Statewide dissemination of a rural, non-chain restaurant intervention: adoption, implementation and maintenance.

The obesity epidemic calls for greater dissemination of nutrition-related programs, yet there remain few studies of the dissemination process. This study, guided by elements of the RE-AIM model, describes the statewide dissemination of a simple, point-of-purchase restaurant intervention. Conducted in rural counties of the Midwest, United States, the study targeted randomly selected, non-chain, family-style restaurants. Owners were recruited through mail, then telephone follow-up. Data were collected through telephone at baseline, and 3, 6, 12 and 18 months post-adoption. Using mixed methods, measures captured the program adoption rate, characteristics of adopters and non-adopters, program implementation and maintenance issues, and owner and customer satisfaction. Analyses involved descriptive statistics and summaries of qualitative data. The program adoption rate was 28%. Adopters were similar to responding non-adopters demographically, but varied in attitudes. The majority of restaurants maintained the program for at least 12 months. Adopters and their customers expressed satisfaction with the program. With some adjustments, the RE-AIM model was helpful in guiding evaluation of this process. Results provide implications for future dissemination of this and other programs with regard to research procedures and potential barriers that may be encountered. Research on alternative strategies for widespread dissemination of such programs is needed in this and other settings.

Alternative methods for validation of cell culture infection with duck hepatitis B virus.

Hepatitis B virus (HBV) is an important virus used in disinfection procedures for blood spillage. However, validation of HBV inactivation is difficult, since there are no feasible infectivity assays. In some countries, the duck HBV (DHBV) is recognized as a suitable model for testing antiviral activity of chemical biocides against HBV. Currently, DHBV-infected ducks are required for preparation of the test virus as well as eggs from DHBV-free flocks for testing DHBV infectivity. To improve the practicality of the system, we suggested to use commercially available embryonated duck eggs for preparation of DHBV-susceptible hepatocyte cultures and to exclude infected hepatocytes by pre-screening with qualitative detection of DHBV DNA using polymerase chain reaction (PCR). A standardized DHBV test virus was prepared from the DHBV DNA-transfected hepatoma cell line D2, which contained 10(11)DHBV DNA molecules per mL detected by light cycler real-time PCR. Infection of cell cultures was most efficient 4 days after plating. The best identification of infected cultures was possible 6 days after infection with immunofluorescence using an antiserum against DHBV surface antigen.

Effect of a combination of clevudine and emtricitabine with adenovirus-mediated delivery of gamma interferon in the woodchuck model of hepatitis B virus infection.

Our aim was to evaluate the antiviral effect of a combination of two nucleoside reverse transcriptase inhibitors, emtricitabine (FTC) and clevudine (L-FMAU), with the addition of an adenovirus-driven delivery of recombinant gamma interferon (IFN-gamma) in the woodchuck model of hepatitis B virus infection. Six woodchuck hepatitis virus (WHV)-infected woodchucks received L-FMAU (10 mg/kg) plus FTC (30 mg/kg) intraperitoneally for 8 weeks; six other animals received in addition an intravenous injection of a recombinant adenovirus vector expressing woodchuck IFN-gamma (Ad-IFN) at weeks 4 and 8. In the control group, two animals received Ad-IFN alone, two received adenovirus vector expressing the green fluorescent protein reporter gene, and one remained untreated. In less than 2 weeks, all woodchucks that received L-FMAU plus FTC showed a rapid and marked inhibition of viral replication, with a 4-log(10) drop in serum WHV DNA. In two animals, viremia remained suppressed for several months after the end of treatment. Similarly, a dramatic decrease in intrahepatic replicative intermediates of viral DNA was observed in the L-FMAU/FTC-treated groups. The additional administration of Ad-IFN led to increased inflammation in the liver but did not enhance the antiviral effect of the L-FMAU/FTC combination. In conclusion, therapies combining L-FMAU and FTC in WHV-infected woodchucks resulted in a potent and sustained antihepadnaviral effect both in the liver and in the blood circulation. However, no extra benefit of adding IFN-gamma gene transduction to the L-FMAU/FTC combination could be detected.

[Case management and functional outcome in persons aged 65 years and over with hip fracture]. / Case-Management und funktionelle Ergebnisse nach proximaler Femurfraktur im höheren Lebensalter.

