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1.  A Phase 2 Trial of Ponatinib in Philadelphia Chromosome–Positive Leukemias 
The New England journal of medicine  2013;369(19):10.1056/NEJMoa1306494.
BACKGROUND
Ponatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor–refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome–positive acute lymphoblastic leukemia (Ph-positive ALL).
METHODS
We enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months.
RESULTS
Among 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3%. A total of 12% of patients discontinued treatment because of an adverse event.
CONCLUSIONS
Ponatinib had significant antileukemic activity across categories of disease stage and mutation status. (Funded by Ariad Pharmaceuticals and others; PACE ClinicalTrials.gov number, NCT01207440.)
doi:10.1056/NEJMoa1306494
PMCID: PMC3886799  PMID: 24180494
2.  Self-injurious behaviour: limbic dysregulation and stress effects in an animal model 
Journal of intellectual disability research : JIDR  2011;56(5):10.1111/j.1365-2788.2011.01485.x.
Background
Self-injurious behaviour (SIB) is prevalent in neurodevelopmental disorders, but its expression is highly variable within, and between diagnostic categories. This raises questions about the factors that contribute to aetiology and expression of SIB. Expression of SIB is generally described in relation to social reinforcement. However, variables that predispose vulnerability have not been as clearly characterised. This study reports the aetiology and expression of self-injury in an animal model of pemoline-induced SIB. It describes changes in gross neuronal activity in selected brain regions after chronic treatment with pemoline, and it describes the impact that a history of social defeat stress has on the subsequent expression of SIB during pemoline treatment.
Methods
Experiment 1
Male Long-Evans rats were injected on each of five consecutive days with pemoline or vehicle, and the expression of SIB was evaluated using a rating scale. The brains were harvested on the morning of the sixth day, and were assayed for expression of cytochrome oxidase, an index of sustained neuronal metabolic activity.
Experiment 2
Male Long-Evans rats were exposed to a regimen of 12 daily sessions of social defeat stress or 12 daily sessions of handling (i.e. controls). Starting on the day after completion of the social defeat or handling regimen, each rat was given five daily injections of pemoline. The durations of self-injurious oral contact and other stereotyped behaviours were monitored, and the areas of tissue injury were quantified.
Results
Experiment 1
Neuronal metabolic activity was significantly lower in a variety of limbic and limbic-associated brain structures in the pemoline-treated rats, when compared with activity in the same regions of vehicle-treated controls. In addition, neuronal activity was low in the caudate–putamen, and in subfields of the hypothalamus, but did not differ between groups for a variety of other brain regions, including nucleus accumbens, substantia nigra, ventral tegmentum, thalamus, amygdala, and cortical regions.
Experiment 2
All the pemoline-treated rats exhibited SIB, and whereas the social defeat regimen did not alter the total amount of self-injurious oral contact or other stereotyped behaviours, it significantly increased the severity of tissue injury.
Conclusions
A broad sampling of regional metabolic activity indicates that the pemoline regimen produces enduring changes that are localised to specific limbic, hypothalamic and striatal structures.The potential role of limbic function in aetiology of SIB is further supported by the finding that pemoline-induced self-injury is exacerbated by prior exposure to social defeat stress. Overall, the results suggest brain targets that should be investigated further, and increase our understanding of the putative role that stress plays in the pathophysiology of SIB.
doi:10.1111/j.1365-2788.2011.01485.x
PMCID: PMC3839243  PMID: 21988194
animal model; Lesch–Nyhan syndrome; pemoline; self-injury; social defeat; stress
3.  Population Genetics of Trypanosoma brucei rhodesiense: Clonality and Diversity within and between Foci 
African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda) and Southern (Malawi) Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics.
Author Summary
Trypanosomes are single-celled organisms transmitted by the biting tsetse fly, which cause sleeping sickness in humans in sub-Saharan Africa, but also infect livestock and other mammals. Most trypanosomes cannot infect humans as they die in human serum, but two mutants of Trypanosoma brucei have evolved the ability to survive in human serum. This survival in human serum is conferred by the presence of one gene in the East African human-infective T. b. rhodesiense. How often trypanosomes exchange genetic material (they can mate in the tsetse fly) is debated, but will impact upon the spread of genes (e.g. that which confers human infectivity) through a population. We studied T. b. rhodesiense populations from different geographic locations (Malawi and two locations in Uganda), and over time (Uganda), to see if the populations are stable over time and space, using a panel of variable genetic markers enabling assessment of diversity. Our results suggest that there is significant difference in diversity between locations; those in Uganda are very closely related, increasingly so over time, whereas the Malawi population is very genetically diverse, consistent with the trypanosomes mating. These findings suggest that a greater understanding of T. b. rhodesiense population evolution will inform on sleeping sickness epidemiology.
doi:10.1371/journal.pntd.0002526
PMCID: PMC3828156  PMID: 24244771
4.  Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs 
Objectives
Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs.
