Quantitative proteomics analysis of BMS‐214662 effects on CD34 positive cells from chronic myeloid leukaemia patients

Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell and is driven by the Bcr‐Abl oncoprotein. Abl kinase inhibitors (protein tyrosine kinase inhibitors) represent standard treatment for CML and induce remission in the majority of patients with early disease, however these drugs do not target leukaemic stem cells (LSCs) effectively, thus preventing cure. Previously, we identified the farnesyl transferase inhibitor BMS‐214662 as a selective inducer of apoptosis in LSCs of CML patients relative to normal controls; however, the mechanism underlying LSC‐specific apoptosis remains unclear. To identify pathways involved in the favourable effects of BMS‐214662 in CML, we employed a proteomic approach (based on iTRAQ) to analyse changes in protein expression in response to drug treatment in the nuclear and cytoplasmic fractions of CD34⁺ CML cells. The study identified 88 proteins as altered after drug treatment, which included proteins known to be involved in nucleic acid metabolism, oncogenesis, developmental processes and intracellular protein trafficking. We found that expression of Ebp1, a negative regulator of proliferation, was upregulated in the nucleus of BMS‐214662‐treated cells. Furthermore, proteins showing altered levels in the cytosol, such as histones, were predominantly derived from the nucleus and BMS‐214662 affected expression levels of nuclear pore complex proteins. Validation of key facets of these observations suggests that drug‐induced alterations in protein localisation, potentially via loss of nuclear membrane integrity, contributes to the LSC specificity of BMS‐214662, possibly via Ran proteins as targets.     

Treatment Response and Mortality among Patients Starting Antiretroviral Therapy with and without Kaposi Sarcoma: A Cohort Study

Background

Improved survival among HIV-infected individuals on antiretroviral therapy (ART) has focused attention on AIDS-related cancers including Kaposi sarcoma (KS). However, the effect of KS on response to ART is not well-described in Southern Africa. We assessed the effect of KS on survival and immunologic and virologic treatment responses at 6- and 12-months after initiation of ART.

Methods

We analyzed prospectively collected data from a cohort of HIV-infected adults initiating ART in South Africa. Differences in mortality between those with and without KS at ART initiation were estimated with Cox proportional hazard models. Log-binomial models were used to assess differences in CD4 count response and HIV virologic suppression within a year of initiating treatment.

Results

Between January 2001–January 2008, 13,847 HIV-infected adults initiated ART at the study clinics. Those with KS at ART initiation (n = 247, 2%) were similar to those without KS (n = 13600,98%) with respect to age (35 vs. 35yrs), presenting CD4 count (74 vs. 85cells/mm³) and proportion on TB treatment (37% vs. 30%). In models adjusted for sex, baseline CD4 count, age, treatment site, tuberculosis and year of ART initiation, KS patients were over three times more likely to have died at any time after ART initiation (hazard ratio[HR]: 3.62; 95% CI: 2.71–4.84) than those without KS. The increased risk was highest within the first year on ART (HR: 4.05; 95% CI: 2.95–5.55) and attenuated thereafter (HR: 2.30; 95% CI: 1.08–4.89). Those with KS also gained, on average, 29 fewer CD4 cells (95% CI: 7–52cells/mm³) and were less likely to increase their CD4 count by 50 cells from baseline (RR: 1.43; 95% CI: 0.99–2.06) within the first 6-months of treatment.

Conclusions

HIV-infected adults presenting with KS have increased risk of mortality even after initiation of ART with the greatest risk in the first year. Among those who survive the first year on therapy, subjects with KS demonstrated a poorer immunologic response to ART than those without KS.     

Genetic segregation of inflammatory lung disease and autoimmune disease severity in SHIP-1-/- mice

