Access to health care among status Aboriginal people with chronic kidney disease

Background

Ethnic disparities in access to health care and health outcomes are well documented. It is unclear whether similar differences exist between Aboriginal and non-Aboriginal people with chronic kidney disease in Canada. We determined whether access to care differed between status Aboriginal people (Aboriginal people registered under the federal Indian Act) and non-Aboriginal people with chronic kidney disease.

Methods

We identified 106 511 non-Aboriginal and 1182 Aboriginal patients with chronic kidney disease (estimated glomerular filtration rate less than 60 mL/min/1.73 m²). We compared outcomes, including hospital admissions, that may have been preventable with appropriate outpatient care (ambulatory-care–sensitive conditions) as well as use of specialist services, including visits to nephrologists and general internists.

Results

Aboriginal people were almost twice as likely as non-Aboriginal people to be admitted to hospital for an ambulatory-care–sensitive condition (rate ratio 1.77, 95% confidence interval [CI] 1.46–2.13). Aboriginal people with severe chronic kidney disease (estimated glomerular filtration rate < 30 mL/min/1.73 m²) were 43% less likely than non-Aboriginal people with severe chronic kidney disease to visit a nephrologist (hazard ratio 0.57, 95% CI 0.39–0.83). There was no difference in the likelihood of visiting a general internist (hazard ratio 1.00, 95% CI 0.83–1.21).

Interpretation

Increased rates of hospital admissions for ambulatory-care–sensitive conditions and a reduced likelihood of nephrology visits suggest potential inequities in care among status Aboriginal people with chronic kidney disease. The extent to which this may contribute to the higher rate of kidney failure in this population requires further exploration.Ethnic disparities in access to health care are well documented;^1,2 however, the majority of studies include black and Hispanic populations in the United States. The poorer health status and increased mortality among Aboriginal populations than among non-Aboriginal populations,^3,4 particularly among those with chronic medical conditions,^5,6 raise the question as to whether there is differential access to health care and management of chronic medical conditions in this population.The prevalence of end-stage renal disease, which commonly results from chronic kidney disease, is about twice as common among Aboriginal people as it is among non-Aboriginal people.^7,8 Given that the progression of chronic kidney disease can be delayed by appropriate therapeutic interventions^9,10 and that delayed referral to specialist care is associated with increased mortality,^11,12 issues such as access to health care may be particularly important in the Aboriginal population. Although previous studies have suggested that there is decreased access to primary and specialist care in the Aboriginal population,^13–15 these studies are limited by the inclusion of patients from a single geographically isolated region,¹³ the use of survey data,¹⁴ and the inability to differentiate between different types of specialists and reasons for the visit.¹⁵In addition to physician visits, admission to hospital for ambulatory-care–sensitive conditions (conditions that, if managed effectively in an outpatient setting, do not typically result in admission to hospital) has been used as a measure of access to appropriate outpatient care.^16,17 Thus, admission to hospital for an ambulatory-care–sensitive condition reflects a potentially preventable complication resulting from inadequate access to care. Our objective was to determine whether access to health care differs between status Aboriginal (Aboriginal people registered under the federal Indian Act) and non-Aboriginal people with chronic kidney disease. We assess differences in care by 2 measures: admission to hospital for an ambulatory-care–sensitive condition related to chronic kidney disease; and receipt of nephrology care for severe chronic kidney disease as recommended by clinical practice guidelines.¹⁸     

Impaired Quality of the Hepatitis B Virus (HBV)-Specific T-Cell Response in Human Immunodeficiency Virus Type 1-HBV Coinfection