The goal of the investigation was to study case management and functional outcome in older patients with hip fracture. A prospective observational survey was performed, including all patients aged 65 years and over presenting with hip fracture in Heidelberg, from 1 July 1999 to 30 June 2000. All patients were reassessed by telephone calls 6 months post-fracture. A total of 331 patients were included (mean age 81.5 years, 81% female,23.8% nursing home residents). Hip fracture incidence per 1,000 was 7.8/year, and nursing home residents had a six times higher incidence rate than those living at home. Prior to the fracture, half of the patients were dependent in ambulation and a third needed support in basic activities. With substantial comorbidity (42% cognitive impairment), complications were common. Geriatric care was needed for 82% of the survivors. In-hospital treatment costs were about 10,000 Euro per fracture. Mortality at 6 months was 19.9%. The majority of survivors showed loss of competence and mobility. Functional outcome in older patients with hip fracture is disappointing. As the majority of the patients are frail, clinical treatment is complicated by "geriatric" problems. Thus, improved interdisciplinary care, with close cooperation between geriatricians and surgeons might result in a better functional outcome.

Central anticholinergic syndrome in a child undergoing circumcision.

We describe one of the few pediatric cases of central anticholinergic syndrome (CAS) in an 8-year-old boy undergoing elective surgery. Deep sedation, inadequate response to stimuli and reduced muscular tone of the upper airway resulting in airway obstruction were the clinical manifestations of CAS. The symptoms resolved immediately after administration of physostigmine. This case illustrates the importance of considering central anticholinergic syndrome as a differential diagnosis in children if prolonged sedation after general anesthesia occurs.

Endotoxin stimulates liver macrophages to release mediators that inhibit an early step in hepadnavirus replication.

Hepadnaviruses are known to be sensitive to various extracellular mediators. Therefore, bacterial endotoxin, which induces the secretion of proinflammatory mediators in the liver, was studied for its effect on hepadnavirus infection in vitro using the duck hepatitis B virus (DHBV) model. In initial experiments, endotoxin was shown to inhibit DHBV replication in primary duck hepatocyte cultures prepared by standard collagenase perfusion. As a primary endotoxin target, hepatic nonparenchymal cells (NPC) contaminating primary hepatocyte cultures, and among these probably macrophages (Kupffer cells), were identified to secrete polypeptide mediators into the cell culture medium. When added during DHBV infection, these mediators elicited the principal antiviral effect in a dose-dependent fashion. On the molecular level, they inhibited accumulation of viral proteins as well as amplification of the nuclear extrachromosomal DHBV DNA templates. In hepatocytes with an established DHBV infection, DHBV protein and progeny virus production was inhibited while the levels of established nuclear DHBV DNA templates and viral transcripts remained unaffected. Finally, in hepatocytes infected with a replication-deficient recombinant DHBV-green fluorescent protein (GFP) virus, the endotoxin-induced mediators markedly reduced GFP expression from chimeric DHBV-GFP transcripts, indicating that the major effect is at a level of translation of viral RNAs. Taken together, the data obtained demonstrate that antiviral mediators, and among these the cytokines alpha interferon (IFN-alpha) and IFN-gamma, are released from hepatic NPC, most probably liver macrophages, upon endotoxin stimulation; furthermore, these mediators act at a posttranscriptional step of hepadnavirus replication.

Elimination of duck hepatitis B virus RNA-containing capsids in duck interferon-alpha-treated hepatocytes.

Evidence is presented that the previously cloned type I duck interferon (DuIFN) cDNA encodes a homologue of mammalian interferon-alpha (IFN-alpha). Recombinant DuIFN-alpha was used to study the inhibition of duck hepatitis B virus (DHBV) replication in primary hepatocytes in order to determine the IFN-sensitive steps of the virus replication cycle. IFN-treated cells accumulated two- to threefold-lower amounts of viral RNA transcripts early during infection, when IFN was added before virus. This reduction was not due to inhibition of virus entry since initial covalently closed circular DNA levels were not decreased in IFN-treated cells. Interestingly, the inhibitory effect of IFN on viral RNA levels was not observed in cells infected with a mutant DHBV that fails to synthesize core protein, suggesting that an uncharacterized core protein-mediated enhancing effect is blocked by IFN. When IFN was added at 4 days postinfection, encapsidated viral RNA pregenomes disappeared from infected cells within 3 days. This depletion was not simply due to conversion of pregenomes to DNA since depletion was not blocked by phosphonoformic acid, an inhibitor of the viral reverse transcriptase. The intracellular concentration of intact nucleocapsids was reduced, suggesting that in the presence of IFN pregenome-containing capsids were selectively depleted in hepatocytes. Thus, two steps in DHBV replication that involve the viral core protein were inhibited by DuIFN-alpha.

