Transformation of hepatitis C antiviral treatment in a national healthcare system following the introduction of direct antiviral agents.

BACKGROUND: Highly effective direct antiviral agents (DAAs) for hepatitis C virus (HCV) were introduced recently. Their utilisation has been limited by high cost and low access to care. AIM: To describe the effect of DAAs on HCV treatment and cure rates in the United States Veterans Affairs (VA) national healthcare system. METHODS: We identified all HCV antiviral treatment regimens initiated from 1 January 1999 to 31 December 2015 (n = 105 369) in the VA national healthcare system, and determined if they resulted in sustained virological response (SVR). RESULTS: HCV antiviral treatment rates were low (1981-6679 treatments/year) in the interferon era (1999-2010). The introduction of simeprevir and sofosbuvir in 2013 and ledipasvir/sofosbuvir and paritaprevir/ombitasvir/ritonavir/dasabuvir in 2014 were followed by increases in annual treatment rates to 9180 in 2014 and 31 028 in 2015. The number of patients achieving SVR was 1313 in 2010, the last year of the interferon era, and increased 5.6-fold to 7377 in 2014 and 21-fold to 28 084 in 2015. The proportion of treated patients who achieved SVR increased from 19.2% in 1999 and 36.0% in 2010 to 90.5% in 2015. Within 2015, monthly treatment rates ranged from 727 in July to 6868 in September correlating with the availability of funds for DAAs. CONCLUSIONS: DAAs resulted in a 21-fold increase in the number of patients achieving HCV cure. Treatment rates in 2015 were limited primarily by the availability of funds. Further increases in funding and cost reductions of DAAs in 2016 suggest that the VA could cure the majority of HCV-infected Veterans in VA care within the next few years.

Can early infection explain the sibling effect in adult atopy?

Atopy is strongly and inversely related to family size, a pattern which is plausibly assumed to reflect a protective effect of early infection. The current study tested this hypothesis by case-referent analysis of an adult cohort in the UK. The study established that atopy, defined by prick tests to common aeroallergens, was less common among those from larger families after adjustment for potentially confounding factors. In particular, a higher number of brothers appeared to offer protection. The current authors attempted to explain this distribution by examining contemporary family-doctor records of early childhood infections; and by a number of other indirect indices of early-life "hygiene". The sibling effect was unexplained by evidence of infection with either hepatitis A or Helicobacter pylori, or by counts of infections or antibiotic prescriptions in early life. There was a significant and independent negative association between the number of gastrointestinal infections before the age of 5 yrs and the odds of atopy. Dog ownership and home moving in early life also displayed potentially protective associations. Although the current study replicates the finding that atopy is inversely associated with family size this could not be explained by documentary or serological evidence of early infection. The findings support the suggestion that the "sibling effect" in atopy may not simply reflect protection by early infection.

Fgf8 is required for outgrowth and patterning of the limbs.

The expression pattern and activity of fibroblast growth factor-8 (FGF8) in experimental assays indicate that it has important roles in limb development, but early embryonic lethality resulting from mutation of Fgf8 in the germ line of mice has prevented direct assessment of these roles. Here we report that conditional disruption of Fgf8 in the forelimb of developing mice bypasses embryonic lethality and reveals a requirement for Fgf8 in the formation of the stylopod, anterior zeugopod and autopod. Lack of Fgf8 in the apical ectodermal ridge (AER) alters expression of other Fgf genes, Shh and Bmp2.

Normal limb development in conditional mutants of Fgf4.