Hepatits B virus (HBV)-specific T cells play a key role both in the control of HBV replication and in the pathogenesis of liver disease. Human immunodeficiency virus type 1 (HIV-1) coinfection and the presence or absence of HBV e (precore) antigen (HBeAg) significantly alter the natural history of chronic HBV infection. We examined the HBV-specific T-cell responses in treatment-naïve HBeAg-positive and HBeAg-negative HIV-1-HBV-coinfected (n = 24) and HBV-monoinfected (n = 39) Asian patients. Peripheral blood was stimulated with an overlapping peptide library for the whole HBV genome, and tumor necrosis factor alpha and gamma interferon cytokine expression in CD8⁺ T cells was measured by intracellular cytokine staining and flow cytometry. There was no difference in the overall magnitude of the HBV-specific T-cell responses, but the quality of the response was significantly impaired in HIV-1-HBV-coinfected patients compared with monoinfected patients. In coinfected patients, HBV-specific T cells rarely produced more than one cytokine and responded to fewer HBV proteins than in monoinfected patients. Overall, the frequency and quality of the HBV-specific T-cell responses increased with a higher CD4⁺ T-cell count (P = 0.018 and 0.032, respectively). There was no relationship between circulating HBV-specific T cells and liver damage as measured by activity and fibrosis scores, and the HBV-specific T-cell responses were not significantly different in patients with either HBeAg-positive or HBeAg-negative disease. The quality of the HBV-specific T-cell response is impaired in the setting of HIV-1-HBV coinfection and is related to the CD4⁺ T-cell count.There are 40 million people worldwide infected with human immunodeficiency virus type 1 (HIV-1), and 6 to 15% of HIV-1-infected patients are also chronically infected with hepatitis B virus (HBV) (13, 20, 35, 38, 40-42, 47, 50, 61, 69). The highest rates of coinfection with HIV-1 and HBV are in Asia and Africa, where HBV is endemic (33, 68). Following the introduction of highly active antiretroviral therapy (HAART), liver disease is now the major cause of non-AIDS-related deaths in HIV-1-infected patients (12, 13, 38, 59, 65).Coinfection of HBV with HIV-1 alters the natural history of HBV infection. Individuals with HIV-1-HBV coinfection seroconvert from HBV e (precore) antigen (HBeAg) to HBV e antibody less frequently and have higher HBV DNA levels but lower levels of alanine aminotransferase (ALT) and milder necroinflammatory activity on histology than those infected with HBV alone (18, 26, 49). Progression to cirrhosis, however, seems to be more rapid and more common, and liver-related mortality is higher, in HIV-1-HBV coinfection than with either infection alone (47, 59). HBeAg is an accessory protein of HBV and is not required for viral replication or infection; however, chronic HBV infection typically is divided into two distinct phases: HBeAg positive and HBeAg negative (reviewed in reference 15). Most natural history studies of HIV-1-HBV coinfection to date have primarily focused on HBeAg-positive patients from non-Asian countries (23, 44, 46).We previously developed an overlapping peptide library for the HBV genome to detect HBV-specific CD4⁺ and CD8⁺ T-cell responses to all HBV gene products from multiple HBV genotypes (17). In a small cross-sectional study of patients recruited in Australia, we found that in coinfected patients, HBV-specific CD4⁺ T-cell responses, as measured by gamma interferon (IFN-γ) production, were diminished compared to those seen in HBV-monoinfected patients (17). However, patients had varying lengths of exposure to anti-HBV-active HAART at the time of analysis. In this study, therefore, we aimed to characterize the HBV-specific T-cell response in untreated HBeAg-positive and HBeAg-negative HIV-1-HBV-coinfected patients and to determine the relationship between the HBV-specific immune response, HBeAg status, and liver disease.     

