The orphan receptor CRF2-4 is an essential subunit of the interleukin 10 receptor.

The orphan receptor CRF2-4 is a member of the class II cytokine receptor family (CRF2), which includes the interferon receptors, the interleukin (IL) 10 receptor, and tissue factor. CRFB4, the gene encoding CRF2-4, is located within a gene cluster on human chromosome 21 that comprises three interferon receptor subunits. To elucidate the role of CRF2-4, we disrupted the CRFB4 gene in mice by means of homologous recombination. Mice lacking CRF2-4 show no overt abnormalities, grow normally, and are fertile. CRF2-4 deficient cells are normally responsive to type I and type II interferons, but lack responsiveness to IL-10. By approximately 12 wk of age, the majority of mutant mice raised in a conventional facility developed a chronic colitis and splenomegaly. Thus, CRFB4 mutant mice recapitulate the phenotype of IL-10-deficient mice. These findings suggest that CRF2-4 is essential for IL-10-mediated effects and is a subunit of the IL-10 receptor.

Neutrophil and B cell expansion in mice that lack the murine IL-8 receptor homolog.

Interleukin-8 (IL-8) is a proinflammatory cytokine that specifically attracts and activates human neutrophils. A murine gene with a high degree of homology to the two known human IL-8 receptors was cloned and then deleted from the mouse genome by homologous recombination in embryonic stem (ES) cells. These mice, although outwardly healthy, had lymphadenopathy, resulting from an increase in B cells, and splenomegaly, resulting from an increase in metamyelocytes, band, and mature neutrophils. Thus, this receptor may participate in the expansion and development of neutrophils and B cells. This receptor was the major mediator of neutrophil migration to sites of inflammation and may provide a potential therapeutic target in inflammatory disease.

Induction of type I diabetes by interferon-alpha in transgenic mice.

Type I diabetes is an autoimmune disease involving an interaction between an epigenetic event (possibly a viral infection), the pancreatic beta cells, and the immune system in a genetically susceptible host. The possibility that the type I interferons could mediate this interaction was tested with transgenic mice in which the insulin-producing beta cells expressed an interferon-alpha. These mice developed a hypoinsulinemic diabetes associated with a mixed inflammation centered on the islets. The inflammation and the diabetes were prevented with a neutralizing antibody to the interferon-alpha. Thus, the expression of interferon-alpha by the beta cells could be causal in the development of type I diabetes, which suggests a therapeutic approach to this disease.

Multiple defects of immune cell function in mice with disrupted interferon-gamma genes.

Interferon-gamma (IFN-gamma) is a pleiotrophic cytokine with immunomodulatory effects on a variety of immune cells. Mice with a targeted disruption of the IFN-gamma gene were generated. These mice developed normally and were healthy in the absence of pathogens. However, mice deficient in IFN-gamma had impaired production of macrophage antimicrobial products and reduced expression of macrophage major histocompatibility complex class II antigens. IFN-gamma-deficient mice were killed by a sublethal dose of the intracellular pathogen Mycobacterium bovis. Splenocytes exhibited uncontrolled proliferation in response to mitogen and alloantigen. After a mixed lymphocyte reaction, T cell cytolytic activity was enhanced against allogeneic target cells. Resting splenic natural killer cell activity was reduced in IFN-gamma-deficient mice. Thus, IFN-gamma is essential for the function of several cell types of the murine immune system.

Control of cell pattern in the neural tube by the zinc finger transcription factor and oncogene Gli-1.

Sonic hedgehog (Shh) is a putative morphogen secreted by the floor plate and notochord, which specifies the fate of multiple cell types in the ventral aspect of the vertebrate nervous system. Since in Drosophila the actions of Hh have been shown to be transduced by Cubitus interruptus (Ci), a zinc finger transcription factor, we examined whether a vertebrate homolog of this protein can mediate the functions of Shh in the vertebrate nervous system. Here, we demonstrate that expression of Gli-1, one of three vertebrate homologs of Ci, can be induced by Shh in the neural tube. Further, ectopic expression of Gli-1 in the dorsal midbrain and hindbrain of transgenic mice mimics the effects of ectopically expressed Shh-N, leading to the activation of ventral neural tube markers such as Ptc, HNF-3beta, and Shh; to the suppression of dorsal markers such as Pax-3 and AL-1; and to the formation of ectopic dorsal clusters of dopaminergic and serotonergic neurons. These findings demonstrate that GLI-1 can reproduce the cell patterning actions of Shh in the developing nervous system and provide support for the hypothesis that it is a mediator of the Shh signal in vertebrates.

