The long road to elimination: malaria mortality in a South African population cohort over 21 years.

BACKGROUND: Malaria elimination is on global agendas following successful transmission reductions. Nevertheless moving from low to zero transmission is challenging. South Africa has an elimination target of 2018, which may or may not be realised in its hypoendemic areas. METHODS: The Agincourt Health and Demographic Surveillance System has monitored population health in north-eastern South Africa since 1992. Malaria deaths were analysed against individual factors, socioeconomic status, labour migration and weather over a 21-year period, eliciting trends over time and associations with covariates. RESULTS: Of 13 251 registered deaths over 1.58 million person-years, 1.2% were attributed to malaria. Malaria mortality rates increased from 1992 to 2013, while mean daily maximum temperature rose by 1.5 °C. Travel to endemic Mozambique became easier, and malaria mortality increased in higher socioeconomic groups. Overall, malaria mortality was significantly associated with age, socioeconomic status, labour migration and employment, yearly rainfall and higher rainfall/temperature shortly before death. CONCLUSIONS: Malaria persists as a small but important cause of death in this semi-rural South African population. Detailed longitudinal population data were crucial for these analyses. The findings highlight practical political, socioeconomic and environmental difficulties that may also be encountered elsewhere in moving from low-transmission scenarios to malaria elimination.

The development of a localised HIV epidemic and the associated excess mortality burden in a rural area of South Africa.

The human immunodeficiency virus (HIV) epidemic in South Africa rapidly developed into a major pandemic. Here we analyse the development of the epidemic in a rural area of the country. The data used were collected between 1992 and 2013 in a longitudinal population survey, the Agincourt Health and Demographic Surveillance Study, in the northeast of the country. Throughout the period of study mortality rates were similar in all villages, suggesting that there were multiple index cases evenly spread geographically. These were likely to have been returning migrant workers. For those aged below 39 years the HIV mortality rate was higher for women, above this age it was higher for men. This indicates the protective effect of greater access to HIV testing and treatment among older women. The recent convergence of mortality rates for Mozambicans and South Africans indicates that the former refugee population are being assimilated into the host community. More than 60% of the deaths occurring in this community between 1992 and 2013 could be attributed directly or indirectly to HIV. Recently there has been an increasing level of non-HIV mortality which has important implications for local healthcare provision. This study demonstrates how evidence from longitudinal analyses can support healthcare planning.

Induced pluripotent stem cell technology and stem cell therapy for diabetes.

Although diabetes can be managed clinically with the use of insulin injections, it remains an incurable and inconvenient disorder. In the long-term, it is associated with a number of clinical complications, such as cardiovascular disease, resulting in a desire for the development of new methodologies to replace defective cells and provide a lasting normality without the need for drug treatment. Stem cells, including induced pluripotent stem cells, offer the possibility of generating cells suitable for transplantation due to their capacity to differentiate into all tissue lineages. However, many issues must be addressed before this type of treatment becomes a reality, including the need for a greater understanding of the underlying biology involved in the onset of diabetes.

Dynamics of the "popcorn" Wolbachia infection in outbred Aedes aegypti informs prospects for mosquito vector control.

Forty percent of the world's population is at risk of contracting dengue virus, which produces dengue fever with a potentially fatal hemorrhagic form. The wMelPop Wolbachia infection of Drosophila melanogaster reduces life span and interferes with viral transmission when introduced into the mosquito Aedes aegypti, the primary vector of dengue virus. Wolbachia has been proposed as an agent for preventing transmission of dengue virus. Population invasion by Wolbachia depends on levels of cytoplasmic incompatibility, fitness effects, and maternal transmission. Here we characterized these traits in an outbred genetic background of a potential target population of Ae. aegypti using two crossing schemes. Cytoplasmic incompatibility was strong in this background, and the maternal transmission rate of Wolbachia was high. The infection substantially reduced longevity of infected adult females, regardless of whether adults came from larvae cultured under high or low levels of nutrition or density. The infection reduced the viability of diapausing and nondiapausing eggs. Viability was particularly low when eggs were laid by older females and when diapausing eggs had been stored for a few weeks. The infection affected mosquito larval development time and adult body size under different larval nutrition levels and densities. The results were used to assess the potential for wMelPop-CLA to invade natural populations of Ae. aegypti and to develop recommendations for the maintenance of fitness in infected mosquitoes that need to compete against field insects.