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Na?ve and Formalin-Inactivated RSV-Immunized BALB/c Mice

We examined whether prophylactically administered anti-respiratory syncytial virus (anti-RSV) G monoclonal antibody (MAb) would decrease the pulmonary inflammation associated with primary RSV infection and formalin-inactivated RSV (FI-RSV)-enhanced disease in mice. MAb 131-2G administration 1 day prior to primary infection reduced the pulmonary inflammatory response and the level of RSV replication. Further, intact or F(ab′)₂ forms of MAb 131-2G administered 1 day prior to infection in FI-RSV-vaccinated mice reduced enhanced inflammation and disease. This study shows that an anti-RSV G protein MAb might provide prophylaxis against both primary infection and FI-RSV-associated enhanced disease. It is possible that antibodies with similar reactivities might prevent enhanced disease and improve the safety of nonlive virus vaccines.Respiratory syncytial virus (RSV) infection in infants and young children causes substantial bronchiolitis and pneumonia (11, 27, 28, 40) resulting in 40,000 to 125,000 hospitalizations in the United States each year (27). RSV is also a prominent cause of respiratory illness in older children; those of any age with compromised cardiac, pulmonary, or immune systems; and the elderly (6, 7, 11, 17, 18, 39). Despite extensive efforts toward vaccine development (3, 5, 8, 20, 30, 38), none is yet available. Currently, only preventive measures are available that focus on infection control to decrease transmission and prophylactic administration of a humanized IgG monoclonal antibody (MAb) directed against the F protein of RSV (palivizumab) that is recommended for high-risk infants and young children (4, 7, 17). To date, no treatment has been highly effective for active RSV infection (17, 21).The first candidate vaccine, a formalin-inactivated RSV (FI-RSV) vaccine developed in the 1960s, not only failed to protect against disease but led to severe RSV-associated lower respiratory tract infection in young vaccine recipients upon subsequent natural infection (8, 16). The experience with FI-RSV has limited nonlive RSV vaccine development for the RSV-naïve infant and young child. Understanding the factors contributing to disease pathogenesis and FI-RSV vaccine-enhanced disease may identify ways to prevent such a response and to help achieve a safe and effective vaccine.The RSV G, or attachment, protein has been implicated in the pathogenesis of disease after primary infection and FI-RSV-enhanced disease (2, 26, 31). The central conserved region of the G protein contains four evolutionarily conserved cysteines in a cysteine noose structure, within which lies a CX3C chemokine motif (9, 29, 34). The G protein CX3C motif is also immunoactive, as suggested by studies with the mouse model that show that G protein CX3C motif interaction with CX3CR1 alters pulmonary inflammation (41), RSV-specific T-cell responses (12), FI-RSV vaccine-enhanced disease, and expression of the neurokinin substance P (14) and also depresses respiratory rates (32). Recent studies demonstrated that therapeutic treatment with a murine anti-RSV G protein monoclonal antibody (MAb 131-2G) which blocks binding to CX3CR1 can reduce pulmonary inflammation associated with primary infection (13, 23). These findings led us to hypothesize that prophylactic administration of this anti-RSV G monoclonal antibody may also diminish pulmonary inflammation associated with RSV infection in naïve and in FI-RSV-vaccinated mice. In this study, we evaluate the impact of prophylactic administration of MAb 131-2G on the pulmonary inflammatory response to primary infection and to RSV challenge following FI-RSV immunization in mice.     

