Distance to treatment as a factor for loss to follow up of hepatitis C patients in North East England.

BACKGROUND: A large proportion of the 200 000 HCV-infected individuals in the UK are undiagnosed or lost to follow-up. Engaging known infected individuals in treatment is essential for elimination. METHODS: Using PHE surveillance data and HCV treatment registers from North East of England (NE) treatment centres for 1997-2016, we estimated the number of HCV cases not linked to treatment and the proportion with active infection. We compared distances of treated and untreated cases to treatment services, and assessed the effect of expanding HCV treatment into existing drug and alcohol treatment centres in the NEE on treatment accessibility. RESULTS: The odds of being treated was associated with distance to treatment services. Confirmatory results for ~50% were not reported to PHE NE. Overall, 3385 patients reported to PHE NE had no record of treatment; we estimated 1621 of these may have been lost to follow-up after confirmation of active infection. CONCLUSIONS: Poor access to healthcare services may contribute to under-diagnosis or loss to follow-up. Expanding HCV treatment delivery into NEE drug and alcohol treatment centres would improve the accessibility of treatment services to people infected with/at risk of HCV. This may increase the proportion receiving treatment and support progress towards elimination.

Large outbreak of multiple gastrointestinal pathogens associated with fresh curry leaves in North East England, 2013.

A total of 592 people reported gastrointestinal illness following attendance at Street Spice, a food festival held in Newcastle-upon-Tyne, North East England in February/March 2013. Epidemiological, microbiological and environmental investigations were undertaken to identify the source and prevent further cases. Several epidemiological analyses were conducted; a cohort study; a follow-up survey of cases and capture re-capture to estimate the true burden of cases. Indistinguishable isolates of Salmonella Agona phage type 40 were identified in cases and on fresh curry leaves used in one of the accompaniments served at the event. Molecular testing indicated entero-aggregative Escherichia coli and Shigella also contributed to the burden of illness. Analytical studies found strong associations between illness and eating food from a particular stall and with food items including coconut chutney which contained fresh curry leaves. Further investigation of the food supply chain and food preparation techniques identified a lack of clear instruction on the use of fresh uncooked curry leaves in finished dishes and uncertainty about their status as a ready-to-eat product. We describe the investigation of one of the largest outbreaks of food poisoning in England, involving several gastrointestinal pathogens including a strain of Salmonella Agona not previously seen in the UK.

An epidemiological review of gastrointestinal outbreaks associated with Clostridium perfringens, North East of England, 2012-2014.

An anecdotal increase in C. perfringens outbreaks was observed in the North East of England during 2012-2014. We describe findings of investigations in order to further understanding of the epidemiology of these outbreaks and inform control measures. All culture-positive (>105 c.f.u./g) outbreaks reported to the North East Health Protection Team from 1 January 2012 to 31 December 2014 were included. Epidemiological (attack rate, symptom profile and positive associations with a suspected vehicle of infection), environmental (deficiencies in food preparation or hygiene practices and suspected vehicle of infection) and microbiological investigations are described. Forty-six outbreaks were included (83% reported from care homes). Enterotoxin (cpe) gene-bearer C. perfringens were detected by PCR in 20/46 (43%) and enterotoxin (by ELISA) and/or enterotoxigenic faecal/food isolates with indistinguishable molecular profiles in 12/46 (26%) outbreaks. Concerns about temperature control of foods were documented in 20/46 (43%) outbreaks. A suspected vehicle of infection was documented in 21/46 (46%) of outbreaks (meat-containing vehicle in 20/21). In 15/21 (71%) identification of the suspected vehicle was based on descriptive evidence alone, in 5/21 (24%) with supporting evidence from an epidemiological study and in 2/21 (10%) with supporting microbiological evidence. C. perfringens-associated illness is preventable and although identification of foodborne outbreaks is challenging, a risk mitigation approach should be taken, particularly in vulnerable populations such as care homes for the elderly.

Case studies of conservation plans that incorporate geodiversity.