Knock-in of nuclear localised beta-galactosidase reveals that the tyrosine phosphatase Ptprv is specifically expressed in cells of the bone collar.

Ptprv is a member of the transmembrane tyrosine phosphatase gene family reported to be expressed in osteoblasts and gonads. To better define the developmental and tissue specificity of Ptprv expression, we generated knock-in mice expressing a nuclear localised beta-galactosidase reporter under the control of resident Ptprv regulatory elements. Histochemical staining of Ptprv-nLacZ mice revealed that Ptprv expression is readily detectable in the foetal gonadal ridge of both sexes and in adult gonads where it is localised to Sertoli cells of the testis and celomic epithelial cells of the ovaries. During early limb development, Ptprv expression is prominent in the apical ectodermal ridge of the limb bud. At latter stages of development, Ptprv is predominantly expressed in the perichondrial and periosteal region of long bones, known as the bone collar. In contrast to previous indications from in vitro studies, there is little if any expression in mature osteoblasts in vivo. Analysis of Ptprv mRNA localisation by in situ hybridization in parallel with molecular markers of chondrocytes and osteoblasts confirmed the specific expression of Ptprv in immature bone collar cells. The specificity of Ptprv expression in these cells may be a useful tool to elucidate their role in the transition of skeletal elements from cartilage template to bone.

An unpaired mouse centromere passes consistently through male meiosis and does not significantly compromise spermatogenesis.

ST1 is an artificial mini-chromosome approximately 4.5 Mb in size containing mouse minor and major satellite DNA, human alphoid DNA and sequences derived from interval 5 of the human Y chromosome. Here we have measured the mitotic and meiotic transmission of ST1 and have used the mini-chromosome to define the ability of mice to monitor the presence of unpaired centromeres during meiosis. ST1 is mitotically stable, remaining intact and autonomous in mice for many generations. Female mice efficiently transmit ST1 to their offspring at a frequency approaching 50%. Male mice also reliably transmit the mini-chromosome, though to only 20% of their offspring. Presence of ST1 in males is not associated with any compromise in the output of the seminiferous epithelium nor with histological or immunocytochemical evidence of increased apoptosis, outcomes predicted for a synapsis checkpoint. These data indicate that the presence of an unpaired centromere is not sufficient to arrest male meiosis, implying that univalents are normally eliminated by a mechanism other than a tension-sensitive spindle checkpoint.

Artificial chromosomes: ideal vectors?

Artificial chromosomes are DNA molecules of predictable structure, which are assembled in vitro from defined constituents that behave with the properties of natural chromosomes. Artificial chromosomes were first assembled in budding yeast and have since been useful in many aspects of yeast genetics. Several attempts have been made at building artificial chromosomes in mammals, although these have been met with limited success. Consequently, mini-chromosomes of defined structure have been developed to address questions regarding mammalian chromosome function and for biotechnological applications. Here we review progress in these areas and consider how it influences plans to build artificial chromosomes in plants and parasites.

A structurally defined mini-chromosome vector for the mouse germ line.

Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.

Cross-talk of NO, superoxide and molecular oxygen, a majesty of aerobic life.