Dynamin Reduces Pyk2 Y402 Phosphorylation and Src Binding in Osteoclasts

Signaling via the Pyk2-Src-Cbl complex downstream of integrins contributes to the assembly, organization, and dynamics of podosomes, which are the transient adhesion complexes of highly motile cells such as osteoclasts and dendritic cells. We previously demonstrated that the GTPase dynamin is associated with podosomes, regulates actin flux in podosomes, and promotes bone resorption by osteoclasts. We report here that dynamin associates with Pyk2, independent of dynamin''s GTPase activity, and reduces Pyk2 Y402 phosphorylation in a GTPase-dependent manner, leading to decreased Src binding to Pyk2. Overexpressing dynamin decreased the macrophage colony-stimulating factor- and adhesion-induced phosphorylation of Pyk2 in osteoclastlike cells, suggesting that dynamin is likely to regulate Src-Pyk2 binding downstream of integrins and growth factor receptors with important cellular consequences. Furthermore, catalytically active Src promotes dynamin-Pyk2 association, and mutating specific Src-phosphorylated tyrosine residues in dynamin blunts the dynamin-induced decrease in Pyk2 phosphorylation. Thus, since Src binds to Pyk2 through its interaction with phospho-Y402, our results suggest that Src activates a negative-feedback loop downstream of integrin engagement and other stimuli by promoting both the binding of dynamin to Pyk2-containing complexes and the dynamin-dependent decrease in Pyk2 Y402 phosphorylation, ultimately leading to the dissociation of Src from Pyk2.Podosomes are specialized transient actin-containing adhesion structures (11, 14, 37, 60) that are found in highly motile cells, such as osteoclasts, macrophages, dendritic cells, transformed metastatic cells, and v-src-transformed cells (37, 43), where they are thought to play important roles in cellular migration and invasion (34). In resorbing osteoclasts on bone, podosomes are concentrated within the sealing zone, a beltlike actin-rich structure that is important for adhesion and which delineates the resorptive region of the cell known as the ruffled border. Unlike focal adhesions, which are relatively stable structures (11, 60), the assembly and disassembly of podosomes occurs within minutes (t_1/2 = 2 to 4 min) and involves the recruitment and activation of integrins, signaling proteins and scaffolding proteins (11, 14, 35, 47, 60). However, the mechanisms of action of key signaling proteins involved in podosome assembly and disassembly are only partially understood.The focal adhesion kinase Pyk2 has been linked to the proliferation, migration, and activity of a variety of mesenchymal, epithelial, and hematopoietic cell types. Several groups, including our own, have reported the importance of Pyk2 in podosome belt organization, cell spreading, and bone-resorbing activity in osteoclasts (18, 26, 31, 40, 65, 66). Pyk2 is recruited to activated β₂ and β₃ integrins (9, 20) at adhesion sites and is autophosphorylated at Y402 (17, 47, 50) via an intermolecular trans-acting mechanism (46). Although Pyk2 is partially activated by integrin-induced Ca²⁺ signaling (20, 50), the induction of Pyk2''s full catalytic activity requires the binding of Src via its SH2 domain to autophosphorylated Pyk2 Y402 and the subsequent phosphorylation of Pyk2 at functionally distinct sites, including Y579, Y580, and Y881 (17, 31, 46). The binding of Src to phosphorylated Pyk2, which leads to the formation of a multiprotein signaling complex at adhesion sites (17, 40, 50), is critical for Pyk2 activity, as demonstrated by the fact that Pyk2 phosphorylation and activity are significantly reduced in osteoclasts derived from Src^−/− mice (17, 40). Src^−/− osteoclasts also exhibit decreased motility (50) and decreased bone-resorbing activity (40, 54, 59), and we recently demonstrated that Src promotes both podosome formation and disassembly, as well as actin flux into existing podosomes and the organization of podosomes into a peripheral belt in osteoclasts (15).We have also demonstrated that the GTP-hydrolyzing protein dynamin-2, which is ubiquitously expressed and well known for its role in endocytosis (53), regulates actin remodeling in the podosomes of osteoclasts and Rous sarcoma virus-transformed baby hamster kidney cells (43). In addition, a dynamin-2 mutant that binds GTP with reduced affinity (dynK44A) (12) decreased the flux of actin into podosomes (43) and disrupted podosome belt formation in osteoclasts, thereby affecting osteoclast migration and bone-resorbing activity (8). The dynamin proteins, of which there are three homologous isoforms (3), contain several protein domains: a GTP-hydrolyzing domain (GTPase), a plextrin homology domain that mediates binding to phosphoinositides, a GTPase effector domain (GED), and a C-terminal proline-rich domain (PRD) (38, 45, 55) through which dynamin binds a number of functionally diverse SH3-containing molecules, such as Src, cortactin, Grb2, and N-Wasp (1, 7, 27, 39, 58). We previously reported that dynamin-2 partially colocalizes and associates with the E3-ubiquitin ligase Cbl within the podosome belt/sealing zone of osteoclasts, as well as in SYF cells, which lack the Src family kinases Src, Yes, and Fyn, and in HEK 293 cells that stably express the vitronectin receptor (293VnR) (8). Protein complexes containing dynamin-2 and Cbl, which are both substrates of Src (1, 2, 23, 50, 56), were disrupted in the presence of activated Src and stabilized in the absence of Src (8), demonstrating a key role of Src in regulating the formation of signaling complexes in osteoclasts downstream of integrins.In the present study, we sought to determine whether dynamin, which regulates podosome actin dynamics and bone resorption in osteoclasts, also associates with Pyk2 and/or regulates Pyk2''s activities in osteoclasts. We report here that dynamin associates with Pyk2 and promotes the dephosphorylation of Pyk2 Y402 and that catalytically active Src promotes both dynamin''s association with Pyk2 and the dynamin-induced dephosphorylation of Pyk2 Y402, resulting, in turn, in the decreased binding of Src to Pyk2. Thus, we propose that dynamin regulates podosome dynamics and osteoclast bone-resorbing activity by promoting the disassembly of the Pyk2-Src-Cbl complex that is formed in osteoclasts downstream of β₃ integrin activation.     

Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast

During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.Yeast sporulation is a developmental process that involves multiple, sequential events that need to be tightly coordinated (59, 68). In the fission yeast Schizosaccharomyces pombe, when cells of opposite mating type (h⁺ and h⁻) are mixed and shifted to conditions of nitrogen starvation, cell fusion and karyogamy occur to form a diploid zygote, which then undergoes premeiotic DNA replication, the two meiotic divisions, formation of the spore envelopes (comprising the plasma membrane and a specialized cell wall), and maturation of the spores (74, 81). At the onset of meiosis II, precursors of the spore envelopes, the forespore membranes (FSMs), are formed by the fusion of vesicles at the cytoplasmic surface of each spindle-pole body (SPB) and then extend to engulf the four nuclear lobes (the nuclear envelope does not break down during meiosis), thus capturing the haploid nuclei, along with associated cytoplasm and organelles, to form the nascent spores (55, 68, 81). How the FSMs recognize and interact with the nuclear envelope, extend in a properly oriented manner, and close to form uniformly sized spherical spores is not understood, and study of this model system should also help to elucidate the more general question of how membranes obtain their shapes in vivo.It has been shown that both the SPB and the vesicle trafficking system play important roles in the formation and development of the FSM and of its counterpart in the budding yeast Saccharomyces cerevisiae, the prospore membrane (PSM). In S. pombe, the SPB changes its shape from a compact dot to a crescent at metaphase of meiosis II (26, 29), and its outer plaque acquires meiosis-specific components such as Spo2, Spo13, and Spo15 (30, 57, 68). This modified outer plaque is required for the initiation of FSM assembly. In S. cerevisiae, it is well established that various secretory (SEC) gene products are required for PSM formation (58, 59). Similarly, proteins presumably involved in the docking and/or fusion of post-Golgi vesicles and organelles in S. pombe, such as the syntaxin-1A Psy1, the SNAP-25 homologue Sec9, and the Rab7 GTPase homologue Ypt7, are also required for proper FSM extension (34, 53, 54). Consistent with this hypothesis, Psy1 disappears from the plasma membrane upon exit from meiosis I and reappears in the nascent FSM.Phosphoinositide-mediated membrane trafficking also contributes to the development of the FSM. Pik3/Vps34 is a phosphatidylinositol 3-kinase whose product is phosphatidylinositol 3-phosphate [PtdIns(3)P] (35, 72). S. pombe cells lacking this protein exhibit defects in various steps of FSM formation, such as aberrant starting positions for extension, disoriented extension and/or failure of closure, and the formation of spore-like bodies near, rather than surrounding, the nuclei, suggesting that Pik3 plays multiple roles during sporulation (61). The targets of PtdIns(3)P during sporulation appear to include two sorting nexins, Vps5 and Vps17, and the FYVE domain-containing protein Sst4/Vps27. vps5Δ and vps17Δ mutant cells share some of the phenotypes of pik3Δ cells (38). sst4Δ cells also share some of the phenotypes of pik3Δ cells but are distinct from vps5Δ and vps17Δ cells, consistent with the hypothesis that Pik3 has multiple roles during sporulation (62).Membrane trafficking processes alone do not seem sufficient to explain how the FSMs and PSMs extend around and engulf the nuclei, suggesting that some other mechanism(s) must regulate and orient FSM/PSM extension. The observation that the FSM is attached to the SPB until formation of the immature spore is complete (68) suggests that the SPB may regulate FSM extension. In addition, the leading edge of the S. cerevisiae PSM is coated with a complex of proteins (the LEPs) that appear to be involved in PSM extension (51, 59). S. pombe Meu14 also localizes to the leading edge of the FSM, and deletion of meu14 causes aberrant FSM formation in addition to a failure in SPB modification (60). However, it has remained unclear whether the SPB- and LEP-based mechanisms are sufficient to account for the formation of closed FSMs and PSMs of proper size and position (relative to the nuclear envelope), and evidence from S. cerevisiae has suggested that the septin proteins may also be involved.The septins are a conserved family of GTP-binding proteins that were first identified in S. cerevisiae by analysis of the cytokinesis-defective cdc3, cdc10, cdc11, and cdc12 mutants (41). Cdc3, Cdc10, Cdc11, and Cdc12 are related to each other in sequence and form an oligomeric complex that localizes to a ring in close apposition to the plasma membrane at the mother-bud neck in vegetative cells (12, 20, 25, 41, 47, 77). The septin ring appears to be filamentous in vivo (12), and indeed, the septins from both yeast (11, 20) and metazoans (31, 36, 69) can form filaments in vitro. The yeast septin ring appears to form a scaffold for the localization and organization of a wide variety of other proteins (8, 22), and it forms a diffusion barrier that constrains movement of membrane proteins through the neck region (7, 8, 73). In metazoan cells, the septins are involved in cytokinesis but are also implicated in a variety of other cellular processes, such as vesicular transport, organization of the actin and microtubule cytoskeletons, and oncogenesis (27, 70).In S. cerevisiae, a fifth septin (Shs1) is also expressed in vegetative cells, but the remaining two septin genes, SPR3 and SPR28, are expressed at detectable levels only during sporulation (15, 17). In addition, at least some of the vegetatively expressed septins are also present in sporulating cells (17, 48), and one of them (Cdc10) is expressed at much higher levels there than in vegetative cells (32). The septins present during sporulation are associated with the PSM (15, 17, 48, 51), and their normal organization there depends on the Gip1-Glc7 protein phosphatase complex (71). However, it has been difficult to gain insight into the precise roles of the septins during sporulation in S. cerevisiae (59), because some septins are essential for viability during vegetative growth, and the viable mutants have only mild phenotypes during sporulation (15, 17), possibly because of functional redundancy among the multiple septins.S. pombe seemed likely to provide a better opportunity for investigating the role of septins during spore formation. There are seven septin genes (spn1⁺ to spn7⁺) in this organism (23, 41, 63). Four of these genes (spn1⁺ to spn4⁺) are expressed in vegetative cells, and their products form a hetero-oligomeric complex that assembles during cytokinesis into a ring at the division site (2, 3, 10, 76, 79). The septin ring is important for proper targeting of endoglucanases to the division site (44), and septin mutants show a corresponding delay in cell separation (10, 41, 44, 76). However, even the spn1Δ spn2Δ spn3Δ spn4Δ quadruple mutant is viable and grows nearly as rapidly as the wild type (our unpublished results), a circumstance that greatly facilitates studies of the septins'' role during sporulation.spn5⁺, spn6⁺, and spn7⁺ are expressed at detectable levels only during sporulation (1, 45, 78; our unpublished results), and spn2⁺, like its orthologue CDC10 (see above), is strongly induced (45), but the roles of the S. pombe septins in sporulation have not previously been investigated. In this study, we show that the septins are important for the orientation of FSM extension, suggesting that the septins may have a more general role in dynamic membrane organization and shape determination.     

