytes activated by these antibodies. Furthermore, the cytolytic activity of their T cells against allogeneic target cells is significantly impaired. These results demonstrate that a moderate decrease in the ability of murine lymphocytes to synthesize guanine nucleotides during stimulation results in significant impairment in T-cell activation and function.

A modified diffuse reflectance infrared Fourier transform spectroscopy cell for depth profiling of ceramic fibers.

A modified diffused reflectance infrared Fourier transformed spectroscopy (DRIFTS) accessory was used to analyze the surface properties of alumino-silicate fibers. The modifications are simple and involve a different way of performing depth-profiling from traditional DRIFTS by removing approximately 2 mm of salt from a full cup prior to placing the sample in for depth profiling. This method proved successful in elucidating the effects of quenching alumino-silicate fibers in mineral oil versus quenching in an air stream.

Association of terminal deoxynucleotidyl transferase with Ku.

Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity. Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair. We provide evidence for a direct interaction between TdT and Ku proteins. Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer. The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins. The protein-protein interaction is DNA independent and occurs at physiological salt concentrations. Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer. This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair. The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize. The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.