Methods
The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants.
Results
All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical.
Conclusions
TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.
doi:10.1093/jac/dkt442
PMCID: PMC3922157  PMID: 24235095
drug transport; protozoan; parasite; resistance mutation; aquaporin
5.  Human and Animal Trypanosomes in Côte d'Ivoire Form a Single Breeding Population 
PLoS ONE  2013;8(7):e67852.
Background
Trypanosoma brucei is the causative agent of African Sleeping Sickness in humans and contributes to the related veterinary disease, Nagana. T. brucei is segregated into three subspecies based on host specificity, geography and pathology. T. b. brucei is limited to animals (excluding some primates) throughout sub-Saharan Africa and is non-infective to humans due to trypanolytic factors found in human serum. T. b. gambiense and T. b. rhodesiense are human infective sub-species. T. b. gambiense is the more prevalent human, causing over 97% of human cases. Study of T. b. gambiense is complicated in that there are two distinct groups delineated by genetics and phenotype. The relationships between the two groups and local T. b. brucei are unclear and may have a bearing on the evolution of the human infectivity traits.
Methodology/Principal Findings
A collection of sympatric T. brucei isolates from Côte d’Ivoire, consisting of T. b. brucei and both groups of T. b. gambiense have previously been categorized by isoenzymes, RFLPs and Blood Incubation Infectivity Tests. These samples were further characterized using the group 1 specific marker, TgSGP, and seven microsatellites. The relationships between the T. b. brucei and T. b. gambiense isolates were determined using principal components analysis, neighbor-joining phylogenetics, STRUCTURE, FST, Hardy-Weinberg equilibrium and linkage disequilibrium.
Conclusions/Significance
Group 1 T. b. gambiense form a clonal genetic group, distinct from group 2 and T. b. brucei, whereas group 2 T. b. gambiense are genetically indistinguishable from local T. b. brucei. There is strong evidence for mating within and between group 2 T. b. gambiense and T. b. brucei. We found no evidence to support the hypothesis that group 2 T. b. gambiense are hybrids of group 1 and T. b. brucei, suggesting that human infectivity has evolved independently in groups 1 and 2 T. b. gambiense.
doi:10.1371/journal.pone.0067852
PMCID: PMC3699513  PMID: 23844111
7.  Differences between Trypanosoma brucei gambiense Groups 1 and 2 in Their Resistance to Killing by Trypanolytic Factor 1 
Background
The three sub-species of Trypanosoma brucei are important pathogens of sub-Saharan Africa. T. b. brucei is unable to infect humans due to sensitivity to trypanosome lytic factors (TLF) 1 and 2 found in human serum. T. b. rhodesiense and T. b. gambiense are able to resist lysis by TLF. There are two distinct sub-groups of T. b. gambiense that differ genetically and by human serum resistance phenotypes. Group 1 T. b. gambiense have an invariant phenotype whereas group 2 show variable resistance. Previous data indicated that group 1 T. b. gambiense are resistant to TLF-1 due in-part to reduced uptake of TLF-1 mediated by reduced expression of the TLF-1 receptor (the haptoglobin-hemoglobin receptor (HpHbR)) gene. Here we investigate if this is also true in group 2 parasites.
Methodology
Isogenic resistant and sensitive group 2 T. b. gambiense were derived and compared to other T. brucei parasites. Both resistant and sensitive lines express the HpHbR gene at similar levels and internalized fluorescently labeled TLF-1 similar fashion to T. b. brucei. Both resistant and sensitive group 2, as well as group 1 T. b. gambiense, internalize recombinant APOL1, but only sensitive group 2 parasites are lysed.
Conclusions
Our data indicate that, despite group 1 T. b. gambiense avoiding TLF-1, it is resistant to the main lytic component, APOL1. Similarly group 2 T. b. gambiense is innately resistant to APOL1, which could be based on the same mechanism. However, group 2 T. b. gambiense variably displays this phenotype and expression does not appear to correlate with a change in expression site or expression of HpHbR. Thus there are differences in the mechanism of human serum resistance between T. b. gambiense groups 1 and 2.
