Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli

A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D- galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta- galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta- galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.     

Impact of intravenous fluid composition on outcomes in patients with systemic inflammatory response syndrome

IntroductionIntravenous (IV) fluids may be associated with complications not often attributed to fluid type. Fluids with high chloride concentrations such as 0.9 % saline have been associated with adverse outcomes in surgery and critical care. Understanding the association between fluid type and outcomes in general hospitalized patients may inform selection of fluid type in clinical practice. We sought to determine if the type of IV fluid administered to patients with systemic inflammatory response syndrome (SIRS) is associated with outcome.MethodsThis was a propensity-matched cohort study in hospitalized patients receiving at least 500 mL IV crystalloid within 48 hours of SIRS. Patient data was extracted from a large multi-hospital electronic health record database between January 1, 2009, and March 31, 2013. The primary outcome was in-hospital mortality. Secondary outcomes included length of stay, readmission, and complications measured by ICD-9 coding and clinical definitions. Outcomes were adjusted for illness severity using the Acute Physiology Score. Of the 91,069 patients meeting inclusion criteria, 89,363 (98 %) received 0.9 % saline whereas 1706 (2 %) received a calcium-free balanced solution as the primary fluid.ResultsThere were 3116 well-matched patients, 1558 in each cohort. In comparison with the calcium-free balanced cohort, the saline cohort experienced greater in-hospital mortality (3.27 % vs. 1.03 %, P <0.001), length of stay (4.87 vs. 4.38 days, P = 0.016), frequency of readmission at 60 (13.54 vs. 10.91, P = 0.025) and 90 days (16.56 vs. 12.58, P = 0.002) and frequency of cardiac, infectious, and coagulopathy complications (all P <0.002). Outcomes were defined by administrative coding and clinically were internally consistent. Patients in the saline cohort received more chloride and had electrolyte abnormalities requiring replacement more frequently (P <0.001). No differences were found in acute renal failure.ConclusionsIn this large electronic health record, the predominant use of 0.9 % saline in patients with SIRS was associated with significantly greater morbidity and mortality compared with predominant use of balanced fluids. The signal is consistent with that reported previously in perioperative and critical care patients. Given the large population of hospitalized patients receiving IV fluids, these differences may confer treatment implications and warrant corroboration via large clinical trials.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT02083198","term_id":"NCT02083198"}}NCT02083198 clinicaltrials.gov; March 5, 2014

Electronic supplementary material

The online version of this article (doi:10.1186/s13054-015-1045-z) contains supplementary material, which is available to authorized users.     

Development of sedentary communities in the Maya lowlands: Coexisting mobile groups and public ceremonies at Ceibal,Guatemala