Fibroblast growth factors (FGFs) mediate multiple developmental signals in vertebrates. Several of these factors are expressed in limb bud structures that direct patterning of the limb. FGF4 is produced in the apical ectodermal ridge (AER) where it is hypothesized to provide mitogenic and morphogenic signals to the underlying mesenchyme that regulate normal limb development. Mutation of this gene in the germline of mice results in early embryonic lethality, preventing subsequent evaluation of Fgf4 function in the AER. A conditional mutant of Fgf4, based on site-specific Cre/loxP-mediated excision of the gene, allowed us to bypass embryonic lethality and directly test the role of FGF4 during limb development in living murine embryos. This conditional mutation was designed so that concomitant with inactivation of the Fgf4 gene by excision of all Fgf4-coding sequences, a reporter gene was activated in Fgf4-expressing cells, allowing assessment of the site-specific recombination reaction. Although a large body of evidence led us to predict that ablation of Fgf4 gene function in the AER of developing mice would result in abnormal limb outgrowth and patterning, we found that Fgf4 conditional mutants had normal limbs. Furthermore, expression patterns of Shh, Bmp2, Fgf8 and Fgf10 were normal in the limb buds of the conditional mutants. These findings indicate that the previously proposed FGF4-SHH feedback loop is not essential for coordination of murine limb outgrowth and patterning. We suggest that some of the roles currently attributed to FGF4 during early vertebrate limb development may be performed by other AER factors in vivo.

Developing the 'health-promoting school'--a national survey of healthy schools awards.

The concept of the 'Health Promoting School' has been widely advocated as an approach to enhancing public health through school based health promotion. In many areas 'Healthy Schools Award' schemes have been set up to support the development of this concept, but there is no information on how widespread this practice is in the UK, how standards are evaluated, and what effect Healthy Schools Awards may have on young peoples' health. This UK national survey aimed to determine the extent and nature of existing award schemes and how they were being evaluated. A postal questionnaire was sent to all 200 health promotion units in the UK; the response rate corrected for mergers of units was 78.5%. Sixty-eight respondents (51%) were involved with an award scheme and 28 (21%) were planning them. Current award schemes were mostly jointly run by the health and education sectors, encompassing 845 participating schools of which two-thirds were primary schools. The most common issues addressed were; standard chronic disease risk behaviour, the environment and health education in the national curriculum; less frequently addressed were mental health, accident prevention, staff health and developing links with the wider community. Evaluation was usually by target setting and assessment of progress over a two year period. However, evaluation was rarely external or independent, raising doubts about the standards obtained and validity of the approaches. This survey highlights the rapid growth of healthy schools award schemes and the need for wider exchange of information on good practice. In particular there is a need for more explicit and measurable standards of achievement to ensure the quality of award schemes, and further research into their effectiveness.

Grasshopper crop and midgut extract effects on plants: an example of reward feedback.

Acid extracts and a resultant fraction from solid-phase extraction (SPE) of Romalea guttata crop and midgut tissues induce sorghum (Sorghum bicolor var. Rio) coleoptile growth in 24-h incubations an average of 49% above untreated controls. When combined with plant auxin, indole-3-acetic acid (IAA), the SPE fraction shows a synergistic reaction, yielding increases in coleoptile growth that average 295% above untreated controls and 8% above IAA standards. The interaction lowered the point of maximum sensitivity of IAA 3 orders of magnitude, resulting in a new IAA physiological set point at 10(-7) g/ml. This synergism suggests that contents in animal regurgitants making their way into plant tissue during feeding may produce a positive feedback in plant growth and development following herbivory. Such a process, also known as reward feedback, may exert major controls on ecosystem-level relationships in nature.

Structure and function of the murine beta-globin locus control region 5' HS-3.