Author Summary
The sub-species of Trypanosoma brucei are important pathogens of humans and animals in sub-Saharan Africa. T. b. brucei is not able to infect humans due to sensitivity to trypanosome lytic factors (TLF) containing the lytic protein apolipoprotein L1 (APOL1). T. b. gambiense is the most prevalent human infective sub-species, although there are two distinct sub-groups of T. b. gambiense that differ genetically and in terms of their human serum resistance phenotype stability. How they resist lysis by TLF is unknown. Previous data indicates that some group 1 T. b. gambiense parasites are able to avoid lysis due in part to reduced expression and activity of the gene encoding a TLF receptor (HpHbR). The authors showed that despite group 1 T. b gambiense displaying avoidance of some TLF particles, this sub-species group is resistant to the lytic protein of TLF, APOL1. This suggests that avoidance is not the complete story in this sub-species, as previously theorised. Group 2 T. b. gambiense is also innately resistant to APOL1 and there is no evidence of avoidance of TLF in resistant parasites. Group 2 T. b. gambiense variably displays the human serum resistance phenotype that does not appear to correlate with expression of HbHpR.
doi:10.1371/journal.pntd.0001287
PMCID: PMC3167774  PMID: 21909441
8.  Heritability of Lumbar Trabecular Bone Mechanical Properties in Baboons 
Bone  2009;46(3):835-840.
Genetic effects on mechanical properties have been demonstrated in rodents, but not confirmed in primates. Our aim was to quantify the proportion of variation in vertebral trabecular bone mechanical properties that is due to the effects of genes. L3 vertebrae were collected from 110 females and 46 male baboons (6–32 years old) from a single extended pedigree. Cranio-caudally oriented trabecular bone cores were scanned with microCT then tested in monotonic compression to determine apparent ultimate stress, modulus, and toughness. Age and sex effects and heritability (h2) were assessed using maximum likelihood-based variance components methods. Additive effects of genes on residual trait variance were significant for ultimate stress (h2=0.58), toughness (h2=0.64), and BV/TV (h2=0.55). When BV/TV was accounted for, the residual variance in ultimate stress accounted for by the additive effects of genes was no longer significant. Toughness, however, showed evidence of a non-BV/TV-related genetic effect. Overall, maximum stress and modulus show strong genetic effects that are nearly entirely due to bone volume. Toughness shows strong genetic effects related to bone volume and shows additional genetic effects (accounting for 10% of the total trait variance) that are independent of bone volume. These results support continued use of bone volume as a focal trait to identify genes related to skeletal fragility, but also show that other focal traits related to toughness and variation in the organic component of bone matrix will enhance our ability to find additional genes that are particularly relevant to fatigue-related fractures.
doi:10.1016/j.bone.2009.11.002
PMCID: PMC3005696  PMID: 19900599
9.  Rapamycin impairs trabecular bone acquisition from high-dose but not low-dose intermittent parathyroid hormone treatment 
Journal of cellular physiology  2009;221(3):579-585.
The osteo-anabolic effects of intermittent parathyroid hormone (PTH) treatment require insulin-like growth factor (IGF) signaling through the IGF-I receptor. A major downstream target of the IGF-I receptor (via Akt) is the mammalian target of rapamycin (mTOR), a kinase involved in protein synthesis. We investigated whether the bone-building effects of intermittent PTH require functional mTOR signaling. Mice were treated with daily PTH 1-34 (0, 10, 30, or 90 μg/kg) for 6 weeks in the presence or absence of rapamycin, a selective inhibitor of mTOR. We found that all PTH doses were effective in enhancing bone mass, whether rapamycin was present or not. Rapamycin had little to no effect on the anabolic response at low (10 μg) PTH doses, small effects in a minority of anabolic measures at moderate doses (30 μg), but the anabolic effects of high dose PTH (90 μg) were consistently and significantly suppressed by rapamycin (~4-36% reduction). Serum levels of Trap5b, a marker of resorption, were significantly enhanced by rapamycin, but these effects were observed whether PTH was absent or present. Our data suggest that intermittent PTH, particularly at lower doses, is effective in building bone mass in the presence of rapamycin. However, the full anabolic effects of higher doses of PTH are significantly suppressed by rapamycin, suggesting that PTH might normally activate additional pathways (including mTOR) for its enhanced high-dose anabolic effects. Clinical doses of intermittent PTH could be an effective treatment for maintaining or increasing bone mass among patients taking rapamycin analogs for unrelated health issues.
doi:10.1002/jcp.21887
PMCID: PMC2755650  PMID: 19639601
parathyroid hormone; mTOR; bone anabolism; Akt; osteoporosis
10.  Role for Parasite Genetic Diversity in Differential Host Responses to Trypanosoma brucei Infection ▿ ‡  
Infection and Immunity  2010;78(3):1096-1108.