Our archaeological investigations at Ceibal, a lowland Maya site located in the Pasión region, documented that a formal ceremonial complex was built around 950 B.C. at the onset of the Middle Preclassic period, when ceramics began to be used in the Maya lowlands. Our refined chronology allowed us to trace the subsequent social changes in a resolution that had not been possible before. Many residents of Ceibal appear to have remained relatively mobile during the following centuries, living in ephemeral post-in-ground structures and frequently changing their residential localities. In other parts of the Pasión region, there may have existed more mobile populations who maintained the traditional lifestyle of the preceramic period. Although the emerging elite of Ceibal began to live in a substantial residential complex by 700 B.C., advanced sedentism with durable residences rebuilt in the same locations and burials placed under house floors was not adopted in most residential areas until 500 B.C., and did not become common until 300 B.C. or the Late Preclassic period. During the Middle Preclassic period, substantial formal ceremonial complexes appear to have been built only at a small number of important communities in the Maya lowlands, and groups with different levels of sedentism probably gathered for their constructions and for public rituals held in them. These collaborative activities likely played a central role in socially integrating diverse groups with different lifestyles and, eventually, in developing fully established sedentary communities.Recent archaeological investigations have shown that the development of agriculture and sedentism was more diverse than the simple model of agriculture leading to sedentism and then to social complexity. In Europe, for example, the farming lifestyle that originated in the Near East spread in complex ways, involving the coexistence of farmers and foragers in relatively small areas and differential adoptions of Neolithic cultural elements in different regions (1–3). Studies of early monuments, such as Göbekli Tepe in Turkey, Watson Brake in Louisiana, and Caral and earlier mounds in the Andes, show that large constructions involving significant collective labor could be built by preceramic people who were still foragers or were at the early stage of farming adaptation (4–7). These emerging understandings lead to important questions about how sedentary and mobile populations interacted and how their relations affected the process of social change. To address these questions, researchers need fine-grained chronological information and a broad spatial coverage, which are not easy to obtain in many cases. A uniquely rich dataset obtained from the Maya site of Ceibal (or Seibal) suggests the possibility that groups with different levels of mobility gathered and collaborated for constructions and public ceremonies, which contrasts with the common assumption that sedentary and mobile groups maintained separate communities.In the areas surrounding the Maya lowlands, including the southern Gulf Coast, the southern Pacific Coast, and northern Honduras, settlements with ceramic use emerged between 1900 and 1400 B.C. (8, 9). Some inhabitants of the Maya lowlands adopted maize and other domesticates possibly as early as 3400 B.C., but did not accept sedentary lifeways and ceramic use for many centuries (10). Once they began to establish villages with ceramic use around 1000 B.C., the subsequent social change was rapid. Within 1,000 y the lowland Maya developed numerous large centers with pyramids and centralized political organizations. Although a few scholars have suggested the possibility that early sedentary villages in the Maya lowlands coexisted with mobile groups (11), the specific social configurations and the process of transition have not been well understood, partly because of the rapidity of social change and partly because of difficulty in investigating early deposits deeply buried under later constructions.Since 2005 we have been conducting archaeological investigations at the site of Ceibal, Guatemala, located in the Pasión region of the southwestern Maya lowlands (Fig. 1). Previous explorations by Harvard University in the 1960s demonstrated that Ceibal was one of the earliest sedentary communities in the Maya lowlands and subsequently became a major center (12–14). Building on this pioneering work, our deep, intensive excavations targeted specific locations to reveal early constructions and deposits. With detailed stratigraphic information and ceramic data, as well as through the Bayesian analysis of radiocarbon dates, we developed a high-resolution chronology of Ceibal, which we correlated with the archaeological sequences in other parts of the Maya lowlands (SI Text, Figs. S1–S3, and Table S1). This refined chronology allowed us to reconstruct social processes on a fine temporal scale that was not possible before.Open in a separate windowFig. 1.Map of the Maya area with a close-up of the Pasión region.In addressing the development of sedentism, we need to examine its multiple dimensions as continuums rather than categorical divisions (15). In this regard, the lowland Maya of the Classic period (A.D. 250–950) possessed a particularly strong sense of attachment to fixed localities. Houses were repeatedly rebuilt over older ones in the same locations, and some of the dead were buried under house floors. In other words, kin-based groups, or at least their central members, were tied to fixed locations physically and symbolically through generations, although this practice did not preclude certain members from moving out to establish new residences. Temples and public plazas were also periodically rebuilt over previous ones, tethering entire communities to fixed locations. In examining the trajectory leading to this system, we need to analyze diverse levels of residential mobility and the possible coexistence of different modes, potentially including seasonal mobility without ceramic use, residential relocations every few years with ceramic use, and the use of durable houses without transgenerational continuity. These levels of mobility were most likely associated with different subsistence strategies and different notions of property and land ownership (16).     

Impact of an Intervention to Improve Weekend Hospital Care at an Academic Medical Center: An Observational Study

Background

Hospital care on weekends has been associated with delays in care, reduced quality, and poor clinical outcomes.

Objective

The purpose of this study was to evaluate the impact of a weekend hospital intervention on processes of care and clinical outcomes. The multifaceted intervention included expanded weekend diagnostic services, improved weekend discharge processes, and increased physician and care management services on weekends.

Design and Patients

This was an interrupted time series observational study of adult non-obstetric patients hospitalized at a single academic medical center between January 2011 and January 2014. The study included 18 months prior to and 19 months following the implementation of the intervention. Data were analyzed using segmented regression analysis with adjustment for confounders.

Main Measures

The primary outcome was average length of stay. Secondary outcomes included percent of patients discharged on weekends, 30-day readmission rate, and in-hospital mortality rate.