Geodiversity has been used as a surrogate for biodiversity when species locations are unknown, and this utility can be extended to situations where species locations are in flux. Recently, scientists have designed conservation networks that aim to explicitly represent the range of geophysical environments, identifying a network of physical stages that could sustain biodiversity while allowing for change in species composition in response to climate change. Because there is no standard approach to designing such networks, we compiled 8 case studies illustrating a variety of ways scientists have approached the challenge. These studies show how geodiversity has been partitioned and used to develop site portfolios and connectivity designs; how geodiversity-based portfolios compare with those derived from species and communities; and how the selection and combination of variables influences the results. Collectively, they suggest 4 key steps when using geodiversity to augment traditional biodiversity-based conservation planning: create land units from species-relevant variables combined in an ecologically meaningful way; represent land units in a logical spatial configuration and integrate with species locations when possible; apply selection criteria to individual sites to ensure they are appropriate for conservation; and develop connectivity among sites to maintain movements and processes. With these considerations, conservationists can design more effective site portfolios to ensure the lasting conservation of biodiversity under a changing climate.

Projected climate-driven faunal movement routes.

Historically, many species moved great distances as climates changed. However, modern movements will be limited by the patterns of human-dominated landscapes. Here, we use a combination of projected climate-driven shifts in the distributions of 2903 vertebrate species, estimated current human impacts on the landscape, and movement models, to determine through which areas in the western hemisphere species will likely need to move to track suitable climates. Our results reveal areas with projected high densities of climate-driven movements - including, the Amazon Basin, the southeastern United States and southeastern Brazil. Some of these regions, such as southern Bolivia and northern Paraguay, contain relatively intact landscapes, whereas others such as the southeastern United States and Brazil are heavily impacted by human activities. Thus, these results highlight both critical areas for protecting lands that will foster movement, and barriers where human land-use activities will likely impede climate-driven shifts in species distributions.

Economic-based projections of future land use in the conterminous United States under alternative policy scenarios.

Land-use change significantly contributes to biodiversity loss, invasive species spread, changes in biogeochemical cycles, and the loss of ecosystem services. Planning for a sustainable future requires a thorough understanding of expected land use at the fine spatial scales relevant for modeling many ecological processes and at dimensions appropriate for regional or national-level policy making. Our goal was to construct and parameterize an econometric model of land-use change to project future land use to the year 2051 at a fine spatial scale across the conterminous United States under several alternative land-use policy scenarios. We parameterized the econometric model of land-use change with the National Resource Inventory (NRI) 1992 and 1997 land-use data for 844 000 sample points. Land-use transitions were estimated for five land-use classes (cropland, pasture, range, forest, and urban). We predicted land-use change under four scenarios: business-as-usual, afforestation, removal of agricultural subsidies, and increased urban rents. Our results for the business-as-usual scenario showed widespread changes in land use, affecting 36% of the land area of the conterminous United States, with large increases in urban land (79%) and forest (7%), and declines in cropland (-16%) and pasture (-13%). Areas with particularly high rates of land-use change included the larger Chicago area, parts of the Pacific Northwest, and the Central Valley of California. However, while land-use change was substantial, differences in results among the four scenarios were relatively minor. The only scenario that was markedly different was the afforestation scenario, which resulted in an increase of forest area that was twice as high as the business-as-usual scenario. Land-use policies can affect trends, but only so much. The basic economic and demographic factors shaping land-use changes in the United States are powerful, and even fairly dramatic policy changes, showed only moderate deviations from the business-as-usual scenario. Given the magnitude of predicted land-use change, any attempts to identify a sustainable future or to predict the effects of climate change will have to take likely land-use changes into account. Econometric models that can simulate land-use change for broad areas with fine resolution are necessary to predict trends in ecosystem service provision and biodiversity persistence.

The functions of thrombospondin-1 and-2.

Considerable progress has been made towards understanding the function of thrombospondin-1 and-2. The description of the phenotype of mice with thrombospondin-1 and-2 knocked-out supports in vitro biochemical and cell-biological data and has opened new avenues of research. Recently, our understanding of the roles of thrombospondins in the activation of TGFbeta, inhibition of angiogenesis and the initiation of signal transduction has advanced.

Increased apoptosis of Huntington disease lymphoblasts associated with repeat length-dependent mitochondrial depolarization.