We previously identified the murine homologue of the human beta-globin Locus Control Region (LCR) 5' HS-2. The lambda clone containing murine 5' HS-2 extends approximately 12 kb upstream from this site; here, we report the sequence of this entire upstream region. The murine homologue of 5' HS-3 is located approximately 16.0 kb upstream from the mouse epsilon y-globin gene, but no region homologous to human 5' HS-4 was present in our clone. Using a reporter system consisting of a human gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo), we tested murine LCR fragments extending from -21 to -9 kb (with respect to the epsilon y-globin gene cap site) for activity in classical enhancer and integration site assays in K562 and MEL cells. 5' HS-2 behaved as a powerful enhancer and increased the number of productive integration events (as measured by a colony assay) in both K562 and MEL cells. 5' HS-3 had no activity in K562 cells or in transiently transfected MEL cells, but was nearly as active as 5' HS-2 in the MEL cell colony assay. Two additional tests confirmed the identification of murine 5' HS-3: first, a DNA fragment containing 5' HS-3 confers copy number-dependent, integration-site independent inducibility on a linked beta-globin gene in the MEL cell environment. Secondly, a strong DNAseI hypersensitive site maps to the location of the 5' HS-3 functional core in chromatin derived from MEL cells. Collectively, these data suggest that we have identified the murine homologue of human 5' HS-3, and that this site is functional when integrated into the chromatin of MEL cells but not K562 cells. 5' HS-3 may therefore contain information that contributes to the development-specific expression of the beta-like globin genes.

Functional properties of the beta-globin locus control region in K562 erythroleukemia cells.

In this report, we compare the function of the human beta-globin locus control region (LCR) in three K562 erythroleukemia cell assays, including (1) a transient transfection assay for "classical" enhancer activity, (2) a colony assay that detects "productive integration events," and (3) an assay that detects the ability of LCR fragments to confer hemin inducibility on linked, stably integrated gamma-globin promoters. Various LCR fragments were inserted into an expression vector consisting of an A gamma-globin promoter driving the neomycin phosphotransferase gene (gamma-neo). Using these vectors, we determined that a 2.5-kb DNA fragment containing LCR sites I through IV (previously named mu locus activation region [mu LAR]) had activity in all three assays; of the individual LCR sites, only site II was highly active in all three assays. One region within site II, consisting of tandem AP-1/NF-E2 consensus elements, had approximately 10% as much colony assay activity as the entire mu LAR. However, this region did not have detectable activity in a transient enhancer assay in uninduced K562 cells, nor was it capable of conferring hemin inducibility on linked gamma-globin promoters in stably transfected cells. Finally, we tested the ability of the mu LAR to activate promoters (beta-globin and cathepsin G) that are not normally expressed in K562 cells. beta-neo was minimally activated by the mu LAR in transient transfection experiments. The mu LAR increased the number of stably transfected colonies produced by beta-neo, but the absolute number of beta-neo colonies, with or without the mu LAR, was approximately 10% to 20% that of gamma-neo. In contrast, a minimal cathepsin G promoter was activated by the mu LAR in K562 cells. Our results suggest that LCR functions are dependent in part on the environments and the promoters with which the LCR is tested.

Conservation of the primary structure, organization, and function of the human and mouse beta-globin locus-activating regions.

DNA sequences located in a region 6-18 kilobases (kb) upstream from the human epsilon-globin gene are known as the locus-activating region (LAR) or dominant control region. This region is thought to play a key role in chromatin organization of the beta-like globin gene cluster during erythroid development. The beta-globin LAR activates linked globin genes in transiently or stably transfected erythroleukemia cells and in erythroid cells of transgenic mice. Since the human beta-globin LAR is functional in mice, we reasoned that critical LAR sequence elements might be conserved between mice and humans. We therefore cloned murine genomic sequences homologous to one portion of the human LAR (site II, positions -11,054 to -10,322 with respect to the human epsilon gene). We found that this murine DNA fragment (mouse LAR site II) and sequences homologous to human LAR sites I and III are located upstream from the mouse beta-like globin gene cluster and determined that their locations relative to the cluster are similar to that of their human counterparts. The homologous site II sequences are 70% identical between mice and humans over a stretch of approximately 800 base pairs. Multiple core sequences with greater than 80% identity were present within this region. Transient and stable transfection assays of K562 erythroleukemia cells demonstrated that both human and mouse LAR elements contain enhancer activity and confer hemin inducibility on a linked human gamma-globin promoter. These results suggest that primary structural elements--and the spatial organization of these elements--are important for function of the beta-globin LAR.