Osteoblast-specific expression of growth hormone stimulates bone growth in transgenic mice.

Growth hormone (GH) is an important regulator of postnatal growth, acting on a wide variety of target tissues. Here, we show that local production of GH in osteoblasts is able to stimulate bone growth directly without significant systemic effects. Mice were made transgenic by microinjection of an osteocalcin-human GH (osteocalcin-hGH) gene construct in which approximately 1,800 bp of the rat osteocalcin promoter was fused to the hGH gene. Five lines of transgenic mice, each with measurable amounts of serum hGH (ranging from 1 to 1,000 ng/ml), were analyzed. Northern (RNA) blot hybridization showed that the hGH transcript was detectable only in the bone. Further characterization of hGH mRNA distribution by in situ hybridization revealed that in neonates the most intense signal was found in periosteal osteoblasts, while in adults, trabecular and endosteal osteoblasts were favored. In one transgenic line (992-1), hGH was expressed at a much lower level and had minimal systemic effects; however, the local concentrations of hGH in bone were sufficient to stimulate bone growth in these animals.

Differential regulation of amino acid exchange and protein dynamics across splanchnic and skeletal muscle beds by insulin in healthy human subjects.

To define the mechanism of insulin's anticatabolic action, the effects of three different dosages of insulin (0.25, 0.5, and 1.0 mU x kg(-1) x min(-1)) versus saline on protein dynamics across splanchnic and skeletal muscle (leg) beds were determined using stable isotopes of phenylalanine, tyrosine, and leucine in 24 healthy subjects. After an overnight fast, protein breakdown in muscle exceeded protein synthesis, causing a net release of amino acids from muscle bed, while in the splanchnic bed protein synthesis exceeded protein breakdown, resulting in a net uptake of these amino acids. Insulin decreased (P < 0.003) muscle protein breakdown in a dose-dependent manner with no effect on muscle protein synthesis, thus decreasing the net amino acid release from the muscle bed. In contrast, insulin decreased protein synthesis (P < 0.03) in the splanchnic region with no effect on protein breakdown, thereby decreasing the net uptake of the amino acids. In addition, insulin also decreased (P < 0.001) leucine nitrogen flux substantially more than leucine carbon flux, indicating increased leucine transamination (an important biochemical process for nitrogen transfer between amino acids and across the organs), in a dose-dependent manner, with the magnitude of effect being greater on skeletal muscle than on the splanchnic bed. In conclusion, muscle is in a catabolic state in human subjects after an overnight fast and provides amino acids for synthesis of essential proteins in the splanchnic bed. Insulin achieves amino acid balance across splanchnic and skeletal muscle beds through its differential effects on protein dynamics in these tissue beds.

Insulin regulation of regional free fatty acid metabolism.

Studies were conducted to determine whether regional free fatty acid (FFA) release is differentially regulated by insulin. Systemic, leg, and splanchnic palmitate rate of appearance ([9,10-(3)H]palmitate) was measured in 26 healthy adults using the euglycemic-hyperinsulinemic clamp technique to achieve a physiological range of plasma insulin concentrations. We found that insulin inhibited systemic, leg, and splanchnic palmitate release in a dose-dependent fashion over the range of insulin infused (0-1.0 mU x kg(-1) x min(-1)). Progressive hyperinsulinemia changed the leg from a net producer to a net FFA consumer, whereas the splanchnic bed converted from a net FFA consumer to a net producer. At the 0.5 mU x kg(-1) x min(-1) insulin infusion rate, leg FFA release was almost completely suppressed, whereas even with the 1.0 mU x kg(-1) x min(-1) insulin infusion rate, splanchnic FFA release decreased by only approximately 65% (P < 0.05 leg vs. splanchnic). These results demonstrate the regional heterogeneity of insulin-regulated FFA release in vivo, and indicate that visceral adipose tissue lipolysis is more resistant to insulin suppression than is leg lipolysis in humans.

When should we thrombolyse patients with pulmonary embolism? A systematic review of the literature.