Recombinant duck interferon gamma inhibits duck hepatitis B virus replication in primary hepatocytes.

Interferon gamma (IFN-gamma), which has been cloned in several mammalian species and recently in birds, plays a critical role in modulating immune system function. IFN-gamma and tumor necrosis factor alpha (TNF-alpha) have been shown to be crucial in the pathogenesis of viral hepatitis and in the transient disappearance of hepatitis B virus (HBV) from the liver after adoptive transfer of HBV-specific cytotoxic T lymphocytes into HBV-transgenic mice. Similar studies in the natural animal hosts of related hepadnaviruses have been limited because the corresponding probes and recombinant cytokines were not available. For this reason, we initiated studies to clone and characterize cytokines from the duck, the natural host of the duck hepatitis B virus (DHBV). We describe here the cDNA cloning and initial characterization of the IFN-gamma homologue of ducks (DuIFN-gamma). The DuIFN-gamma cDNA codes for a predicted mature protein of 145 amino acids with a molecular mass of 16.6 kDa. The precursor protein has 67% identity with the previously cloned chicken IFN-gamma and 21 to 34% identity with mammalian IFN-gamma. Recombinant DuIFN-gamma induces the transcription of several IFN-inducible genes including IFN regulatory factor 1 and guanylate-binding protein, and it exhibits antiviral activity that protects duck cells from vesicular stomatitis virus-mediated lysis. Importantly, treatment of primary duck hepatocytes with recombinant DuIFN-gamma inhibits DHBV replication in a dose-dependent fashion. Time course analysis revealed that IFN-gamma treatment does not affect initial covalently closed circular DNA (cccDNA) conversion but inhibits the synthesis of progeny cccDNA by amplification.

Reference values for height and weight in Prader-Willi syndrome based on 315 patients.

UNLABELLED: The spontaneous growth of 315 patients (109 girls and 208 boys) with Prader-Willi syndrome (PWS) was analysed in a mixed longitudinal and cross-sectional manner. 33 patients were seen in the department between 1970 and 1994; height and weight of 76 patients from Germany were evaluated by means of a questionnaire with detailed measuring instructions, and 206 definite cases were added from the literature. Mean ( SD) length of newborn babies with PWS was 50.2+/-2.8 cm (145 boys) and 48.9 3.3 cm (79 girls). Mean weight at birth was 2945 570 g in boys and 2782+/-594 g in girls. During the 1st year, the children's growth was nearly normal, thereafter short stature was present in approximately 50% of PWS patients. Between 3 and 13 years of age, the 50th percentile for height in PWS is roughly identical with the 3rd percentile in healthy controls. Body weight was normal for all boys and girls during the first 2 years. Thereafter, a rapid weight gain occurred; after an age of 10 years weight-for-height index in nearly all patients exceeded the normal range. The extent of pubertal growth was reduced for the group. Mean adult height was 161.6+/-8.1 cm (23 males) and 150.2+/-5.5 cm (21 females). Head circumference for age was normal for boys and girls. CONCLUSION: Reference data on spontaneous development of growth and weight gain of children with Prader-Willi syndrome are described allowing a better counselling of patients and parents.

Biological efficacy and signal transduction through STAT proteins of recombinant duck interferon in duck hepatitis B virus infection.

The aim of this study was to characterize the interferon induced intracellular signals in duck hepatocytes and to investigate the effects of duck interferon on virus replication in duck hepatitis B virus (DHBV) infected ducks. Interestingly, duck interferon was found to activate intracellular signal transduction pathways similar to those of its mammalian counterparts. An interferon stimulated gel shift activity like that of gene factor 3 is induced, as well as serum inducible element binding factors homologous to serum inducible factor A (SIF-A), SIF-B and SIF-C. Duck interferon induced signal transducer and activator of transcription activation is not inhibited by DHBV infection of hepatocytes. DHBV infected ducks treated for 10 days with recombinant duck interferon show a decrease in viral DNA in hepatocytes, and in many cases disappearance of viraemia. These findings confirm the usefulness of the DHBV infection model for the study of human hepatitis B virus infection.

