A spirochete surface protein uncouples store-operated calcium channels in fibroblasts: a novel cytotoxic mechanism

The cytotoxicity of infectious agents can be mediated by disruption of calcium signaling in target cells. Outer membrane proteins of the spirochete Treponema denticola, a periodontal pathogen, inhibit agonist-induced Ca(2+) release from internal stores in gingival fibroblasts, but the mechanism is not defined. We determined here that the major surface protein (Msp) of T. denticola perturbs calcium signaling in human fibroblasts by uncoupling store-operated channels. Msp localized in complexes on the cell surface. Ratio fluorimetry showed that in cells loaded with fura-2 or fura-C18, Msp induced cytoplasmic and near-plasma membrane Ca(2+) transients, respectively. Increased conductance was confirmed by fluorescence quenching of fura-2-loaded cells with Mn(2+) after Msp treatment. Calcium entry was blocked with anti-Msp antibodies and inhibited by chelating external Ca(2+) with EGTA. Msp pretreatment reduced the amplitude of [Ca(2+)](i) transients upon challenge with ATP or thapsigargin. In experiments using cells loaded with mag-fura-2 to report endoplasmic reticulum Ca(2+), Msp reduced Ca(2+) efflux from endoplasmic reticulum stores when ATP was used as an agonist. Msp alone did not induce Ca(2+) release from these stores. Msp inhibited store-operated influx of extracellular calcium following intracellular Ca(2+) depletion by thapsigargin and also promoted the assembly of subcortical actin filaments. This actin assembly was blocked by chelating intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester. The reduced amplitude of agonist-induced transients and inhibition of store-operated Ca(2+) entry due to Msp were reversed by latrunculin B, an inhibitor of actin filament assembly. Thus, Msp retards Ca(2+) release from endoplasmic reticulum stores, and it inhibits subsequent Ca(2+) influx by uncoupling store-operated channels. Actin filament rearrangement coincident with conformational uncoupling of store-operated calcium fluxes is a novel mechanism by which surface proteins and toxins of pathogenic microorganisms may damage host cells.     

When does it pay to break the matches for analysis of a matched-pairs design?

Two methods of analysis are compared to estimate the treatment effect of a comparative study where each treated individual is matched with a single control at the design stage. The usual matched-pairs analysis accounts for the pairing directly in its model, whereas regression adjustment ignores the matching but instead models the pairing using a set of covariates. For a normal linear model, the estimated treatment effect from the matched-pairs analysis (paired t-test) is more efficient. For a Bernoulli logistic model, matched-pairs analysis performs better when the sample size is small, but is inferior to logistic regression for large sample sizes.     

Effects of mitogens and co-mitogens on the formation of small-cell colonies in primary cultures of rat hepatocytes

Degradation of collagen by fibroblast phagocytosis is an important pathway for physiological remodelling of soft connective tissues. Perturbations of this pathway may provide a mechanism for the development of fibrotic lesions. As collagen phagocytosis may be regulated by either a change of the proportions or the activity of phagocytic cells, we quantified phagocytosis with an in vitro model system. Collagen-coated fluorescent latex beads were incubated with human gingival fibroblasts and the fluorescence associated with internalized beads was measured by flow cytometry. Cells from normal tissues that had been incubated with beads for 3 hours contained a mean of 64% phagocytic cells; however, a small subpopulation (10% of phagocytic cells) contained more than threefold higher numbers of beads per cell than the mean. In contrast, cells from fibrotic lesions exhibited a large reduction of the proportions of phagocytic cells (mean = 13.8%) and there were no cells with high numbers of beads. On the basis of ³H-Tdr labeling, cells from fibrotic lesions that had internalized beads failed to proliferate, in contrast to phagocytic cells from normal tissues, which underwent repeated cell divisions. This result was not due to variations of cell cycle phase as there was no preferential internalization of beads during different phases of the cell cycle. The low phagocytic rate of cells from fibrotic lesions was also not due to asymmetric partitioning of phagosomes at mitosis as videocinemicrography of bead-labeled phagosomes in single, pre-mitotic cells demonstrated that > 90% of phagocytic cells equally partitioned beads to daughter cells. To investigate if inhibition of phagocytosis could be replicated in vitro, cells were incubated with the fibrosis-inducing drugs nifedipine or dilantin. These cultures exhibited marked (15–75%), dose-dependent reductions in the proportions of phagocytic cells, but there was no reduction in bead number per cell. Fibrotic lesions appear to contain fibroblasts with marked deficiencies in phagocytosis and the reduced phagocytic activity of these cells may contribute to unbalanced degradation and fibrosis. © 1993 Wiley-Liss, Inc.     