The early mortality in pulmonary embolism (PE) is largely predicted by the associated cardiovascular response, with progressive right ventricular failure, hypotension, shock, and circulatory arrest being associated with increasing mortality. Thrombolysis may improve the prognosis of PE associated with these varying degrees of circulatory collapse, but has no place in the treatment of small emboli with no cardiovascular compromise, as it carries a significant risk of haemorrhage. This review sets out to guide the emergency physician in deciding which patients with PE may benefit from thrombolysis.

Entomological surveillance following a long-lasting insecticidal net universal coverage campaign in Midwestern Uganda.

BACKGROUND: A universal coverage campaign (UCC) with long-lasting insecticidal nets (LLINs) was implemented in four districts in Midwestern Uganda in 2009-2010. Entomological surveys were carried out to monitor changes in vector density, behaviour and malaria transmission following this intervention. METHODS: Anopheles mosquitoes were collected using CDC light traps quarterly and human landing catch twice a year in four sites. Collections were done at baseline before the campaign and over a three-year period following the campaign. Plasmodium falciparum circumsporozoite enzyme-linked immunosorbent assays were performed. A subset of anophelines were molecularly identified to species, and kdr L1014S frequencies were determined. RESULTS: The prevailing malaria vector in three sites was Anopheles gambiae s.l. (>97 %), with An. funestus s.l. being present in low numbers only. An. gambiae s.s. dominated (> 95 %) over An. arabiensis within A. gambiae s.l. In the remaining site, all three vector species were observed, although their relative densities varied among seasons and years. Vector densities were low in the year following the UCC but increased over time. Vector infectivity was 3.2 % at baseline and 1.8 % three years post-distribution (p = 0.001). The daily entomological inoculation rate (EIR) in 2012 varied between 0.0-0.98 for the different sites compared to a baseline EIR that was between 0.0-5.8 in 2009. There was no indication of a change in indoor feeding times, and both An. gambiae s.l. and An. funestus s.l. continued to feed primarily after midnight with vectors being active until the early morning. Kdr L1014S frequencies were already high at baseline (53-85 %) but increased significantly in all sites over time. CONCLUSIONS: The entomological surveys indicate that there was a reduction in transmission intensity coinciding with an increase in use of LLINs and other antimalarial interventions in areas of high malaria transmission. There was no change in feeding behaviour, and human-vector contact occurred indoors and primarily after midnight constantly throughout the study. Although the study was not designed to evaluate the effectiveness of the intervention compared to areas with no such intervention, the reduction in transmission occurred in an area with previously stable malaria, which seems to indicate a substantial contribution of the increased LLIN coverage.

An evaluation of the functions of the 22-kilodalton (kDa), the 20-kDa, and the N-terminal polypeptide forms of human growth hormone using transgenic mice.

Multiple peptide hormones can be derived from the single human GH gene. In addition to the full-length 191-amino acid 22-kilodalton (kDa) form, a 20-kDa variant can be produced by alternative splicing, and a 5-kDa variant can be produced by posttranslational cleavage. To more fully appreciate the physiological roles of these proteins, we have made a comparison of transgenic mice that constitutively overexpress one or another of these variants. We have found that both the 22-kDa and the 20-kDa forms of human GH stimulate linear growth and liver hypertrophy. The increase in linear growth in 22-kDa transgenic mice does not, however, correlate with an increase in circulating IGF-I; rather, the increase in IGF-I that does finally occur correlates with marked liver pathology. Both groups of mice also develop glomerulosclerosis and suffer from hyperinsulinemia. Although there are histologically obvious lesions in the livers of both the 22-kDa and the 20-kDa transgenic mice, only the former exhibit hyperalbuminemia and hypercholesterolemia. Both forms of GH lead to anemia, which is normocytic in the 20-kDa transgenic mice and macrocytic in the 22-kDa transgenic mice. Despite the presence of high levels of the 5-kDa N-terminal form of human GH, the transgenic mice that express this protein are indistinguishable from their nontransgenic littermates.

Isolation and characterisation of the retinoic acid receptor-alpha gene in the Japanese pufferfish, F. rubripes.