Promoter structures and differential responses to viral and nonviral inducers of chicken type I interferon genes.

Two serologically distinct type I interferons (IFNs), designated ChIFN1 and ChIFN2, are known in the chicken. ChIFN1 is encoded by a family of 10 or more genes, whereas ChIFN2 is encoded by a single gene. We show here that ChIFN1 and ChIFN2 transcripts are both strongly induced by Newcastle disease virus in primary chicken macrophages. By contrast, oral administration of the imidazoquinoline S-28463, which selectively induces IFN-alpha in mammals, led to a rapid accumulation of ChIFN1 (but not ChIFN2) transcripts in adult chicken spleen and thymus. The 5'-upstream region of the ChIFN2 gene contains a NF-kappaB consensus motif flanked by a sequence element that could serve as a binding site for transcription factor IRF-1, reminiscent of mammalian IFN-beta promoters, and it mediated powerful virus inducibility in a duck fibroblast cell line when cloned in front of a promoterless luciferase reporter gene. The 5'-upstream region of the cloned ChIFN1 gene contains two putative binding sites for IRF-1, but lacks NF-kappaB-binding sites, and it did not respond well to virus in transfected cells. Thus, the promoters of ChIFN1 and ChIFN2 genes not only exhibited differential responses to nonviral inducers in vivo, but also differed in structure and response to virus in transfected cells. These findings indicate that ChIFN2 represents the avian homolog of mammalian IFN-beta, whereas ChIFN1 seems to correspond to mammalian IFN-alpha.

Biological properties of recombinant chicken interferon-gamma.

Supernatants of the chicken T cell line 855 contain antiviral and macrophage activating factor activity and strongly activate transcription of the guanylate binding protein (GBP) gene in chicken cells. To characterize the cytokine responsible for the GBP-inducing activity, we chose a cDNA expression cloning strategy in COS cells. Sequencing a positive clone revealed that it encode chicken interferon-gamma (ChIFN-gamma). Histidine-tagged ChIFN-gamma was expressed in Escherichia coli and purified by nickel chelate affinity chromatography. ChIFN-gamma from COS cells and E. coli both potently induced GBP RNA synthesis but were rather poor antiviral agents. In macrophages, recombinant ChIFN-strongly stimulated secretion of nitric oxide and enhanced expression of major histocompatibility complex class II antigen. A rabbit antiserum to E. coli derived ChIFN-gamma effectively neutralized the macrophage-activating factor activity secreted by concanavalin A-induced spleen cells and various T cell lines, suggesting that IFN-gamma is the major macrophage-activating factor of the chicken.

Chicken guanylate-binding protein. Conservation of GTPase activity and induction by cytokines.

To gain further insights into the cytokine network of birds, we used polymerase chain reaction technology to clone a cDNA that codes for a chicken homolog of the interferon-induced guanylate-binding proteins (GBPs). In its N-terminal moiety, the 64-kDa chicken GBP contains two sequence blocks of 100 and 19 amino acids, respectively, that are about 70% identical to mammalian GBPs. The first region includes two motifs of the canonical GTP-binding consensus element. The other parts of chicken GBP are poorly conserved, except for a CAAX motif at the extreme C terminus which might signal isoprenylation. Like mammalian GBPs, recombinant chicken GBP specifically bound to agarose-immobilized guanine nucleotides and hydrolyzed GTP to both GDP and GMP. Regulation by interferons was also conserved: chicken GBP RNA was barely detectable in uninduced chicken cells. Low GBP RNA levels were found in cells treated with type I interferon, whereas very high levels were observed in cells treated with supernatant of a chicken T cell line that secretes a gamma-interferon-like activity. Together with recent phylogenetic studies of interferon genes, these results suggest that in spite of low sequence conservation, the various components of the avian interferon system are functionally well conserved.

A family of genes coding for two serologically distinct chicken interferons.