Huntington disease (HD) is a genetically dominant condition caused by expanded CAG repeats coding for glutamine in the HD gene product huntingtin. Although HD symptoms reflect preferential neuronal death in specific brain regions, huntingtin is expressed in almost all tissues, so abnormalities outside the brain might be expected. Although involvement of nuclei and mitochondria in HD pathophysiology has been suggested, specific intracellular defects that might elicit cell death have been unclear. Mitochondria dysfunction is reported in HD brains; mitochondria are organelles that regulates apoptotic cell death. We now report that lymphoblasts derived from HD patients showed increased stress-induced apoptotic cell death associated with caspase-3 activation. When subjected to stress, HD lymphoblasts also manifested a considerable increase in mitochondrial depolarization correlated with increased glutamine repeats.

Meta-analysis of the impact of postoperative infective complications on oncological outcomes in colorectal cancer surgery.

BACKGROUND: Cancer outcomes are complex, involving prevention, early detection and optimal multidisciplinary care. Postoperative infection and surgical site-infection (SSI) are not only uncomfortable for patients and costly, but may also be associated with poor oncological outcomes. A meta-analysis was undertaken to assess the oncological effects of SSI in patients with colorectal cancer. METHODS: An ethically approved PROSPERO-registered meta-analysis was conducted following PRISMA guidelines. PubMed and Scopus databases were searched for studies published between 2007 and 2017 reporting the effects of postoperative infective complications on oncological survival in colorectal cancer. Results were separated into those for SSI and those concerning anastomotic leakage. Articles with a Methodological Index for Non-Randomized Studies score of at least 18 were included. Hazard ratios (HRs) with 95 per cent confidence intervals were computed for risk factors using an observed to expected and variance fixed-effect model. RESULTS: Of 5027 articles were reviewed, 43 met the inclusion criteria, with a total of 154 981 patients. Infective complications had significant negative effects on overall survival (HR 1·37, 95 per cent c.i. 1·28 to 1·46) and cancer-specific survival (HR 2·58, 2·15 to 3·10). Anastomotic leakage occurred in 7·4 per cent and had a significant negative impact on disease-free survival (HR 1·14, 1·09 to 1·20), overall survival (HR 1·34, 1·28 to 1·39), cancer-specific survival (HR 1·43, 1·31 to 1·55), local recurrence (HR 1·18, 1·06 to 1·32) and overall recurrence (HR 1·46, 1·27 to 1·68). CONCLUSION: This meta-analysis identified a significant negative impact of postoperative infective complications on overall and cancer-specific survival in patients undergoing colorectal surgery.

ANTECEDENTES: Los resultados del cáncer son complejos, implican prevención, detección precoz y atención multidisciplinaria óptima. La infección postoperatoria y la infección del sitio quirúrgico (surgical site infection, SSI) no solo son inconvenientes para los pacientes y costosas, sino que también pueden estar asociadas con malos resultados oncológicos. Este estudio realizó un metaanálisis para evaluar los efectos oncológicos de la SSI en pacientes con cáncer colorrectal. MÉTODOS: Se realizó un metaanálisis registrado en PROSPERO, aprobado por el comité ético, siguiendo las pautas de PRISMA y utilizando las bases de datos PubMed y Scopus para estudios entre 2007-2017 que describían los efectos de las complicaciones infecciosas postoperatorias en la supervivencia oncológica en el cáncer colorrectal. Los resultados se separaron para el grupo de infección del sitio quirúrgico (SSI) y de fuga anastomótica. Se incluyeron los artículos con una puntuación ≥ 18 según el índice MINORS. Para los factores de riesgo se calcularon los cocientes de riesgos instantáneos (hazard ratios, HR) mediante un modelo de efectos aleatorios y el método de Mantel-Haenszel con los i.c. del 95% utilizando el programe RevMan5. RESULTADOS: Se revisaron 5.027 artículos de los cuales 43 cumplieron con los criterios de inclusión. En total fueron 154.981 pacientes en los cuales las complicaciones infecciosas tuvieron efectos negativos significativos en la supervivencia global (HR: 1,37 i.c. del 95%: 1,28-1,46) y la supervivencia específica relacionada con el cáncer (HR: 2,58 i.c. del 95%: 2,15-3,10). La fuga anastomótica ocurrió en un 7,4% de los casos e impactó negativa y significativamente en la supervivencia libre de enfermedad (HR: 1,14 i.c. del 95%: 1,09-1,20), en la supervivencia global (HR: 1,34 i.c. del 95%: 1,28-1,39), en la supervivencia específica relacionada con el cáncer (HR: 1,43 i.c. del 95% 1.31-1.55), en la recidiva local (HR: 1,18 i.c. del 95%: 1,06-1,32) y en la recidiva global (HR: 1,46 i.c. del 95%: 1,27-1,68). CONCLUSIÓN: Este metaanálisis identificó un impacto negativo significativo en la supervivencia global y en la supervivencia específica relacionada con el cáncer en pacientes con complicaciones postoperatorias infecciosas sometidos a cirugía colorrectal.