Nuclear hormone receptors (NRs) are ligand-inducible transcription factors that mediate critical functions in many species. The majority of novel NRs have hitherto been cloned from cDNA libraries by virtue of their homology to previously identified receptors. In this study, we validate a genomic DNA-based approach to isolating NRs by cloning the retinoic acid receptor-alpha (RARalpha) gene from the genome of the Japanese pufferfish, Fugu rubripes. The fRARalpha gene is more compact than its human and murine counterparts and demonstrates a highly conserved genomic organisation and amino acid sequence, generating two isoforms (fRARalpha1 and fRARalpha2) with divergent aminoterminal domains. In addition, a conserved regulatory element containing a retinoic acid response element was identified upstream of the fRARalpha2-specific exon, implying that retinoid induction of this isoform is evolutionarily conserved and critical to its function in vivo. We propose two uses for the Fugu genome in the study of NRs: the isolation of novel NRs that exhibit restricted spatio-temporal expression from genomic DNA and the identification of evolutionarily conserved promoter or intragenic regulatory DNA elements.

Sequence scanning chicken cosmids: a methodology for genome screening.

The chicken genome is relatively poorly studied at the molecular level. The karyotype 2n=78 is divided into three main chromosomal sub-groups: the macrochromosomes (six pairs), the intermediate microchromosomes (four pairs) and the microchromosomes (29 pairs). Whilst the microchromosome group comprise only 25% of the DNA, increasing evidence is proving that this is disproportionate to their gene content. This paper demonstrates the utility of cosmid sequence scanning as a potential method for analysing the chicken genome, providing an economical method for the production of a molecular map. The GC content, gene density and repeat distribution are analysed relative to chromosomal origin. Results indicate that gene density is higher on the microchromosomes. During the scanning process an example of conserved linkage between chicken and human (12q34.2) has been demonstrated.

Microcystin affinity purification of plant protein phosphatases: PP1C, PP5 and a regulatory A-subunit of PP2A.

Proteins of approximately 35, 55 and 65kDa were purified from cauliflower extracts by microcystin-Sepharose chromatography and identified by amino acid sequencing as plant forms of protein (serine/threonine) phosphatase 1 (PP1) catalytic subunit, PP5 and a regulatory A-subunit of PP2A, respectively. Peptides that corresponded both to the tetratricopeptide (TPR) repeat and catalytic domains of PP5 were identified. Similar to mammalian PP5,the casein phosphatase activity of plant PP5 was activated >10-fold by arachidonic acid, with half-maximal stimulation occurring at approximately 100 microM lipid.

Murine typhus among Khmers living at an evacuation site on the Thai-Kampuchean border.

An outbreak of febrile disease involving 170 Khmer adults at an evacuation site in Thailand occurred during the dry season of 1986, only 8 months after the camp was constructed. The illnesses were characterized by persistent fever, retro-orbital headache, myalgias, and clinical response to tetracycline within 2-3 days. The symptoms, effectiveness of tetracycline, and presence of a large rat population raised the suspicion of murine typhus. Fourteen (74%) of 19 patients had elevated or rising antibody titers against Rickettsia typhi, confirming the clinical diagnosis. Rats were caught, and they and their fleas were identified. In agreement with the known Thai host and vector, 80 (93%) of 86 rats were Rattus exulans, and all of 32 fleas were Xenopsylla cheopis. This first reported outbreak of murine typhus in Thailand is notable for its occurrence in a new human settlement only 8 months after construction.

Treatment of falciparum malaria with quinne and tetracycline or combined mefloquine/sulfadoxine/pyrimethamine on the Thai-Kampuchean border.

Three different regimens were compared for treatment of falciparum malaria in displaced Kampucheans living in encampments on the Thai-Kampuchean border in 1983: single dose 750 mg mefloquine, 1.5 g sulfadoxine, 75 mg pyrimethamine (MSP); 600 mg quinine 8-hourly for 3 days and 500 mg tetracycline 8-hourly for 7 days (Q3T7); or 600 mg quinine 8-hourly for 7 days and 500 mg tetracycline 8-hourly for 7 days (Q7T7). Radical cure rates were 98% (40/41) for MSP, 76% (32/42) for Q3T7 and 92% (33/36) for Q7T7. The criterion for treatment failure was reappearance of parasites by 35 days after commencement of treatment or no parasite clearance. Treatment failures comprised one case of reduction but no clearance of parasites (RII resistance) for MSP, 10 recrudescences (RI) for Q3T7 and 3 recrudescences (RI) for Q7T7. The radical cure rate for Q3T7 was significantly lower than that for MSP (P less than 0.01), whilst Q7T7 significantly from the other groups. Parasite clearance time was shorter (2.4 days) with MSP than with Q3T7 (3.5 days) and Q7T7 (3.3 days). There was little difference in side effects between the regimens, and tolerance was good. The MSP and Q7T7 regimens are both effective for treatment, but the single dose of MSP is much easier to manage than 7 days of quinine and tetracycline.

