Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.     

Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony- stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte- CSF

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte- macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM- CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G- CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM- CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)     

Humoral immune function in pediatric patients treated with autologous bone marrow transplantation for B cell non-Hodgkin's lymphoma. The influence of ex vivo marrow decontamination with anti-Y 29/55 monoclonal antibody and complement

Elimination of neoplastic B cell populations from autologous bone marrow grafts also removes normal B lymphocytes. This is potentially hazardous for the reconstitution of the immune system in patients undergoing high-dose chemotherapy and total body irradiation followed by autologous marrow rescue. Five pediatric patients with B cell non- Hodgkin's lymphoma in first remission undergoing such a regimen were studied. They received bone marrow pretreated with anti-Y 29/55 monoclonal antibody and complement. B and T lymphocyte subpopulations reached normal levels within 6 months after autologous bone marrow transplantation (ABMT), and serum immunoglobulin levels became normal within 4 to 9 months. Vaccination with diphtheria and tetanus toxoid, trivalent poliomyelitis vaccine of the Salk type, and pneumococcal capsular antigens (38 to 54 months after transplantation) gave rise to specific antibody production. ABO isoagglutinins could be demonstrated in all patients. The response pattern was similar to that of patients who received unmanipulated autologous bone marrow. It is concluded that ex vivo anti-Y 29/55 depletion of the marrow graft does not induce relevant disturbances of humoral immune functions.     