The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins.

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.

Cell attachment to thrombospondin: the role of ARG-GLY-ASP, calcium, and integrin receptors.

Thrombospondin is a 420,000-D glycoprotein that has recently been shown to have several properties in common with the members of a class of adhesive proteins. To characterize further the adhesive properties of thrombospondin, we have studied its ability to support cell attachment. Thrombospondin adsorbed to plastic dishes supports the attachment of human endothelial and smooth muscle cells and the monocyte-like cell line (U937) as well as normal rat kidney cells. The majority of attached cells do not spread on the solid-phase thrombospondin. The attachment of all four cell types to thrombospondin is abolished if the assay is performed in the presence of EGTA, although the cells still attach to fibronectin. If thrombospondin is adsorbed to the dishes in the presence of EGTA and then washed with buffer containing calcium before addition of the cells, attachment is still markedly inhibited, indicating that calcium affects the conformation and function of thrombospondin. Attachment of all four cell types is also markedly inhibited by the synthetic peptides gly-arg-gly-asp-ser-pro (GRG-DSP) and gly-arg-gly-asp-ala-cys (GRGDAC) but not by the control peptide gly-arg-gly-glu-ser-pro (GRG-ESP). Affinity chromatography of n-octylglucoside extracts of surface-labeled endothelial cells or smooth muscle cells on thrombospondin-Sepharose and GRG-DSP-Affigel columns was used to identify an integrin complex related to glycoprotein IIb-IIIa as an RGD-dependent receptor for thrombospondin. In addition, a monoclonal antibody (LM609) that blocks attachment of endothelial cells to vitronectin, fibrinogen, and von Willebrand factor also inhibits attachment of endothelial cells to thrombospondin. These data indicate that the attachment of cells to thrombospondin is mediated by RGD and calcium-dependent mechanisms and is consistent with the hypothesis that the GRGDAC sequence in thrombospondin is a site for interaction with an integrin receptor of the beta 3 subclass.

Effects of heat shock on the expression of thrombospondin by endothelial cells in culture.

Heat-shock proteins from confluent primary cultures of bovine aortic endothelial cells were analyzed by SDS-polyacrylamide gels. In addition to the increased synthesis of the classical heat-shock proteins, there is an increase of a 180,000-mol wt polypeptide in the growth media of heat-shocked cells. Immunoprecipitation with specific antiserum indicates that the 180,000-mol wt polypeptide is thrombospondin. Assay of mRNA levels coding for thrombospondin after brief hyperthermic treatment (45 degrees C, 10 min), followed by a recovery of 2 h at 37 degrees C, results in a twofold increase in mRNA abundance. In contrast, the activation level of the 71,000-mol wt heat-shock protein mRNA occurs at an earlier time than for thrombospondin mRNA. Immunofluorescence microscopy was used to study the intracellular and extracellular distribution of thrombospondin. Thrombospondin is localized to a prominent pattern of granules of intracellular fluorescence in a perinuclear distribution in cells not exposed to heat. Upon heat treatment, the pattern of granules of intracellular fluorescence appears more pronounced, and the fluorescence appears to be clustered more about the nucleus. There are at least three pools of extracellular forms of thrombospondin: (a) the fine fibrillar extracellular matrix thrombospondin; (b) the punctate granular thrombospondin; and (c) the thrombospondin found in the conditioned medium not associated with the extracellular matrix. When bovine aortic endothelial cells are exposed to heat, the extracellular matrix staining of a fibrillar nature is noticeably decreased, with an increase in the number and degree of fluorescence of focal areas where the punctate granule thrombospondin structures are highly localized. No gross morphological changes in extracellular matrix staining of fibronectin was noted. However, the intermediate filament network was very sensitive and collapsed around the nucleus after heat shock. We conclude that the expression of thrombospondin is heat-shock stimulated.