Because nitric oxide (NO) reacts with various molecules, such as hemeproteins, superoxide and thiols including glutathione (GSH) and cysteine residues in proteins, biological effects and metabolic fate of this gaseous radical are affected by these reactants. Although the lifetime of NO is short particularly under air atmospheric conditions (where the oxygen tension is unphysiologically high), it increases significantly under physiologically low oxygen concentrations. Because oxygen tensions in human body differ from one tissue to another and change depending on their metabolism, biological activity of NO in various tissues might be affected by local oxygen tensions. To elucidate the role of NO and related radicals in the regulation of circulation and energy metabolism, their effects on arterial resistance and energy metabolism in mitochondria, mammalian cells and enteric bacteria were studied under different oxygen tensions. Kinetic analysis revealed that NO-dependent generation of cGMP in resistance arteries and their relaxation were strongly enhanced by lowering oxygen tensions in the medium. NO reversibly suppressed the respiration and ATP synthesis of isolated mitochondria and intact cells particularly under low oxygen tensions. Kinetic analysis revealed that cross-talk between NO and superoxide generated in and around endothelial cells regulates arterial resistance particularly under physiologically low oxygen tensions. NO also inhibited the respiration and ATP synthesis of E. coli particularly under low oxygen tensions. Because concentrations of NO and H+ in gastric juice are high, most ingested bacteria are effectively killed in the stomach. However, the inhibitory effects of NO on the respiration and ATP synthesis of H. pylori are extremely small. Kinetic analysis revealed that H. pylori generates the superoxide radical thereby inhibiting the bactericidal action of NO in gastric juice. Based on such observations, critical roles of the cross-talk of NO, superoxide and molecular oxygen in the regulation of energy metabolism and survival of aerobic and microaerophilic organisms are discussed.

Greatly reduced efficiency of both positive and negative selection of thymocytes in CD45 tyrosine phosphatase-deficient mice.

The T cell repertoire is shaped by positive and negative selection of thymocytes. TCR-mediated signals that determine these selection processes are only partly understood. The CD45 tyrosine phosphatase has been shown to be important for signal transduction through the TCR, but there has been disagreement about whether CD45 is a positive or negative regulator of TCR signaling. Using CD45-deficient mice expressing transgenic TCR, we show that in the absence of CD45 there is a large increase in the thresholds of TCR stimulation required for both positive and negative selection. Our results conclusively demonstrate that in double-positive thymocytes CD45 is a positive regulator of the TCR signals that drive thymic selection events.

Transchromosomal mouse embryonic stem cell lines and chimeric mice that contain freely segregating segments of human chromosome 21.

At least 8% of all human conceptions have major chromosome abnormalities and the frequency of chromosomal syndromes in newborns is >0.5%. Despite these disorders making a large contribution to human morbidity and mortality, we have little understanding of their aetiology and little molecular data on the importance of gene dosage to mammalian cells. Trisomy 21, which results in Down syndrome (DS), is the most frequent aneuploidy in humans (1 in 600 live births, up to 1 in 150 pregnancies world-wide) and is the most common known genetic cause of mental retardation. To investigate the molecular genetics of DS, we report here the creation of mice that carry different human chromosome 21 (Hsa21) fragments as a freely segregating extra chromosome. To produce these 'transchromosomal' animals, we placed a selectable marker into Hsa21 and transferred the chromosome from a human somatic cell line into mouse embryonic stem (ES) cells using irradiation microcell-mediated chromosome transfer (XMMCT). 'Transchromosomal' ES cells containing different Hsa21 regions ranging in size from approximately 50 to approximately 0.2 Mb have been used to create chimeric mice. These mice maintain Hsa21 sequences and express Hsa21 genes in multiple tissues. This novel use of the XMMCT protocol is applicable to investigations requiring the transfer of large chromosomal regions into ES or other cells and, in particular, the modelling of DS and other human aneuploidy syndromes.

The Rho-family GTP exchange factor Vav is a critical transducer of T cell receptor signals to the calcium, ERK, and NF-kappaB pathways.

Vav is a GTP/GDP exchange factor (GEF) for members of the Rho-family of GTPases that is rapidly tyrosine-phosphorylated after engagement of the T cell receptor (TCR), suggesting that it may transduce signals from the receptor. T cells from mice made Vav-deficient by gene targeting (Vav-/-) fail to proliferate in response to TCR stimulation because they fail to secrete IL-2. We now show that this is due at least in part to the failure to initiate IL-2 gene transcription. Furthermore, we analyze TCR-proximal signaling pathways in Vav-/- T cells and show that despite normal activation of the Lck and ZAP-70 tyrosine kinases, the mutant cells have specific defects in TCR-induced intracellular calcium fluxes, in the activation of extracellular signal-regulated mitogen-activated protein kinases and in the activation of the NF-kappaB transcription factor. Finally, we show that the greatly reduced TCR-induced calcium flux of Vav-deficient T cells is an important cause of their proliferative defect, because restoration of the calcium flux with a calcium ionophore reverses the phenotype.