Key Results

The study included 57,163 hospitalizations. Following implementation of the intervention, average length of stay decreased by 13 % (95 % CI 10–15 %) and continued to decrease by 1 % (95 % CI 1–2 %) per month as compared to the underlying time trend. The proportion of weekend discharges increased by 12 % (95 % CI 2–22 %) at the time of the intervention and continued to increase by 2 % (95 % CI 1–3 %) per month thereafter. The intervention had no impact on readmissions or mortality. During the post-implementation period, the hospital was evacuated and closed for 2 months due to damage from Hurricane Sandy, and a new hospital-wide electronic health record was introduced. The contributions of these events to our findings are not known. We observed a lower inpatient census and found differences in patient characteristics, including higher rates of Medicaid insurance and comorbidities, in the post-Hurricane Sandy period as compared to the pre-Sandy period.

Conclusions

The intervention was associated with a reduction in length of stay and an increase in weekend discharges. Our longitudinal study also illuminated the challenges of evaluating the effectiveness of a large-scale intervention in a real-world hospital setting.KEY WORDS: Health care delivery, Hospital medicine, Variations, Natural disaster     

Tumor necrosis factor is the major monocyte product that increases complement receptor expression on mature human neutrophils

Interleukin-1 (IL-1) and other monocyte products have several important effects on the systemic response to infection in addition to their roles in lymphocyte stimulation. The present studies were carried out to determine whether products of stimulated monocytes activated circulating neutrophils (PMN) to increase expression of receptors for C3b (CR1) and C3bi (CR3), which are necessary for optimal margination, migration, and phagocytosis. Supernatants of human mononuclear cells that had been stimulated with lipopolysaccharide (LPS) or purified protein derivative (PPD) contained both tumor necrosis factor (TNF) and IL-1 and increased CR1 and CR3 expression on isolated PMNs. Supernatants of unstimulated cultures, media alone, or LPS or PPD alone had little or no effect. Supernatant effects were detectable at 1:3,000 final dilution and appeared to have a characteristic slow time course. These supernatants also caused dose- and time-dependent secretion of PMN granular constituents, but maximal receptor expression was accompanied by secretion of less than 10% of the cells' content of lysozyme and less than 16% of the B12 binding protein. Immunoadsorption studies showed that the supernatant's activity could be removed by anti- TNF but not by anti-IL-1. Recombinant IL-1 had no effect on receptor expression, but recombinant TNF increased CR1 and CR3 expression with kinetics similar to the supernatants. These results thus indicate that TNF is the major monocyte product that increases CR1 and CR3 expression on mature blood neutrophils. This would result in increased margination and phagocytic activity and may be an important systemic effect that would help the host eradicate infection.     

Granulocyte colony-stimulating factor after allogeneic bone marrow transplantation

Hematopoietic growth factors have been shown to be effective in reducing the period of neutropenia after autologous bone marrow transplantation (BMT). Initial concerns over potential aggravation of graft-versus-host disease (GVHD) and increase in the incidence of relapse in patients with myeloid leukemias influenced the number of studies using hematopoietic growth factors after allogeneic BMT. We report the experience with 50 patients treated at a single institution using granulocyte colony-stimulating factor (G-CSF) after allogeneic sibling (n = 30) and matched unrelated (n = 20) BMT. The time to an absolute neutrophil count > or = 500/microL was significantly faster in patients who received G-CSF and cyclosporine and prednisone for GVHD prophylaxis when compared with historical control patients receiving the same GVHD prophylaxis (10 v 13 days, P < .01). A similar accelerated myeloid engraftment was observed for those patients who received the addition of methotrexate for GVHD prophylaxis when compared with historical control patients receiving the same GVHD prophylaxis regimen (16 v 19 days, P < .05). The median time to engraftment for patients receiving a matched unrelated BMT and G-CSF was 17 days (range 13 to 26). We did not observe any increase in GVHD or early mortality in the matched related sibling BMT. The incidence of acute GVHD in the matched unrelated BMT recipients was also low at 21%; however, 9 patients (45%) died within 100 days of the date of BMT, similar to the experience reported with granulocyte-macrophage CSF. This study confirms the efficacy of G-CSF in accelerating myeloid engraftment after allogeneic matched sibling BMT. The higher early mortality associated with patients receiving matched unrelated BMT suggests that randomized controlled trials using G-CSF after allogeneic BMT should be performed.