Alternatively activated M2 macrophages are implicated as both regulators and agents of lung disease, but their control is poorly understood. SHIP-1 is a 5' inositol phosphatase that negatively regulates the PI3K signaling pathway implicated in inflammation. SHIP-1-deficient mice have defects in hematopoiesis and B cell development, and die prematurely due to consolidation of lungs with M2-skewed macrophages. SHIP-1 is thought to restrain M2 macrophage polarization, with deregulated M2 skewing coinciding with severe lung disease in SHIP-1-deficient mice. To determine the influence of genetic background on the lung phenotype in SHIP-1(-/-) mice, we backcrossed the SHIP-1 null mutation onto C57BL/6 (Th2-resistant) and BALB/c (Th2-prone) backgrounds. Remarkably, we found that inflammatory lung disease was severe in C57.SHIP-1(-/-) mice, but absent in BALB.SHIP-1(-/-) mice. C57.SHIP-1(-/-), but not BALB.SHIP-1(-/-) mice had greatly increased myeloid progenitors, myeloid hyperplasia, markedly enhanced numbers of activated alveolar macrophages, and elevated amounts of Th2 and proinflammatory cytokines in bronchoalveolar lavage fluid and serum, suggesting that deregulated cytokine production induced disease. C57.SHIP-1(-/-) mice also developed severe B cell-dependent autoimmune disease, which was markedly attenuated on the BALB/c background. These data demonstrate that, contrary to current concepts, loss of SHIP-1 alone is not sufficient to cause lung inflammation, with disease only manifest on a permissive genetic background. This finding questions the nature of the lung disease in SHIP-1(-/-) mice, suggesting that its M2 classification is not strictly correct. Future identification of disease-promoting loci might reveal determinants of comorbid lung disease and autoimmunity and uncover potentially useful therapeutic targets.     

How can diagnostic assessment programs be implemented to enhance inter-professional collaborative care for cancer?

Background

Inter-professional collaborative care (ICC) for cancer leads to multiple system, organizational, professional, and patient benefits, but is limited by numerous challenges. Empirical research on interventions that promote or enable ICC is sparse so guidance on how to achieve ICC is lacking. Research shows that ICC for diagnosis could be improved. Diagnostic assessment programs (DAPs) appear to be a promising model for enabling ICC. The purpose of this study was to explore how DAP structure and function enable ICC, and whether that may be associated with organizational and clinical outcomes.

Methods

A case study approach will be used to explore ICC among eight DAPs that vary by type of cancer (lung, breast), academic status, and geographic region. To describe DAP function and outcomes, and gather information that will enable costing, recommendations expressed in DAP standards and clinical guidelines will be assessed through retrospective observational study. Data will be acquired from databases maintained by participating DAPs and the provincial cancer agency, and confirmed by and supplemented with review of medical records. We will conduct a pilot study to explore the feasibility of estimating the incremental cost-effectiveness ratio using person-level data from medical records and other sources. Interviews will be conducted with health professionals, staff, and referring physicians from each DAP to learn about barriers and facilitators of ICC. Qualitative methods based on a grounded approach will be used to guide sampling, data collection and analysis.

Discussion

Findings may reveal opportunities for unique structures, interventions or tools that enable ICC that could be developed, implemented, and evaluated through future research. This information will serve as a formative needs assessment to identify the nature of ongoing or required improvements, which can be directly used by our decision maker collaborators, and as a framework by policy makers, cancer system managers, and DAP managers elsewhere to strategically plan for and implement diagnostic cancer services.     

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.     

Four-Stage Audit Demonstrating Increased Uptake of HIV Testing in Acute Neurology Admissions Using Staged Practical Interventions

Background

UK National Guidelines (UKNG) advise HIV testing in clinically indicated neurological presentations. We audited the impact of our practical strategies to increase uptake of HIV testing at a regional acute neurology admissions unit.

Methods

We audited HIV testing in 4 periods over 2 years: before we designed a UKNG-based “HIV testing in Neurology” protocol (“pre-protocol”); after dissemination of the protocol alone (“post-protocol”); post-protocol dissemination combined with both a tailored departmental admissions clerking proforma to prompt for HIV testing & consenting, and regular focussed tutorials to doctors on HIV testing in neurological patients (“post-proforma”); and finally one year after the post-proforma period (“+1 year”). We also looked at the total number of HIV tests sent from the unit during the two-year period. We assessed significance using Fisher’s exact test.

Results

47.8% of all acute neurology non-stroke admissions were eligible for HIV testing during all the audit periods. Testing rates were as follows: pre-protocol 21.9%; post-protocol 36.6%; post-proforma 83.3%; and at +1 year 65.4% (p<0.05 for both post-protocol and +1 year when compared to pre-protocol). Documentation of consent for HIV testing improved from 25% to 67.6% with the HIV-tailored clerking proforma. The total number of HIV tests requested from the unit doubled in the post-proforma period compared to pre-protocol (p<0.05).