Coinfecting Prion Strains Compete for a Limiting Cellular Resource

Prion strain interference can influence the emergence of a dominant strain from a mixture; however, the mechanisms underlying prion strain interference are poorly understood. In our model of strain interference, inoculation of the sciatic nerve with the drowsy (DY) strain of the transmissible mink encephalopathy (TME) agent prior to superinfection with the hyper (HY) strain of TME can completely block HY TME from causing disease. We show here that the deposition of PrP^Sc, in the absence of neuronal loss or spongiform change, in the central nervous system corresponds with the ability of DY TME to block HY TME infection. This suggests that DY TME agent-induced damage is not responsible for strain interference but rather prions compete for a cellular resource. We show that protein misfolding cyclic amplification (PMCA) of DY and HY TME maintains the strain-specific properties of PrP^Sc and replicates infectious agent and that DY TME can interfere, or completely block, the emergence of HY TME. DY PrP^Sc does not convert all of the available PrP^C to PrP^Sc in PMCA, suggesting the mechanism of prion strain interference is due to the sequestering of PrP^C and/or other cellular components required for prion conversion. The emergence of HY TME in PMCA was controlled by the initial ratio of the TME agents. A higher ratio of DY to HY TME agent is required for complete blockage of HY TME in PMCA compared to several previous in vivo studies, suggesting that HY TME persists in animals coinfected with the two strains. This was confirmed by PMCA detection of HY PrP^Sc in animals where DY TME had completely blocked HY TME from causing disease.Prions are infectious agents of animals, including humans, which are comprised of PrP^Sc, a misfolded isoform of the noninfectious host encoded protein PrP^C (17, 24, 50, 63). Prion diseases of humans are unique neurodegenerative disorders in that they can have either a sporadic, familial, or infectious etiology. Prions cause disease in economically important domestic and wild animal species such as bovine spongiform encephalopathy in cattle and chronic wasting disease in wild and captive cervids (20, 62). Prion diseases can be zoonotic as illustrated by the transmission of bovine spongiform encephalopathy to humans that resulted in the emergence of variant Creutzfeldt-Jacob disease (14, 19, 22, 23, 46, 61, 68). Prion diseases are inevitably fatal and there are currently no effective treatments (21).Prion strains are defined by a characteristic set of features that breed true upon experimental passage (33, 34). Strain-specific differences have been identified in incubation period, clinical signs, agent distribution, overdominance, host range, neuropathology, and biochemical properties of PrP^Sc (5, 10, 11, 13, 28, 34, 42, 44). Strain-specific conformations of PrP^Sc are hypothesized to encode prion strain diversity; however, it is not understood how these differences result in the distinct strain properties (11, 19, 40, 47, 59, 66).Prion strain interference may be involved in the emergence of a dominant strain from a mixture as could occur during prion adaptation to a new host species or during prion evolution (4, 36, 43, 48, 56). In the natural prion diseases, there are examples where an individual host may be infected with more than one prion strain (15, 25, 55, 57, 58). Experimentally, coinfection or superinfection of prion strains can result in interference where a blocking, long incubation period strain extends the incubation period or completely blocks a superinfecting, short incubation period strain from causing disease (26, 27). Prion interference has been described in experimental studies of mice and hamsters infected with a wide variety of prion strains and routes of inoculation, suggesting it may be a common property of prion disease (3, 27, 52, 53, 60).It has been proposed that prion strains compete for a shared “replication site”; however, mechanistic details are not known, and it is unclear whether the blocking strain destroys or occupies the replication sites required for the superinfecting strain (28). The transport to and relative onset of replication of interfering strains in a common population of neurons is an important factor that can determine which strain will emerge (8). In the present study, we sought to determine whether the blocking strain disables transport and spread of the superinfecting strain or whether prion interference is due to competition for a cellular resource.     

Does delivery volume of family physicians predict maternal and newborn outcome?

Background

The number of births attended by individual family physicians who practice intrapartum care varies. We wanted to determine if the practice–volume relations that have been shown in other fields of medical practice also exist in maternity care practice by family doctors.