The postgenomic era has revolutionized approaches to defining host-pathogen interactions and the investigation of the influence of genetic variation in either protagonist upon infection outcome. We analyzed pathology induced by infection with two genetically distinct Trypanosoma brucei strains and found that pathogenesis is partly strain specific, involving distinct host mechanisms. Infections of BALB/c mice with one strain (927) resulted in more severe anemia and greater erythropoietin production compared to infections with the second strain (247), which, contrastingly, produced greater splenomegaly and reticulocytosis. Plasma interleukin-10 (IL-10) and gamma interferon levels were significantly higher in strain 927-infected mice, whereas IL-12 was higher in strain 247-infected mice. To define mechanisms underlying these differences, expression microarray analysis of host genes in the spleen at day 10 postinfection was undertaken. Rank product analysis (RPA) showed that 40% of the significantly differentially expressed genes were specific to infection with one or the other trypanosome strain. RPA and pathway analysis identified LXR/RXR signaling, IL-10 signaling, and alternative macrophage activation as the most significantly differentially activated host processes. These data suggest that innate immune response modulation is a key determinant in trypanosome infections, the pattern of which can vary, dependent upon the trypanosome strain. This strongly suggests that a parasite genetic component is responsible for causing disease in the host. Our understanding of trypanosome infections is largely based on studies involving single parasite strains, and our results suggest that an integrated host-parasite approach is required for future studies on trypanosome pathogenesis. Furthermore, it is necessary to incorporate parasite variation into both experimental systems and models of pathogenesis.
doi:10.1128/IAI.00943-09
PMCID: PMC2825911  PMID: 20086091
11.  Epigenotype–phenotype correlations in Silver–Russell syndrome 
Journal of Medical Genetics  2010;47(11):760-768.
Background
Silver–Russell syndrome (SRS) is characterised by intrauterine growth restriction, poor postnatal growth, relative macrocephaly, triangular face and asymmetry. Maternal uniparental disomy (mUPD) of chromosome 7 and hypomethylation of the imprinting control region (ICR) 1 on chromosome 11p15 are found in 5–10% and up to 60% of patients with SRS, respectively. As many features are non-specific, diagnosis of SRS remains difficult. Studies of patients in whom the molecular diagnosis is confirmed therefore provide valuable clinical information on the condition.
Methods
A detailed, prospective study of 64 patients with mUPD7 (n=20) or ICR1 hypomethylation (n=44) was undertaken.
Results and conclusions
The considerable overlap in clinical phenotype makes it difficult to distinguish these two molecular subgroups reliably. ICR1 hypomethylation was more likely to be scored as ‘classical’ SRS. Asymmetry, fifth finger clinodactyly and congenital anomalies were more commonly seen with ICR1 hypomethylation, whereas learning difficulties and referral for speech therapy were more likely with mUPD7. Myoclonus-dystonia has been reported previously in one mUPD7 patient. The authors report mild movement disorders in three further cases. No correlation was found between clinical severity and level of ICR1 hypomethylation. Use of assisted reproductive technology in association with ICR1 hypomethylation seems increased compared with the general population. ICR1 hypomethylation was also observed in affected siblings, although recurrence risk remains low in the majority of cases. Overall, a wide range of severity was observed, particularly with ICR1 hypomethylation. A low threshold for investigation of patients with features suggestive, but not typical, of SRS is therefore recommended.
doi:10.1136/jmg.2010.079111
PMCID: PMC2976034  PMID: 20685669
Silver Russell syndrome; mUPD7; 11p15 hypomethylation; phenotype; imprinting; clinical genetics
12.  Multiple Genetic Mechanisms Lead to Loss of Functional TbAT1 Expression in Drug-Resistant Trypanosomes ▿  
Eukaryotic Cell  2010;9(2):336-343.
The P2 aminopurine transporter, encoded by TbAT1 in African trypanosomes in the Trypanosoma brucei group, carries melaminophenyl arsenical and diamidine drugs into these parasites. Loss of this transporter contributes to drug resistance. We identified the genomic location of TbAT1 to be in the subtelomeric region of chromosome 5 and determined the status of the TbAT1 gene in two trypanosome lines selected for resistance to the melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), and in a Trypanosoma equiperdum clone selected for resistance to the diamidine, diminazene aceturate. In the Trypanosoma brucei gambiense STIB 386 melarsamine hydrochloride-resistant line, TbAT1 is deleted, while in the Trypanosoma brucei brucei STIB 247 melarsamine hydrochloride-resistant and T. equiperdum diminazene-resistant lines, TbAT1 is present, but expression at the RNA level is no longer detectable. Further characterization of TbAT1 in T. equiperdum revealed that a loss of heterozygosity at the TbAT1 locus accompanied loss of expression and that P2-mediated uptake of [3H]diminazene is lost in drug-resistant T. equiperdum. Adenine-inhibitable adenosine uptake is still detectable in a ΔTbat1 T. b. brucei mutant, although at a greatly reduced capacity compared to that of the wild type, indicating that an additional adenine-inhibitable adenosine permease, distinct from P2, is present in these cells.
doi:10.1128/EC.00200-09
PMCID: PMC2823006  PMID: 19966032
13.  P2X7 receptor at the heart of disease 
Hippokratia  2010;14(3):155-163.