Identification and characterization of thrombospondin-4, a new member of the thrombospondin gene family.

A new member of the thrombospondin gene family, designated thrombospondin-4, has been identified in the Xenopus laevis genome. The predicted amino acid sequence indicates that the protein is similar to the other members of this gene family in the structure of the type 3 repeats and the COOH-terminal domain. Thrombospondin-4 contains four type 2 repeats and lacks the type 1 repeats that are found in thrombospondin-1 and 2. The amino-terminal domain of thrombospondin-4 has no significant homology with the other members of the thrombospondin gene family or with other proteins in the database. RNAse protection analysis establishes that the initial expression of Xenopus thrombospondin-4 is observed during neurulation. Levels of mRNA expression increase twofold during tailbud stages but decrease by the feeding tadpole stage. The size of the thrombospondin-4 message is 3.3 Kb and 3.4 Kb in the frog and human, respectively. Northern blot analysis of human tissues reveals high levels of thrombospondin-4 expression in heart and skeletal muscle, low levels in brain, lung and pancreas and undetectable levels in the placenta, liver and kidney. These data establish the existence of a new member of the thrombospondin gene family that may participate in the genesis and function of cardiac and skeletal muscle.

Transition Probabilities of Co ii Weak Lines to the Ground and Low Metastable Levels.

New branching fraction (BF) measurements based primarily on data from a cross-dispersed echelle spectrometer are reported for 84 lines of Co ii. The BFs for 82 lines are converted to absolute atomic transition probabilities using radiative lifetimes from laser-induced fluorescence (LIF) measurements on 19 upper levels of the lines. A lifetime of 3.3(2) ns for the z5D0 level is used based on LIF measurements for lifetimes of the four other levels in the z5D term. Twelve of the eighty-four lines are weak transitions connecting to the ground and low metastable levels of Co+. Another 46 lines are strong transitions connecting to the ground and low metastable levels of Co+. For these lines, log(gf) values were measured in earlier studies and, with a few exceptions, are confirmed in this study. Such lines, if unblended in stellar spectra, have the potential to yield Co abundance values unaffected by any breakdown of the local thermodynamic equilibrium approximation in stellar photospheres because the ground and low metastable levels of Co+ are the primary population reservoirs of Co in the photospheres of interest. Weak lines, if unblended, are useful in photospheres with high Co abundance, and strong lines are useful in metal-poor photospheres. New hyperfine structure A constants for 28 levels of ionized Co from least-squares fits to Fourier transform spectra line profiles are reported. These laboratory data are applied to re-determine the Co abundance in the metal-poor halo star HD 84937. BFs and transition probabilities for 19 lines are reported for the first time.

Longitudinal thermal heterogeneity in rivers and refugia for coldwater species: effects of scale and climate change.

Climate-change driven increases in water temperature pose challenges for aquatic organisms. Predictions of impacts typically do not account for fine-grained spatiotemporal thermal patterns in rivers. Patches of cooler water could serve as refuges for anadromous species like salmon that migrate during summer. We used high-resolution remotely sensed water temperature data to characterize summer thermal heterogeneity patterns for 11,308 km of 2nd- to 7th-order rivers throughout the Pacific Northwest and northern California (USA). We evaluated (1) water temperature patterns at different spatial resolutions, (2) the frequency, size, and spacing of cool thermal patches suitable for Pacific salmon (i.e., contiguous stretches ≥0.25 km, ≤15°C and ≥2°C cooler than adjacent water), and (3) potential influences of climate change on availability of cool patches. Thermal heterogeneity was nonlinearly related to the spatial resolution of water temperature data, and heterogeneity at fine resolution (<1 km) would have been difficult to quantify without spatially continuous data. Cool patches were generally >2.7 and <13.0 km long, and spacing among patches was generally >5.7 and <49.4 km. Thermal heterogeneity varied among rivers, some of which had long uninterrupted stretches of warm water ≥20°C, and others had many smaller cool patches. Our models predicted little change in future thermal heterogeneity among rivers, but within-river patterns sometimes changed markedly compared to contemporary patterns. These results can inform long-term monitoring programs as well as near-term climate-adaptation strategies.