Molecular requirements for lineage commitment in the thymus--antibody-mediated receptor engagements reveal a central role for lck in lineage decisions.

Recent experiments in our laboratory have focused on the receptor engagements required for the differentiation of fully mature, single positive thymocytes from their double positive precursors. We have used a novel approach which involves the ligation of surface receptors on immature thymocytes with genetically engineered F(ab1)2 reagents, which, unlike conventional antibodies, do not aggregate the CD3 complex to such an extent as to induce extensive deletion of these cells. The experimental data presented in this review indicate that differentiation of the two mature CD4 and CD8 lineages occurs in response to distinct intracellular signals induced by particular receptor engagements. The data suggest that the tyrosine kinase p56lck (lck) plays a crucial role in determining lineage choice, in that maturation of thymocytes into the CD4 lineage occurs upon recruitment of active lck to the T-cell receptor (TCR)/CD3 complex, whereas CD8 maturation can be induced by CD3 ligation in the absence of co-receptor-mediated lck recruitment. A central role for lck activity in determining the threshold for differentiation of the CD4 lineage is revealed in experiments with thymi deficient for a regulator of lck activity, CD45. A model of thymocyte differentiation is presented in which we propose that the relative balance of signals delivered by TCR engagement and lck activation determines lineage choice.

Hierarchical interactions of control elements determine CD8alpha gene expression in subsets of thymocytes and peripheral T cells.

CD4 and CD8 are crucial for the development and function of T cells. An intergenic deoxyribonuclease I hypersensitive site region (cluster CIII) directs expression in mature CD8 T cells only. Here, we show that two further independent regions from the CD8 gene locus in conjunction with cluster CIII restore transgene expression in appropriate immature thymocytes. Deletion of two of the intergenic cluster CIII DNaseI-HSS in homozygous mutant mice affects expression of CD8alphaalpha homodimers on intraepithelial T cells (IEL), particularly on the gammadeltaTCR+ subset. Surprisingly, none of the thymocyte or peripheral alphabetaTCR T cell subsets are affected by this mutation, indicating hierarchical activation of these elements within the different T cell subsets.

Tumorigenesis and a DNA repair defect in mice with a truncating Brca2 mutation.

Germline mutation of the BRCA2 gene carries a high risk of developing breast cancer. To study the function of this gene, we generated a mutation in Brca2 in mice. Unlike other mutations in the Brca2 gene, which are lethal early in embryogenesis when homozygous, some of our homozygous mutant mice survive to adulthood. These animals have a wide range of defects, including small size, improper differentiation of tissues, absence of germ cells and the development of lethal thymic lymphomas. Fibroblasts cultured from BrcaZ-/-embryos have a defect in proliferation that may be mediated by over-expression of p53 and p21Waf1/CIP1. We show that Brca2 is required for efficient DNA repair, and our results suggest that loss of the p53 checkpoint may be essential for tumour progression triggered by mutations in BRCA2.

A requirement for the Rho-family GTP exchange factor Vav in positive and negative selection of thymocytes.

The T cell repertoire is shaped by positive and negative selection of thymocytes that express low levels of T cell receptor (TCR) and both CD4 and CD8. TCR-mediated signals that determine these selection processes are only partly understood. Vav, a GDP-GTP exchange factor for Rho-family proteins, is tyrosine phosphorylated following TCR stimulation, suggesting that it may transduce TCR signals. We now demonstrate that mice lacking Vav are viable and display a profound defect in the positive selection of both class I- and class II-restricted T cells. In contrast, Vav is not essential for negative selection, though in its absence negative selection is much less effective. Vav may influence the efficiency of TCR-induced selection events by regulating the intracellular calcium flux of thymocytes.