Purinergic signaling is a crucial component of disease whose pathophysiological basis is now well established. This review focuses on P2X7, a unique bifunctional purinoreceptor that either opens a non selective cation channel or forms a large, cytolytic pore depending on agonist application and leading to membrane blebbing and to cell death either by necrosis or apoptosis.
Activation of P2X7 receptor has been shown to stimulate the release of multiple proinflammatory cytokines by activated macrophages, with the IL-1b to be the most extensively studied among them. These findings were verified by the use of knockout P2X7 (-/-) mice.
Update information coming from all fields of research implicate this receptor at the very heart of diseases such as rheumatoid arthritis, multiple sclerosis, depression, Alzheimer disease, and to kidney damage, in renal fibrosis and experimental nephritis.
Clinical studies are currently underway with the newly developed selective antagonists for P2X7 receptor, the results of which are eagerly anticipated. These studies together with data from in-vivo experiments with the P2X7 knockout mice and in-vitro experiments will shed light in this exciting area.
PMCID: PMC2943352  PMID: 20981163
P2X7; NLRP3 inflammasome; IL-1b ATP; purinergic signaling; purinergic receptors; review
14.  A Major Genetic Locus in Trypanosoma brucei Is a Determinant of Host Pathology 
The progression and variation of pathology during infections can be due to components from both host or pathogen, and/or the interaction between them. The influence of host genetic variation on disease pathology during infections with trypanosomes has been well studied in recent years, but the role of parasite genetic variation has not been extensively studied. We have shown that there is parasite strain-specific variation in the level of splenomegaly and hepatomegaly in infected mice and used a forward genetic approach to identify the parasite loci that determine this variation. This approach allowed us to dissect and identify the parasite loci that determine the complex phenotypes induced by infection. Using the available trypanosome genetic map, a major quantitative trait locus (QTL) was identified on T. brucei chromosome 3 (LOD = 7.2) that accounted for approximately two thirds of the variance observed in each of two correlated phenotypes, splenomegaly and hepatomegaly, in the infected mice (named TbOrg1). In addition, a second locus was identified that contributed to splenomegaly, hepatomegaly and reticulocytosis (TbOrg2). This is the first use of quantitative trait locus mapping in a diploid protozoan and shows that there are trypanosome genes that directly contribute to the progression of pathology during infections and, therefore, that parasite genetic variation can be a critical factor in disease outcome. The identification of parasite loci is a first step towards identifying the genes that are responsible for these important traits and shows the power of genetic analysis as a tool for dissecting complex quantitative phenotypic traits.
Author Summary
Trypanosomes are single-celled organisms that are transmitted between animal hosts by the tsetse fly. These parasites infect a wide range of mammals and in sub-Saharan Africa are extensively debilitating to livestock, and some species are also able to infect humans causing a disease, sleeping sickness, that is usually fatal unless treated. Some trypanosome strains cause more severe disease than others, and studying these differences may allow the identification of how serious disease is caused. We approached this problem by looking at how differences in disease symptoms (enlarged spleen and liver, and reduced blood cell numbers) that are caused in infections in mice with two strains of Trypanosoma brucei, TREU927 and STIB247. These disease manifestations are clinically relevant in human and livestock trypanosome infections. Examining how the symptoms are inherited in infections with offspring of a cross between the two strains allowed the identification of a region of the T. brucei genome that contains a gene (or several genes) that contributes significantly towards the enlarged spleen and liver observed in infected mice. This is a first step towards identifying the parasite genes that cause disease in the host (virulence factors), which may provide routes for developing novel therapies against the disease.
doi:10.1371/journal.pntd.0000557
PMCID: PMC2780326  PMID: 19956590
15.  SIGNAL TRANSDUCTION AND GENE EXPRESSION IN CULTURED ACCESSORY OLFACTORY BULB NEURONS 
Neuroscience  2008;157(2):340-348.