Function of transfected globin promoters and the globin locus activator in K562 erythroleukemia cells.

We have examined the importance of cis-acting regulatory elements within the human gamma-globin gene promoter and the globin locus activating region in K562 cells. A gamma-globin or beta-globin promoter fragments were fused with the neomycin phosphotransferase gene in a plasmid-based vector (gamma-neo or beta-neo) and transiently transfected by electroporation into K562 cells. Correctly initiated gamma-neo or beta-neo transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation and then determined that a gamma-globin promoter fragment extending from -299 and +36 was active in the assay but that a beta-globin promoter extending from -375 to +46 was inactive. Deletion of the gamma-globin promoter to -199 did not affect promoter function, but deletion to -160 reduced promoter strength to 70% of that of control. Additional deletion to position -130 reduced promoter strength to 19% of the control value, and to position -61, 8.7% of the control value. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (HPFH), -202 C----G, -196 C----T and -117 G----A, were not overexpressed in K562 cells, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only in adult red cells. When the beta-globin locus activating region (LAR) was added to a wild-type or an HPFH gamma-neo plasmid, the abundance of correctly initiated gamma-neo transcripts increased dramatically. However, beta-neo expression could not be activated by the LAR in K562 cells. These studies should allow us to further dissect the interactive roles of globin promoters and enhancers in K562 cells.

Cloning and characterization of an abundant Plasmodium knowlesi antigen which cross reacts with Gambian sera.

A 110 kDa Plasmodium knowlesi antigen, termed PK110, has been identified on the basis of messenger RNA abundance in late schizonts. Most Plasmodium genes previously cloned have been identified by immune sera, which have selected immunodominant antigens composed of repeating epitopes. Although PK110 was not selected by immune sera, it also contains amino acid repeats, indicating that this structure may be a common feature of malarial proteins. Determination of 296 codons in the PK110 gene revealed the presence of thirteen tandem repeats of twelve amino acids whose consensus sequence is E E T Q K T V E P E Q T. A termination codon interrupts the fourteenth repeat, indicating that these repeats are at the C-terminus of the protein. Indirect immunofluorescence experiments with sera raised against the lambda gt11 fusion protein indicate that PK110 is present in intra-erythrocytic late schizonts. Cloned PK110 is recognized by Gambian sera, and shares epitopes with Plasmodium ovale. PK110 does not cross react immunologically or by DNA hybridization with Plasmodium falciparum.

Metacyclic variant surface glycoprotein genes of Trypanosoma brucei subsp. rhodesiense are activated in situ, and their expression is transcriptionally regulated.

During the metacyclic stage in the life cycle of Trypanosoma brucei subsp. rhodesiense, the expression of variant surface glycoproteins (VSGs) is restricted to a small subset of antigenic types. Previously we identified cDNAs for the VSGs expressed in metacyclic variant antigen types (MVATs) 4 and 7 and found that these VSG genes do not rearrange when expressed at the metacyclic stage (M. J. Lenardo, A. C. Rice-Ficht, G. Kelly, K. Esser, and J. E. Donelson, Proc. Nathl. Acad Sci. USA 81:6642-6646, 1984). We now provide further evidence that these genes do not rearrange and demonstrate that their 5' upstream regions lack the 72 to 76-base-pair repeats which are considered the substrate for duplication and transposition events. Pulsed field gradient electrophoresis showed that the MVAT VSG genes were located on the largest chromosome-sized DNA molecules, and the lack of the MVAT 4 gene in one of two different serodemes suggested that one mechanism for the evolution of MVAT repertoires is gene deletion. When MVATs were inoculated into the bloodstream of a mammalian host by a bite from the insect vector, they rapidly switched into nonmetacyclic VSG types. We found that this switch was accomplished by a loss of MVAT RNA concomitant with the loss of metacyclic VSGs. Transcription studies with isolated metacyclic nuclei showed that the MVAT genes were expressed in situ from a single locus and were regulated at the level of transcription.