A molecular defect of spectrin in a subset of patients with hereditary elliptocytosis. Alterations in the alpha-subunit domain involved in spectrin self-association.

Hereditary elliptocytosis (HE) is a clinically and biochemically heterogenous group of diseases characterized by elliptically shaped erythrocytes and an autosomal dominant mode of inheritance. Whereas the self-association of spectrin heterodimers to tetramers is defective in a subpopulation of HE patients, designated HE[SpD-SpD], it is normal in others. We have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from patients with HE[SpD-SpD] to determine if the functional defects in spectrin self-association could be correlated with structural changes in the spectrin molecule. Although the peptide pattern produced by limited tryptic digestion of spectrin extracts from those HE patients with normal spectrin self-association was indistinguishable from the pattern from control normal volunteers, digestion of the spectrin extracts from the HE[SpD-SpD] patients showed a reproducible diminution in the 80,000-D domain of the alpha-subunit, which is involved in spectrin dimer self-association. The decrease in the 80,000-D fragment was associated with an increase in a 74,000-D fragment in eight of nine families, or, in one family, with an increase of fragments at 46,000 and 17,000 D. These atypical peptide patterns were similar to those previously reported in two variants of hereditary pyropoikilocytosis (HPP), which also had defective self-association of spectrin. These data indicate that two distinct structural variants of spectrin alpha-subunit are associated with the defective spectrin heterodimer self-association in a subpopulation of HE patients.

Molecular defect of spectrin in hereditary pyropoikilocytosis. Alterations in the trypsin-resistant domain involved in spectrin self-association.

In hereditary pyropoikilocytosis (HPP) the erythrocyte membrane skeleton exhibits mechanical instability that can be correlated to defective self-association of spectrin heterodimers. To detect structural changes in the functional domains of HPP spectrin we have examined the peptide pattern produced by limited tryptic digestion of spectrin extracts from two families that contain three HPP patients. Limited tryptic digestion of all three HPP patients revealed a similar and reproducible decrease in the staining intensity of an 80,000-, and 22,000-, and an 88,000-dalton polypeptide with a concomitant increase in a 74,000- and a 90,000-dalton polypeptide as compared with controls. Only changes in the 80,000-, and 74,000-, and 22,000-dalton polypeptides could be correlated to defective spectrin self-association and the amount of spectrin dimers in 0 degrees C extracts of the HPP patients and their affected kindred. Similar results were obtained when the tryptic digests were analyzed by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the affected 74,000- and 80,000-dalton polypeptides focusing into multiple spots ranging in isoelectric point from 5.3-5.4. When HPP spectrin dimers and tetramers were separated and subjected to trypsin digestion, changes in the 80,000-, 74,000-, and 22,000-dalton polypeptides were found predominantly in the spectrin dimer pool. Similar results were obtained for spectrin from two of the probands' mother, whom we have identified as an HPP carrier. We conclude that these HPP patients contain a population of normal, (principally tetrameric) and mutant (principally dimeric) spectrin. The latter is characterized by a defective spectrin dimer self-association due to conformational changes that affect the 80,000-dalton domain.

Platelet-derived growth factor promotes proliferation of erythropoietic progenitor cells in vitro.

To investigate serum requirements for optimal erythropoiesis in vitro, we studied the response of erythroid progenitor cell proliferation in culture to platelet-derived growth factor (PDGF). Human bone marrow cells cultured with platelet-poor plasma-derived serum (PDS) form fewer erythroid colonies than do cells cultured with human whole blood serum or fetal calf serum (P less than 0.05). Treatment of washed platelets with thrombin releases a low molecular weight (less than 100,000) factor that enhances colony growth. This secreted factor appears to be PDGF, based upon the ability of partially purified and electrophoretically pure PDGF to restore colony-forming capacity of PDS-containing cultures to 70-96% of the level found in control cultures with whole blood serum or fetal calf serum. Enhancement of colony growth by PDGF was noted only in marrow cultures supplemented with erythropoietin and PDS. Presence of bioactive erythropoietin in PDGF preparations was excluded by assay in hypertransfused, polycythemic mice, and in fasted rats. Although PDGF stimulates erythroid burst formation in marrow cultures containing optimal concentrations of burst-promoting activity (BPA), it does not influence proliferation of circulating erythroid bursts, regardless of BPA concentration added to culture. We conclude that PDGF is a serum determinant of optimal erythroid progenitor cell proliferation in marrow culture. The activity of PDGF is distinct from that of the apparent erythroid specific growth factors erythropoietin and BPA.

Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia.

The thrombospondins are a family of extracellular calcium-binding proteins that modulate cellular phenotype. Thrombospondin-1 (TSP-1) reportedly regulates cellular attachment, proliferation, migration, and differentiation in vitro. To explore its function in vivo, we have disrupted the TSP-1 gene by homologous recombination in the mouse genome. Platelets from these mice are completely deficient in TSP-1 protein; however, thrombin-induced platelet aggregation is not diminished. TSP-1-deficient mice display a mild and variable lordotic curvature of the spine that is apparent from birth. These mice also display an increase in the number of circulating white blood cells, with monocytes and eosinophils having the largest percent increases. The brain, heart, kidney, spleen, stomach, intestines, aorta, and liver of TSP-1-deficient mice showed no major abnormalities. However, consistent with high levels of expression of TSP-1 in lung, we observe abnormalities in the lungs of mice that lack the protein. Although normal at birth, histopathological analysis of lungs from 4-wk-old TSP-1-deficient mice reveals extensive acute and organizing pneumonia, with neutrophils and macrophages. The macrophages stain for hemosiderin, indicating that diffuse alveolar hemorrhage is occurring. At later times, the number of neutrophils decreases and a striking increase in the number of hemosiderin-containing macrophages is observed associated with multiple-lineage epithelial hyperplasia and the deposition of collagen and elastin. A thickening and ruffling of the epithelium of the airways results from increasing cell proliferation in TSP-1-deficient mice. These results indicate that TSP-1 is involved in normal lung homeostasis.

Acetylated lipoproteins impair erythroid growth factor release from endothelial cells.

Endothelial cells are a known source of hematopoietic growth-enhancing factors, including platelet-derived growth factor (PDGF). In addition, endothelium interacts directly with plasma lipoproteins which have been shown to modulate hematopoiesis. To determine the relationship of these properties, we measured the release of an erythroid growth-enhancing factor from bovine endothelial cells under lipid-loaded and control conditions. Human bone marrow cells cultured under serum-free conditions form more erythroid, granulocyte/macrophage, and mixed hematopoietic colonies when supplemented with endothelial cell-conditioned medium (ECCM) than do controls (P less than 0.05). The activity is expressed over a wide range of erythropoietin, lymphocyte-conditioned medium (LCM), recombinant human interleukin-3, and colony-stimulating factor (CSF) concentrations, and is related to ECCM dose. In contrast, enhancing activity in ECCM prepared with 0-400 micrograms/ml acetylated low density lipoproteins (AcLDL) or native LDL is diminished to 0% in a dose-dependent fashion (relative to ECCM from unexposed cells or from cells incubated with very low density lipoproteins, P less than 0.05). Upon dilution, medium prepared from cells incubated with LDL shows a rightward shift in the dose-response curve for erythroid colony formation, while that prepared from AcLDL loaded cells demonstrates a downward shift, indicating that the inhibitory activities are kinetically distinct. Delipidation of ECCM prior to addition to marrow culture removes the inhibitory action of native LDL (P less than 0.05) but not that of AcLDL (P greater than 0.10). Immunochemical analysis suggests that the erythropoietic activity in ECCM is unrelated to that of PDGF, recombinant human CSF, and erythroid burst-promoting activity (BPA) present in LCM. This conclusion is supported by Northern blot analysis of endothelial cells using a cDNA probe for the v-sis homologue of the PDGF beta chain and by immunoprecipitation of metabolically labeled PDGF. The relative amounts of c-sis transcripts and of secreted PDGF were similar in endothelial cells incubated with or without AcLDL. We conclude that AcLDL impair the synthesis or release of an erythropoietic growth-enhancing factor(s) which is biologically distinct from PDGF and BPA present in LCM.