Glutamate and norepinephrine (NE) are believed to mediate the long-lasting synaptic plasticity in the accessory olfactory bulb (AOB) that underlies pheromone recognition memory. The mechanisms by which these neurotransmitters bring about the synaptic changes are not clearly understood. In order to study signals that mediate synaptic plasticity in the AOB, we used AOB neurons in primary culture as a model system. Because induction of pheromone memory requires coincident glutamatergic and noradrenergic input to the AOB, and requires new protein synthesis, we reasoned that glutamate and NE must induce gene expression in the AOB. We used a combination of agonists that stimulate α-1 and α-2 adrenergic receptors in combination with NMDA and tested expression of the immediate-early gene c-Fos. We found that the glutamatergic and noradrenergic stimulation caused significant induction of c-Fos mRNA and protein. Induction of c-Fos was significantly reduced in the presence of inhibitors of protein kinase C, MAP kinase and phospholipase C. These results suggest that glutamate and NE induce gene expression in the AOB through a signaling pathway mediated by PKC and MAPK.
doi:10.1016/j.neuroscience.2008.09.016
PMCID: PMC2615302  PMID: 18848604
glutamate; norepinephrine; protein kinase C; MAP kinase; immediate-early gene; c-Fos
16.  Inflammatory Lung Injury After Cardiopulmonary Bypass is Attenuated by Adenosine A2A Receptor Activation 
Objectives
Cardiopulmonary bypass has been shown to exert an inflammatory response within the lung, often resulting in postoperative pulmonary dysfunction. Several studies have shown that adenosine A2A receptor (A2AR) activation attenuates lung ischemia-reperfusion injury, however the effect of A2AR activation on cardiopulmonary bypass-induced lung injury has not been studied. We hypothesized that specific A2AR activation by ATL313 would attenuate inflammatory lung injury following cardiopulmonary bypass.
Methods
Adult male Sprague-Dawley rats were randomly divided into three groups: 1) SHAM group (underwent cannulation+heparinization only); 2) CONTROL group (underwent 90-minutes of normothermic cardiopulmonary bypass with normal whole-blood priming solution; 3) ATL group (underwent 90-minutes of normothermic cardiopulmonary bypass with ATL313 added to the normal priming solution).
Results
There was significantly less pulmonary edema and lung injury in the ATL group compared to the CONTROL group. The ATL group had significant reductions in bronchoalveolar lavage interleukin-1, interleukin-6, interferon-γ and myeloperoxidase levels compared to the CONTROL group. Similarly, lung tissue interleukin-6, tumor necrosis factor-α, and interferon-γ were significantly decreased in the ATL group compared to the CONTROL group. There was no significant difference between the SHAM and ATL groups in the amount of pulmonary edema, lung injury, or levels of pro-inflammatory cytokines.
Conclusions
The addition of a potent A2AR agonist to the normal priming solution prior to the initiation of CPB significantly protects the lung from the inflammatory effects of CPB and reduces the amount of lung injury. A2AR agonists could represent a new therapeutic strategy for reducing the potentially devastating consequences of the inflammatory response associated with CPB.
Ultra-mini Abstract
Pharmacologic activation of the adenosine A2A receptor during cardiopulmonary bypass resulted in substantial decreases in pulmonary pro-inflammatory cytokines as well as clinically significant reductions in both postoperative pulmonary edema and lung injury severity in a rat model of cardiopulmonary bypass.
doi:10.1016/j.jtcvs.2008.07.010
PMCID: PMC2652167  PMID: 19026816
17.  Teaching and learning in clinical settings 
Archives of Disease in Childhood  2006;91(10):874-875.
doi:10.1136/adc.2006.100263
PMCID: PMC2066012
18.  Timing of Stroke After Cardiopulmonary Bypass Determines Mortality 
The Annals of thoracic surgery  2008;85(5):1556-1563.
Background
Stroke is an important complication of cardiopulmonary bypass (CPB). This study determined if the timing of stroke events after CPB predicted stroke-related mortality or rehabilitation needs at hospital discharge.
Methods
We performed a retrospective review of 7201 consecutive cardiac surgical patients during a 10-year period and identified 202 strokes. Postoperative stroke after CPB was classified as early (≤ 24 hours) or late (> 24 hours). Data were collected on patient characteristics, intraoperative variables and outcomes, postoperative course, stroke severity, and discharge status, including death from stroke. Logistic regression analysis was used to assess the relationship between the timing of stroke and discharge status after adjusting for clinically relevant factors.
Results
The stroke incidence was 2.8%. Postoperative strokes occurred within 24 hours in 22.8% (46 of 202) and after 24 hours in 77.2% (156 of 202). Factors found in logistic regression analysis to be independently associated with stroke-related death included stroke within 24 hours postoperatively (odds ratio [OR], 9.16; p < 0.0001), preoperative chronic renal insufficiency (OR, 4.46; p = 0.01), and National Institute of Health Stroke Scale (NIHSS) score (OR, 1.16 per NIHSS point increase; p < 0.0001). Among survivors, early stroke was associated with greater rehabilitation needs (p < 0.001).
Conclusions
Early stroke after CPB is independently associated with higher stroke-related death and is associated with increased need for skilled rehabilitation at discharge. Neuroprotective strategies aimed at reducing early postoperative stroke may positively impact death and neurologic disability after CPB.
doi:10.1016/j.athoracsur.2008.02.035
PMCID: PMC2749222  PMID: 18442537
19.  Discovery of Mating in the Major African Livestock Pathogen Trypanosoma congolense 
PLoS ONE  2009;4(5):e5564.