Methods

For the period April 1997 to August 1998, we analyzed all singleton births at a major maternity teaching hospital for which the family physician was the responsible physician. Physicians were grouped into 3 categories on the basis of the number of births they attended each year: fewer than 12, 12 to 24, and 25 or more. Physicians with a low volume of deliveries (72 physicians, 549 births), those with a medium volume of deliveries (34 physicians, 871 births) and those with a high volume of deliveries (46 physicians, 3024 births) were compared in terms of maternal and newborn outcomes. The main outcome measures were maternal morbidity, 5-minute Apgar score and admission of the baby to the neonatal intensive care unit or special care unit. Secondary outcomes were obstetric procedures and consultation patterns.

Results

There was no difference among the 3 volume cohorts in terms of rates of maternal complications of delivery, 5-minute Apgar scores of less than 7 or admissions to the neonatal intensive care unit or the special care unit, either before or after adjustment for parity, pregnancy-induced hypertension, diabetes, ethnicity, lone parent status, maternal age, gestational age, newborn birth weight and newborn head circumference at birth. High- and medium-volume family physicians consulted with obstetricians less often than low-volume family physicians (adjusted odds ratio [OR] 0.586 [95% confidence interval, CI, 0.479–0.718] and 0.739 [95% CI 0.583–0.935] respectively). High- and medium-volume family physicians transferred the delivery to an obstetrician less often than low-volume family physicians (adjusted OR 0.668 [95% CI 0.542–0.823] and 0.776 [95% CI 0.607–0.992] respectively). Inductions were performed by medium-volume family physicians more often than by low-volume family physicians (adjusted OR 1.437 [95% CI 1.036–1.992].

Interpretation

Family physicians'' delivery volumes were not associated with adverse outcomes for mothers or newborns. Low-volume family physicians referred patients and transferred deliveries to obstetricians more frequently than high- or medium-volume family physicians. Further research is needed to validate these findings in smaller facilities, both urban and rural.More than 20 years ago, Luft and associates¹ conducted one of the earliest volume–outcome studies. Since then, many studies addressing the relation between volume of procedures and patient outcomes have been published.^2,3 In some of these studies, either the hospital size or the physician procedural volume was used as a surrogate for physician expertise. Among studies analyzing hospital volumes and outcomes, better outcomes have been associated with higher patient volumes in some instances^4,5,6,7 but not others.^3,8,9 Some studies of individual provider volume have shown a positive relation between volume and outcomes,^10,11 whereas others have shown no relation or inconsistent results.^3,12 Finally, a few studies analyzing both hospital volume and provider volume have reported a positive volume–outcome relation.^13,14Criticism levelled at the methods used in volume–outcome studies have addressed the lack of adjustment for case mix, different cutoff points for volume categories and retrospective design.³ Other factors that have an effect on patient outcomes but that have not been included in previous volume analyses include health maintenance organization status, physician certification and years since graduation, and patient socioeconomic status, age and ethnicity. Furthermore, most of the studies on volume have covered surgical or oncology specialities.The few studies that have been done on volume and outcome in maternity care have shown variable effects. Rural health care is often associated with lower volumes of obstetric procedures. However, no differences in maternal or newborn outcomes have been shown in some comparisons of births in urban and rural locations.^15,16,17,18 Other studies have shown poorer maternal and newborn outcomes in low-volume hospitals, neonatal intensive care units (NICUs) and rural locations.^19,20,21,22 Conversely, higher volume (hospitals with more than 1000 deliveries per year) has been associated with more maternal lacerations or complications.²³When the health care provider has been the unit of analysis, a relation between volume and maternal or newborn outcome has been demonstrated in at least one study²⁴ but not in others.^25,26 Low volume has been defined as 20 to 24 deliveries per year.^24,26 Hass and colleagues²⁴ reported an adjusted odds ratio (OR) of 1.4 for low birth weight for infants delivered by low-volume non-board-certified physicians relative to high-volume non-board-certified physicians; the adjusted OR was 1.56 for low-volume board-certified physicians relative to high-volume board-certified physicians (98.7% of whom were obstetricians).Possible explanations for the differences among studies include differences in health care delivery systems, insurance coverage, experience and training of providers, maternal risk factors, triage or transfer of high-risk cases, choice of outcome measures, and changes over time in access to care, quality assurance and standard of living. Relations have been reported between maternal or newborn outcomes and smoking, maternal history of low birth weight (for previous pregnancies), pregnancy–induced hypertension, diabetes, prepregnancy weight, gestational weight gain, maternal height and age, multiple gestation, previous vaginal birth after cesarean section, history of previous delivery problems, parity, large-for-date fetus, ethnicity and fetal sex.^25,27,28,29 Few studies of the relation between volume of births and obstetric outcome have been able to control for these potentially confounding variables and adjust for maternal risk factors.Our database of detailed accounts of births in one hospital setting allowed us to examine this issue more rigorously. We posed 2 research questions: Is there a relation between the volume of deliveries attended by individual family physicians and maternal and newborn outcomes? If there are differences in outcomes, are they related to different physician practice styles and consultation patterns?     