Southern blot analysis and screening of a genomic lambda phage library with the previously cloned chicken interferon (IFN) cDNA indicated that the chicken genome contains at least 10 IFN genes. A particularly strongly hybridizing phage clone that we analyzed in more detail carried a head to tail arrangement of three intron-less IFN genes that differed from each other and from the cloned chicken IFN cDNA by only a few base changes. The primary translation products of these three IFN genes consist of 193 amino acids, and the mature proteins are composed of 162 amino acids. All three genes of this IFN family, designated IFN1, yielded active chicken IFN when expressed individually in transfected COS7 cells. A weakly hybridizing phage clone contained an additional intron-less chicken IFN gene, designated IFN2, whose product was 57% identical to chicken IFN1. Southern blot analysis suggested that the chicken genome contains a single IFN2 gene. The primary translation product of IFN2 consists of 203 amino acids, and the mature protein is composed of 176 amino acids. Purified recombinant chicken IFN2 from Escherichia coli had a specific antiviral activity of about 10(6) units/mg, which was about 20-fold lower than that of chicken IFN1 purified in parallel. The antiviral activity of chicken IFN2 from E. coli or from transfected COS7 cells could not be neutralized by antiserum to recombinant chicken IFN1. Thus, like mammals, the chicken has a large number of type I IFN genes that code for at least two serologically distinct antiviral activities.

Recombinant duck interferon: a new reagent for studying the mode of interferon action against hepatitis B virus.

Although interferon is widely used to treat chronic hepatitis B virus infections, its mode of action against hepadnaviruses is largely unknown. This deficit is due mainly to the lack of suitable model systems. The duck system could not be used because purified duck interferon was not available in sufficient quantities. We have now cloned a DNA fragment that contains an intronless gene for duck interferon. The primary translation product consists of 191 amino acids, the N-terminal 30 residues of which constitute a signal peptide. Mature duck interferon is 50% identical to the recently cloned chicken interferon. Sequence homology to mammalian interferons is marginal, but conservation of four cysteine residues and inducibility by virus indicate a distant relationship between duck interferon and mammalian type I interferons. Purified recombinant duck interferon from Escherichia coli is biologically active: it activates the interferon-inducible Mx gene, prevents cell destruction by cytolytic RNA viruses, and has a strong inhibitory effect on duck hepatitis B virus in cultured primary duck hepatocytes. This new reagent should help to define the interferon-sensitive step of the hepadnavirus life cycle. Furthermore, the duck system can now be used for systematic studies of the in vivo effectiveness of interferon in chronic hepatitis B virus infections.

Recombinant chicken interferon from Escherichia coli and transfected COS cells is biologically active.

We have expressed a cDNA for virus-induced chick interferon in Escherichia coli. The product, a 19-kDa protein lacking the signal peptide, was purified to homogeneity from the bacterial inclusion bodies. Proteins in the insoluble fraction of bacterial lysates were dissolved in guanidine hydrochloride and subjected to chromatography on Q-Sepharose and MonoS columns. Purified recombinant chick interferon has a specific antiviral activity of approximately 10(8) IU/mg and is a powerful inducer of the interferon-responsive promoter of the chicken Mx gene. Culture medium of transfected COS cells expressing full-length chick interferon cDNA contained up to 5 x 10(4) IU antiviral activity/ml that could be neutralized by antibodies to purified recombinant chick interferon. The antibodies precipitated proteins of 23-28 kDa from the supernatants of transfected COS cells. Treatment with endoglycosidase F reduced the size of the immunoprecipitated proteins to approximately 20 kDa, demonstrating that chick interferon is a glycoprotein.

Chicken interferon consensus sequence-binding protein (ICSBP) and interferon regulatory factor (IRF) 1 genes reveal evolutionary conservation in the IRF gene family.

Members of the IRF family mediate transcriptional responses to interferons (IFNs) and to virus infection. So far, proteins of this family have been studied only among mammalian species. Here we report the isolation of cDNA clones encoding two members of this family from chicken, interferon consensus sequence-binding protein (ICSBP) and IRF-1. The predicted chicken ICSBP and IRF-1 proteins show high levels of sequence similarity to their corresponding human and mouse counterparts. Sequence identities in the putative DNA-binding domains of chicken and human ICSBP and IRF-1 were 97% and 89%, respectively, whereas the C-terminal regions showed identities of 64% and 51%; sequence relationships with mouse ICSBP and IRF-1 are very similar. Chicken ICSBP was found to be expressed in several embryonic tissues, and both chicken IRF-1 and ICSBP were strongly induced in chicken fibroblasts by IFN treatment, supporting the involvement of these factors in IFN-regulated gene expression. The presence of proteins homologous to mammalian IRF family members, together with earlier observations on the occurrence of functionally homologous IFN-responsive elements in chicken and mammalian genes, highlights the conservation of transcriptional mechanisms in the IFN system, a finding that contrasts with the extensive sequence and functional divergence of the IFNs.