Conclusion

In conclusion: the combination of an HIV testing protocol, a tailored departmental clerking proforma and regular focussed teaching to doctors on indications for HIV testing led to a sustained increase in HIV testing uptake in our regional acute neurology admissions unit.     

Trait‐mediated indirect interactions in a marine intertidal system as quantified by functional responses

Studies of trait‐mediated indirect interactions (TMIIs) typically focus on effects higher predators have on per capita consumption by intermediate consumers of a third, basal prey resource. TMIIs are usually evidenced by changes in feeding rates of intermediate consumers and/or differences in densities of this third species. However, understanding and predicting effects of TMIIs on population stability of such basal species requires examination of the type and magnitude of the functional responses exhibited towards them. Here, in a marine intertidal system consisting of a higher‐order fish predator, the shanny Lipophrys pholis, an intermediate predator, the amphipod Echinogammarus marinus, and a basal prey resource, the isopod Jaera nordmanni, we detected TMIIs, demonstrating the importance of habitat complexity in such interactions, by deriving functional responses and exploring consequences for prey population stability. Echinogammarus marinus reacted to fish predator diet cues by reducing activity, a typical anti‐predator response, but did not alter habitat use. Basal prey, Jaera nordmanni, did not respond to fish diet cues with respect to activity, distribution or aggregation behaviour. Echinogammarus marinus exhibited type II functional responses towards J. nordmanni in simple habitat, but type III functional responses in complex habitat. However, while predator cue decreased the magnitude of the type II functional response in simple habitat, it increased the magnitude of the type III functional response in complex habitat. These findings indicate that, in simple habitats, TMIIs may drive down consumption rates within type II responses, however, this interaction may remain de‐stabilising for prey populations. Conversely, in complex habitats, TMIIs may strengthen regulatory influences of intermediate consumers on prey populations, whilst potentially maintaining prey population stability. We thus highlight that TMIIs can have unexpected and complex ramifications throughout communities, but can be unravelled by considering effects on intermediate predator functional response types and magnitudes. Synthesis Higher‐order predators and habitat complexity can influence behaviour of intermediate species, affecting their consumption of prey through trait‐mediated indirect interactions (TMIIs). However, it is not clear how these factors interact to determine prey population stability. Using functional responses (FRs), relating predator consumption to prey density, we detected TMIIs in a marine system. In simple habitats, TMIIs reduced consumption rates, but FRs remained de‐stabilising for prey populations. In complex habitats, TMIIs strengthened prey regulation with population stabilizing FRs. We thus demonstrate that FRs can assess interactions of environmental and biological cues that result in complex and unexpected outcomes for prey populations.     

Distinct macrophage subpopulations characterize acute infection and chronic inflammatory lung disease

Although great progress has been made in delineating lung dendritic cell and lymphocyte subpopulations, similar advances in lung macrophages (MΦs) have been hampered by their intrinsic autofluorescence, cell plasticity, and the complexities of monocyte-MΦ compartmentalization. Using spectral scanning, we define alveolar MΦ autofluorescence characteristics, which has allowed us to develop an alternative flow cytometry method. Using this methodology, we show that mouse lung MΦs form distinct subpopulations during acute inflammation after challenge with LPS or influenza virus, and in chronic inflammatory lung disease consequent to SHIP-1 deletion. These subpopulations are distinguished by differential Mac-1 and CD11c integrin expression rather than classical M1 or M2 markers, and display differential gene signatures ex vivo. Whereas the resolution of acute inflammation is characterized by restoration to a homogenous population of CD11c(high)Mac-1(neg/low) MΦs reflective of lung homeostasis, chronic inflammatory lung disease associated with SHIP-1 deficiency is accompanied by an additional subpopulation of CD11c(high)Mac-1(pos) MΦs that tracks with lung disease in susceptible genetic background SHIP-1(-/-) animals and disease induction in chimeric mice. These findings may help better understand the roles of MΦ subpopulations in lung homeostasis and disease.     

Dynamic life and death interactions between Mycobacterium smegmatis and J774 macrophages

After internalization into macrophages non-pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phago-lysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill 'non-pathogenic'Mycobacterium smegmatis. This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V-ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process.