The protozoan parasite, Trypanosoma congolense, is one of the most economically important pathogens of livestock in Africa and, through its impact on cattle health and productivity, has a significant effect on human health and well being. Despite the importance of this parasite our knowledge of some of the fundamental biological processes is limited. For example, it is unknown whether mating takes place. In this paper we have taken a population genetics based approach to address this question. The availability of genome sequence of the parasite allowed us to identify polymorphic microsatellite markers, which were used to genotype T. congolense isolates from livestock in a discrete geographical area of The Gambia. The data showed a high level of diversity with a large number of distinct genotypes, but a deficit in heterozygotes. Further analysis identified cryptic genetic subdivision into four sub-populations. In one of these, parasite genotypic diversity could only be explained by the occurrence of frequent mating in T. congolense. These data are completely inconsistent with previous suggestions that the parasite expands asexually in the absence of mating. The discovery of mating in this species of trypanosome has significant consequences for the spread of critical traits, such as drug resistance, as well as for fundamental aspects of the biology and epidemiology of this neglected but economically important pathogen.
doi:10.1371/journal.pone.0005564
PMCID: PMC2679202  PMID: 19440370
20.  The genome of the simian and human malaria parasite Plasmodium knowlesi 
Nature  2008;455(7214):799-803.
Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax​4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6-8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.
doi:10.1038/nature07306
PMCID: PMC2656934  PMID: 18843368
21.  Audio computer assisted interviewing to measure HIV risk behaviours in a clinic population 
Sexually Transmitted Infections  2005;81(6):501-507.
Methods: 1350 clinic patients were assigned to complete a detailed behavioural survey on sexual risk practices, previous STIs and symptoms, condom use, and drug and alcohol use using either ACASI or IAQ.
Results: Respondents assigned to ACASI were more likely to report recent risk behaviours such as sex without a condom in the past 24 hours (adjusted OR = 1.9), anal sex (adjusted OR = 2.0), and one or more new partners in the past 6 months (adjusted OR = 1.5) compared to those interviewed by IAQ. The impact of ACASI varied by sex but, contrary to expectations, not by whether the patient had previously visited an STI clinic. Mode of survey administration made little difference within this population in reports of STI knowledge, previous STIs, STI symptoms, or illicit drug use.
Conclusion: ACASI provides a useful tool for improving the quality of behavioural data in clinical environments.
doi:10.1136/sti.2004.014266
PMCID: PMC1745071  PMID: 16326855
22.  Metabolic syndrome and risk of coronary heart disease in a Pakistani cohort 
Heart  2005;91(8):1003-1007.
Objective: To assess the relation of the metabolic insulin resistance syndrome (M-IRS) with coronary heart disease (CHD) in Pakistani patients.
Subjects: 200 patients with angiographic disease (CHD(+)) matched with 200 patients with chest pain without occlusive disease (CHD(−)).
Design: Prospective case–control study.
Setting: Tertiary referral cardiology unit in Pakistan.
Results: M-IRS was present in 37% of CHD(+) versus 27% of CHD(−) patients by criteria for white patients or 47% versus 42%, respectively, by Asian criteria (p < 0.001). After adjustment for other risk factors, M-IRS was not a significant predictor for CHD or angiographic disease. Age (p  =  0.03), smoking (p < 0.001), diabetes-years (p = 0.003), sialic acid (p  =  0.01), and creatinine (p  =  0.008) accounted for the excess risk of CHD. Similarly, age (p  =  0.005), creatinine (p < 0.001), cigarette pack-years (p  =  0.02), diabetes-years (p  =  0.003), and sialic acid (p  =  0.08) were predictors of greater angiographic disease. M-IRS differed between Pakistani and white patients, as waist circumference correlated weakly (r  =  −0.03–0.08, p  =  0.45–0.52) with triglycerides, high density lipoprotein cholesterol, systolic blood pressure, or glucose. Sialic acid was the only inflammatory marker associated with M-IRS.