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (10⁷ CFU/cm²) resulted in significant reduction or complete killing of the pathogen inoculated at 10² to 10⁴ CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.Antagonistic interactions among marine bacteria are well documented, and secretion of antagonistic compounds is common among bacteria that colonize particles or surfaces (8, 13, 16, 21, 31). These marine bacteria may be interesting as sources for new antimicrobial drugs or as probiotic bacteria for aquaculture.Aquaculture is a rapidly growing sector, but outbreaks of bacterial diseases are a limiting factor and pose a threat, especially to young fish and invertebrates that cannot be vaccinated. Because regular or prophylactic administration of antibiotics must be avoided, probiotic bacteria are considered an alternative (9, 18, 34, 38, 39, 40). Several microorganisms have been able to reduce bacterial diseases in challenge trials with fish or fish larvae (14, 24, 25, 27, 33, 37, 39, 40). One example is Phaeobacter strain 27-4 (17), which inhibits Vibrio anguillarum and reduces mortality in turbot larvae (27). The antagonism of Phaeobacter 27-4 and the closely related Phaeobacter inhibens is due mainly to the sulfur-containing tropolone derivative tropodithietic acid (TDA) (2, 5), which is also produced by other Phaeobacter strains and Ruegeria mobilis (28). Phaeobacter and Ruegeria strains or their DNA has been commonly found in marine larva-rearing sites (6, 17, 28).Phaeobacter and Ruegeria (Alphaproteobacteria, Roseobacter clade) are efficient surface colonizers (7, 11, 31, 36). They are abundant in coastal and eutrophic zones and are often associated with algae (3, 7, 41). Surface-attached Phaeobacter bacteria may play an important role in determining the species composition of an emerging biofilm, as even low densities of attached Phaeobacter strain SK2.10 bacteria can prevent other marine organisms from colonizing solid surfaces (30, 32).In continuation of the previous research on roseobacters as aquaculture probiotics, the purpose of this study was to determine the antagonistic potential of Phaeobacter and Ruegeria against Vibrio anguillarum in liquid systems that mimic a larva-rearing environment. Since production of TDA in liquid marine broth appears to be highest when roseobacters form an air-liquid biofilm (5), we addressed whether they could be applied as biofilms on solid surfaces.     

Identification of a Novel Class of Membrane-Bound [NiFe]-Hydrogenases in Thermococcus onnurineus NA1 by In Silico Analysis

In silico analysis of group 4 [NiFe]-hydrogenases from a hyperthermophilic archaeon, Thermococcus onnurineus NA1, revealed a novel tripartite gene cluster consisting of dehydrogenase-hydrogenase-cation/proton antiporter subunits, which may be classified as the new subgroup 4b of [NiFe]-hydrogenases-based on sequence motifs.Hydrogenases are the key enzymes involved in the metabolism of H₂, catalyzing the following chemical reaction: 2H⁺ + 2e⁻ ↔ H₂. Hydrogenases can be classified into [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-hydrogenases, based on their distinctive functional core containing the catalytic metal center (11, 17).The genomic analysis of Thermococcus onnurineus NA1, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent area, revealed the presence of several distinct gene clusters encoding seven [NiFe]-hydrogenases and one homolog similar to Mbx (membrane-bound oxidoreductase) from Pyrococcus furiosus (1, 6, 8, 12). According to the classification system of hydrogenases by Vignais et al. (17), three hydrogenases (one F₄₂₀-reducing and two NADP-reducing hydrogenases) belong to group 3 [NiFe]-hydrogenases, and four hydrogenases belong to group 4 [NiFe]-hydrogenases. The group 4 hydrogenases are widely distributed among bacteria and archaea (17), with Hyc and Hyf (hydrogenase 3 and 4, respectively) from Escherichia coli (19), Coo (CO-induced hydrogenase) from Rhodospirillum rubrum (4), Ech (energy-converting hydrogenase) from Methanosarcina barkeri (7), and Mbh (membrane-bound hydrogenase) from P. furiosus (6, 10, 12) being relatively well-characterized hydrogenases in this group. One of the four group 4 hydrogenases from T. onnurineus NA1 was found to be similar in sequence to that of P. furiosus Mbh (10).