Quantitation of human shoulder anatomy for prosthetic arm control--II. Anatomy matrices.

Part I presented mathematically continuous surfaces and origin-to-insertion centroidal trajectory data for the muscles of the human shoulder. Part II presents linear trajectory data for the same muscles, in addition to kinematic descriptions of the joints. 'Anatomy' matrices for musculature, which convert muscle forces (as estimated by cutaneously monitored EMG signals) to moments, within a prosthetic arm controller, are developed for both the linear and non-linear (centroidal) data, and then compared. Graphical analyses of the muscle functions are also presented via computer-generated 'circle diagrams'.

Quantitation of human shoulder anatomy for prosthetic arm control--I. Surface modelling.

Anatomical data and models for the human shoulder musculo-skeletal system are developed with the intent of quantifying physiological subcomponents of a model-based multi-axis prosthetic limb control scheme which has heretofore been implemented empirically. Part I presents the controller formulation, the surface descriptions of the muscles (and bones), and the centroidal trajectory data of the muscles. The data partially quantify the muscle modelling components of the controller, and set the stage for the analysis of the force-to-moment anatomical conversion factors of Part II.

Use of the alveolar dead space fraction (Vd/Vt) and plasma D-dimers to exclude acute pulmonary embolism in ambulatory patients.

OBJECTIVE: To evaluate the utility of a modified calculation of the alveolar dead space fraction (Vd/Vt), combined with plasma D-dimers, to aid in the exclusion of acute pulmonary embolism (PE). METHODS: A prospective comparison of screening modalities was performed in a metropolitan teaching ED. Ambulatory patients evaluated for PE underwent simultaneous end-tidal CO2 and arterial blood gas determinations, as well as venous latex-agglutination D-dimer quantification. The modified Bohr equation was used to calculate Vd/Vt as an index of alveolar dead space. Acute PE was diagnosed or excluded using appropriate combinations of clinical suspicion, ventilation-perfusion lung scanning, lower-extremity venous Doppler ultrasonography, pulmonary angiography, and comprehensive follow-up. RESULTS: Of 170 subjects studied, PE was confirmed (PE+) in 26 (15%) and excluded (PE-) in 144 (85%). In the PE+ group, Vd/Vt was 0.31 +/- 0.13 (mean +/- SD), and in the PE- group, Vd/Vt was 0.06 +/- 0.10 (p < 0.05, t-test). Regarding false-negative rates, Vd/Vt was normal (i.e., < 0.2) in 3/26 PE+ patients and D-dimer concentrations were normal (< 0.5 microgram/L) in 4/26 patients in the PE+ group. The combination of a normal Vd/Vt and D-dimer concentration was 100% sensitive (95% CI = 88-100%) in excluding PE. False-positive testing (either test positive) occurred in 49/144 subjects (specificity 65%, 95% CI = 52-73%). The age-adjusted alveolar-arterial O2 gradient was 33 +/- 38 torr in the PE+ group vs 13 +/- 37 torr in the PE- group (p = 0.11). CONCLUSIONS: In ambulatory patients, the finding of Vd/Vt < 0.2 and D-dimers < 0.5 microgram/L lowers the probability of acute PE.

Fatigue compensation of the electromyographic signal for prosthetic control and force estimation.

During a sustained muscle contraction, the amplitude of electromyographic (EMG) signals increases and the spectrum of the EMG signal shifts toward lower frequencies. These effects are due to muscular fatigue and can cause problems in the control of myoelectric prostheses and in the estimation of contraction level from the EMG signal. It has been well known that the fatigue effects can be explained by the conduction velocity changes during the fatigue process and by the idea that the conduction velocity is linearly proportional to the median frequency of EMG signals. Hence the fatigue process can be monitored by measuring the median frequency. A fatigue compensation preprocessor has been developed. It uses the widely accepted power spectrum density model of EMG signals that contains the conduction velocity as a measure of fatigue. It was verified that the preprocessor scales down the amplitude of the fatigued EMG signal and decompresses the spectrum. Hence, the preprocessor eliminates the increase in amplitude and the shift in frequency and enables consistent EMG signals to be used to control prostheses.