SHORT-TERM CULTURES OF MOUSE MARROW CELLS SEPARATED BY VELOCITY SEDIMENTATION

Our experiments were designed to identify the source of the increased number of cells forming colonies in culture (CFU-C) detected after short-term culture of mouse marrow cells over 'feeders' of renal tubules. Accordingly, marrow cell suspensions were fractionated by velocity sedimentation and aliquots of each fraction cultured for 2 days over tubule 'feeders'. We found a greater increase of CFU-C in suspensions of slowly sedimenting cells as compared to rapidly sedimenting cells. Colcemid and vinblastine were used to alter the peak sedimentation velocity of marrow suspensions by changing the distribution of cells in the cell cycle. After such manipulations, the peak increase in CFU-C continued to be associated with fractions containing slowly sedimenting cells. Such fractions also contained most of the pluripotent stem cells identified by the spleen colony technique.     

Metabolism of inositol 1- and 4-monophosphates in HL60 promyelocytic leukaemia cells

The metabolism of inositol 1- and 4-monophosphates in HL60 promyelocytic leukaemia cells was studied. LiCl, BeCl2 and NaF inhibited the hydrolysis of both monophosphates with half maximal inhibition occurring at 1.2 mM, 0.3 microM, 0.25 mM (Ins 1P) and 0.14 mM, 0.56 microM, 0.28 mM (Ins 4P) respectively. Lithium was an uncompetitive inhibitor with respect to both substrates. Ins 4P inhibited the hydrolysis of Ins 1P in a concentration dependent manner, suggesting that it acts as a competing substrate for the same enzyme. Half maximal inhibition occurred at 120 microM Ins 4P. The lithium sensitive activity responsible for the metabolism of both monophosphates was present in a soluble fraction made from the cells. Taken together these data suggest that Ins 1P and Ins 4P are hydrolysed by a single soluble enzyme activity which is sensitive to inhibition by lithium, beryllium and fluoride.     

Quality Improvement in Surgery Combining Lean Improvement Methods with Teamwork Training: A Controlled Before-After Study

Background

To investigate the effectiveness of combining teamwork training and lean process improvement, two distinct approaches to improving surgical safety. We conducted a controlled interrupted time series study in a specialist UK Orthopaedic hospital incorporating a plastic surgery team (which received the intervention) and an Orthopaedic theatre team acting as a control.

Study Design

We used a 3 month intervention with 3 months data collection period before and after it. A combined teamwork training and lean process improvement intervention was delivered by an experienced specialist team. Before and after the intervention we evaluated team non-technical skills using NOTECHS II, technical performance using the glitch rate and WHO checklist compliance using a simple 3 point scale. We recorded complication rate, readmission rate and length of hospital stay data for 6 months before and after the intervention.

Results

In the active group, but not the control group, full compliance with WHO Time Out (T/O) increased from 14 to 71% (p = 0.032), Sign Out attempt rate (S/O) increased from 0% to 50% (p<0.001) and Oxford NOTECHS II scores increased after the intervention (P = 0.058). Glitch rate decreased in the active group and increased in the control group (p = 0.001). Complications and length of stay appeared to rise in the control group and fall in the active group.

Conclusions

Combining teamwork training and systems improvement enhanced both technical and non-technical operating team process measures, and were associated with a trend to better safety outcome measures in a controlled study comparison. We suggest that approaches which address both system and culture dimensions of safety may prove valuable in reducing risks to patients.     

The within-host dynamics of African trypanosome infections

African trypanosomes are single-celled protozoan parasites that are capable of long-term survival while living extracellularly in the bloodstream and tissues of mammalian hosts. Prolonged infections are possible because trypanosomes undergo antigenic variation—the expression of a large repertoire of antigenically distinct surface coats, which allows the parasite population to evade antibody-mediated elimination. The mechanisms by which antigen genes become activated influence their order of expression, most likely by influencing the frequency of productive antigen switching, which in turn is likely to contribute to infection chronicity. Superimposed upon antigen switching as a contributor to trypanosome infection dynamics is the density-dependent production of cell-cycle arrested parasite transmission stages, which limit the infection while ensuring parasite spread to new hosts via the bite of blood-feeding tsetse flies. Neither antigen switching nor developmental progression to transmission stages is driven by the host. However, the host can contribute to the infection dynamic through the selection of distinct antigen types, the influence of genetic susceptibility or trypanotolerance and the potential influence of host-dependent effects on parasite virulence, development of transmission stages and pathogenicity. In a zoonotic infection cycle where trypanosomes circulate within a range of host animal populations, and in some cases humans, there is considerable scope for a complex interplay between parasite immune evasion, transmission potential and host factors to govern the profile and outcome of infection.