Conclusions: Despite strong associations between individual risk factors associated with M-IRS and a univariate association between M-IRS and CHD in native Pakistanis, the principal discriminant risk factors in this group are age, smoking, inflammation, diabetes-years, and impaired renal function. The poor sensitivity of M-IRS for CHD reflects the high underlying prevalence of M-IRS, thus reducing sensitivity, confounding by other urban lifestyle traits, or a lack of association of waist circumference with M-IRS risk factors. The definition of M-IRS may have to be revised to increase its power as a discriminant risk factor for CHD in Pakistani populations.
doi:10.1136/hrt.2003.033092
PMCID: PMC1769029  PMID: 16020583
coronary heart disease; Indian Asian; insulin resistance; metabolic syndrome; risk
23.  The Role of CXCR2 in Cigarette Smoke-Induced Lung Inflammation 
It has been hypothesized that the destruction of lung tissue observed in smokers with chronic obstructive pulmonary disease and emphysema is mediated by neutrophils recruited to the lungs by smoke exposure. This study investigated the role of the chemokine receptor CXCR2 in mediating neutrophilic inflammation in the lungs of mice acutely exposed to cigarette smoke. Exposure to dilute mainstream cigarette smoke for 1 hour, twice per day for 3 days induced acute inflammation in the lungs of C57BL/6 mice, with increased neutrophils and neutrophil chemotactic CXC chemokines MIP-2 and KC. Treatment with SCH-N, an orally active small molecule inhibitor of CXCR2, reduced the influx of neutrophils into the bronchoalveolar lavage (BAL) fluid. Histologic changes were seen, with drug treatment reducing perivascular inflammation and the number of tissue neutrophils. β-glucuronidase activity was reduced in the BAL fluid of mice treated with SCH-N, indicating that the reduction in neutrophils was associated with a reduction in tissue damaging enzymes. Interestingly, while MIP-2 and KC were significantly elevated in the BAL fluid of smoke exposed mice, they were further elevated in mice exposed to smoke and treated with drug. The increase in MIP-2 and KC with drug treatment may be due to the decrease in lung neutrophils which either are not present to bind these chemokines or which fail to provide a feedback signal to other cells that produce these chemokines. Overall, these results demonstrate that inhibiting CXCR2 reduces neutrophilic inflammation and associated lung tissue damage due to acute cigarette smoke exposure.
doi:10.1152/ajplung.00039.2005
PMCID: PMC2491909  PMID: 15833762
neutrophil chemokines; emphysema; COPD; MIP-2; KC
24.  Genetic analysis of the human infective trypanosome Trypanosoma brucei gambiense: chromosomal segregation, crossing over, and the construction of a genetic map 
Genome Biology  2008;9(6):R103.
A high-resolution genetic linkage map of the STIB 386 strain of Trypanosoma brucei gambiense is presented.
Background
Trypanosoma brucei is the causative agent of human sleeping sickness and animal trypanosomiasis in sub-Saharan Africa, and it has been subdivided into three subspecies: Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense, which cause sleeping sickness in humans, and the nonhuman infective Trypanosoma brucei brucei. T. b. gambiense is the most clinically relevant subspecies, being responsible for more than 90% of all trypanosomal disease in humans. The genome sequence is now available, and a Mendelian genetic system has been demonstrated in T. brucei, facilitating genetic analysis in this diploid protozoan parasite. As an essential step toward identifying loci that determine important traits in the human-infective subspecies, we report the construction of a high-resolution genetic map of the STIB 386 strain of T. b. gambiense.
Results
The genetic map was determined using 119 microsatellite markers assigned to the 11 megabase chromosomes. The total genetic map length of the linkage groups was 733.1 cM, covering a physical distance of 17.9 megabases with an average map unit size of 24 kilobases/cM. Forty-seven markers in this map were also used in a genetic map of the nonhuman infective T. b. brucei subspecies, permitting comparison of the two maps and showing that synteny is conserved between the two subspecies.
Conclusion
The genetic linkage map presented here is the first available for the human-infective trypanosome T. b. gambiense. In combination with the genome sequence, this opens up the possibility of using genetic analysis to identify the loci responsible for T. b. gambiense specific traits such as human infectivity as well as comparative studies of parasite field populations.
doi:10.1186/gb-2008-9-6-r103
PMCID: PMC2481433  PMID: 18570680
25.  Monitoring STI prevalence using telephone surveys and mailed urine specimens: a pilot test 
Sexually Transmitted Infections  2005;81(3):236-238.
Objectives: This pilot test assessed the feasibility of a cost effective population based approach to STI monitoring using automated telephone interviews, urine specimen collection kits sent out and returned by US Postal Service mail, and monetary incentives to motivate participation.
Methods: 100 residents of Baltimore, MD, USA, completed an automated telephone survey and agreed to mail in a urine specimen to be tested for chlamydia and gonorrhoea. Participants were paid $10 for completing the survey and $40 for mailing the specimen.
Results: 86% of survey participants mailed in a urine specimen for testing.
Conclusions: Automated telephone surveys linked with testing of mailed-in urine specimens may be a feasible lower cost (relative to household surveys) method of estimating infection prevalences in a population.
doi:10.1136/sti.2004.011080
PMCID: PMC1744988  PMID: 15923293

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