Hematopoiesis in mice lacking the entire granulocyte-macrophage colony-stimulating factor/interleukin-3/interleukin-5 functions.

Interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 are major hematopoietic cytokines produced by activated T cells and exhibit similar biologic activities by signaling through a common receptor subunit (beta c). Mice lacking beta c show a pulmonary alveolar proteinosis-like disease and reduced numbers of peripheral eosinophils, which are explained by the lack of GM-CSF and IL-5 function, respectively. However, beta c-deficient hematopoietic cells do respond to IL-3 normally, probably through an additional beta subunit of the IL-3 receptor (beta IL3) that is present in the mouse. Thus, almost normal hematopoiesis in beta c-deficient mice may be caused by functional redundancy between IL-3 and GM-CSF. To clarify the role of the entire IL-3/GM-CSF/IL-5 system in hematopoiesis in vivo, we crossed the beta c mutant mice with mice deficient for IL-3 ligand to generate mice lacking the entire IL-3/GM-CSF/IL-5 functions. The double-mutant mice were apparently normal and fertile. The severity of the lung pathology in the beta c/IL-3 double-mutant mice showed normal hemodynamic parameters except for reduced numbers of eosinophils and the lack of eosinophilic response to parasites, which were also found in beta c mutant mice. The immune response of the beta c/IL-3 double-mutant mice to Listeria mono-cytogenes was normal, as was hematopoietic recovery after administration of the cytotoxic drug, 5-fluorouracil. Although it has been believed that IL-3/GM-CSF/IL-5 produced by activated T cells play a major role in expansion of hematopoietic cells in emergency, our results indicate that the entire function of IL-3/GM-CSF/IL-5 is dispensable for hematopoiesis in emergency as well as in the steady state. Thus, there must be an alternative mechanism to produce blood cells in both situations.

Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk.

The tyrosine kinase Syk (relative molecular mass 72,000), which is widely expressed in haematopoietic cells, becomes associated with and activated by engagement of the B-cell antigen receptor. Furthermore, it has been implicated in signalling through the receptors for interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF) and Fc, the T cell receptor, as well as through receptors for several platelet agonists. A homologous kinase, ZAP-70, is crucial in signalling through the T-cell receptor and in T-cell development. Using homologous recombination in embryonic stem cells, we created mice null for the syk gene which showed petechiae in utero and died shortly after birth. Irradiated mice reconstituted with Syk-deficient fetal liver showed a block in B-cell development at the pro-B to pre-B cell transition, consistent with a key role for Syk in pre-B-cell receptor signalling. Despite the production of small numbers of immature B cells, Syk-deficient radiation chimaeras failed to accumulate mature B cells, indicating a possible role for this protein in the production or maintenance of mature B cells. In addition, whereas the development of alpha beta T cells proceeded normally, Syk-deficient mice showed impaired development of thymocytes using the V gamma 3 variable region gene (V gamma 3+ thymocytes). Finally, we show that Syk is not required for signalling through the IL-2 and G-CSF receptors.

Defective antigen receptor-mediated proliferation of B and T cells in the absence of Vav.

Crosslinking of B- or T-cell antigen receptors results in the rapid tyrosine phosphorylation of a number of proteins, including Vav, a protein expressed in cells of the haematopoietic system. Vav contains an array of structural motifs that include Src-homology domains SH2/SH3 and regions of homology to the guanine-nucleotide-exchange protein Dbl, pleckstrin and protein kinase C (refs 5-9). Using the RAG-complementation approach, we have analysed in vivo differentiation and in vitro responses of B- and T-lineage cells generated by injection of embryonic stem cells homozygous for a null mutation in the vav gene into blastocysts of RAG-1- or RAG-2-deficient mice. Here we report that antigen receptor-mediated proliferative responses of B and T cells in vitro are severely reduced in the absence of Vav. We also suggest a direct link between the low proliferative response of Vav-deficient B and T cells and the reduced number of these cells in peripheral lymphoid organs of chimaeric mice.