Tau mutants bind tubulin heterodimers with enhanced affinity

Tau is a microtubule binding protein that forms pathological aggregates in the brain in Alzheimer’s disease and other tauopathies. Disease etiology is thought to arise from loss of native interactions between tau and microtubules, as well as from gain of toxicity tied to tau aggregation, although neither mechanism is well understood. Here we investigate the link between function and disease using disease-associated and disease-motivated mutants of tau. We find that mutations to highly conserved proline residues in repeats 2 and 3 of the microtubule binding domain have differential effects on tau binding to tubulin and the capacity of tau to enhance tubulin polymerization. Notably, mutations to these residues result in an increased affinity for tubulin dimers while having a negligible effect on binding to stabilized microtubules. We measure conformational changes in tau on binding to tubulin that provide a structural framework for the observed altered affinity and function. Additionally, we find that these mutations do not necessarily enhance aggregation, which could have important implications for tau therapeutic strategies that focus solely on searching for tau aggregation inhibitors. We propose a model that describes tau binding to tubulin dimers and a mechanism by which disease-relevant alterations to tau impact its function. Together, these results draw attention to the interaction between tau and free tubulin as playing an important role in mechanisms of tau pathology.Tau is a microtubule (MT)-associated protein that plays a critical role in the pathology of several neurodegenerative disorders including Alzheimer’s disease, frontotemporal dementias, and traumatic brain injury (1). Normally, tau is found primarily in the axons of neurons where it regulates the dynamic instability of MTs (2, 3) and plays an important role in axonal transport (4, 5). Both in vitro and in vivo measurements find that tau increases the rate of MT polymerization, as well as decreasing rates of catastrophe (2, 6, 7). In disease, tau is found as aggregated, filamentous deposits that are the defining feature of a diverse class of neurodegenerative diseases, called tauopathies (1, 8). Pathological mutations to tau are thought to alter the native interactions of tau with MTs, in addition to increasing the propensity of tau to aggregate (9–11). Although the precise cause or mechanism by which tau contributes to toxicity in disease is unknown, both a loss of native function and a gain of toxic function are implicated (1, 12, 13).Tau consists of a C-terminal microtubule binding domain (MTBR) composed of imperfect repeats (R1–R4; Fig. 1A) (14), a flanking proline-rich region that enhances MT binding and assembly (3), and an N-terminal projection domain with putative roles in MT spacing (15) and membrane anchoring (16). Alternative splicing results in the expression of six isoforms of tau in the adult human brain, with zero, one, or two N-terminal inserts and three or four MT binding repeat units. The repeat units contain an 18-residue imperfect repeat sequence, which terminates with a highly conserved proline-glycine-glycine-glycine (PGGG), and are linked by a 13–14 residue interrepeat sequence (Fig. 1C). Early biochemical studies depicted the conserved regions as binding weakly to MTs, with the interrepeats acting as spacers between them (14, 17). More recently, it was shown that the interrepeats are also directly involved in binding and polymerization (18, 19), with the N-terminal proline-rich region playing a regulatory role (20). Tau binds MTs with a biphasic pattern indicative of two distinct binding sites on the MT lattice with unequal affinities (21, 22). Binding to MTs is negatively regulated by phosphorylation on sites in the MTBR and adjacent regions (23). Tau derived from aggregates in the brain is hyperphosphorylated (24), suggesting a role for aberrant phosphorylation in disease.Open in a separate windowFig. 1.Schematic of tau constructs and an MTBR repeat. The functional domains of tau are indicated on the longest full-length isoform with alternatively spliced regions marked by dashed lines (A). The interrepeat regions that link the conserved repeat sequences are indicated by cross-hatching. On the fragments used in this study (B), K16 (residues 198–372) and K18 (residues 244–372), the residues mutated to cysteine for attachment of fluorophores (residues 244, 322, and 354), and proline to leucine/serine mutation sites (301 and 332) are indicated. A schematic of a repeat within the MTBR (C) illustrates interrepeat and repeats sequences, with the conserved residues shown.Attempts to obtain resolution structural information of tau have been hindered by the fact that it is intrinsically disordered under physiological conditions (25) and remains largely disordered even on binding to MTs. Contrasting models derived from cryo-EM suggest tau binds either along the outer protofilament ridge (26) or to the inner surface (27) of MTs. The MTBR carries a net positive charge, and binding of tau to the acidic carboxy termini of α and β tubulin has been observed both in the context of MTs (28) and for the isolated peptides corresponding to these tubulin sequences (29, 30). In addition, a secondary binding site has been mapped to an independent region within the C-terminal third of tubulin (30). Cleavage of the C-terminal tail of tubulin is sufficient to increase polymerization rates (30), and it has been suggested that tau may promote MT polymerization through a similar charge neutralization mechanism.The MTBR also plays a central role in tau pathology in that it forms the core of the aggregates found in disease (31) and contains the minimum sequence necessary to recapitulate relevant features of aggregation in vitro (32). Moreover, the majority of mutations to tau implicated in tauopathies are either point mutations within the MTBR or mutations that interfere with alternative splicing, altering the normal ∼1:1 ratio of 3R:4R tau (8, 9). Notable among the point mutants is P301L (tau_P301L) (Fig. 1B), implicated in frontotemporal dementia with Parkinsonism-17, which provided one of the first genetic links between tau and neurodegenerative disease (33). The P301L variant has emerged as a particularly reliable model for tau-based neurodegenerative disease, having successfully reproduced aspects of pathology in animal models of Alzheimer’s disease and other tauopathies (34).Although significant work has focused on tau interactions with MTs, very little is known about the mechanistic details of tau-mediated MT polymerization, including interactions of tau with tubulin dimers during the assembly process. Although many alterations to tau implicated in disease have been shown to affect MT polymerization both in vitro and in vivo, virtually nothing is known about the mechanism by which these changes occur. Tau_P301L exhibits impaired tubulin polymerization (35), as well as increased aggregation propensity relative to WT protein (tau_WT) (32) and thus serves as a model for both loss of function and gain of toxic function aspects of disease. This mutation is in the highly conserved PGGG sequence of R2 (Fig. 1C). To broaden our understanding of the impact of this point mutation on pathology, we designed a tau variant with the analogous proline to leucine substitution in the PGGG sequence of R3, tau_P332L, as well as a double mutant, tau_P301L/P332L. We use single molecule fluorescence to investigate structural and functional aspects of the interaction between tau and tubulin. In combination with ensemble polymerization and aggregation assays, this work provides insight into the relationship between functional and dysfunctional roles of tau.     

Characterization of Adverse Events Detected in a Large Health Care Delivery System Using an Enhanced Global Trigger Tool over a Five‐Year Interval

Objective

To report 5 years of adverse events (AEs) identified using an enhanced Global Trigger Tool (GTT) in a large health care system.

Study Setting

Records from monthly random samples of adults admitted to eight acute care hospitals from 2007 to 2011 with lengths of stay ≥3 days were reviewed.

Study Design

We examined AE incidence overall and by presence on admission, severity, stemming from care provided versus omitted, preventability, and category; and the overlap with commonly used AE-detection systems.

Data Collection

Professional nurse reviewers abstracted 9,017 records using the enhanced GTT, recording triggers and AEs. Medical record/account numbers were matched to identify overlapping voluntary reports or AHRQ Patient Safety Indicators (PSIs).

Principal Findings

Estimated AE rates were as follows: 61.4 AEs/1,000 patient-days, 38.1 AEs/100 discharges, and 32.1 percent of patients with ≥1 AE. Of 1,300 present-on-admission AEs (37.9 percent of total), 78.5 percent showed NCC-MERP level F harm and 87.6 percent were “preventable/possibly preventable.” Of 2,129 hospital-acquired AEs, 63.3 percent had level E harm, 70.8 percent were “preventable/possibly preventable”; the most common category was “surgical/procedural” (40.5 percent). Voluntary reports and PSIs captured <5 percent of encounters with hospital-acquired AEs.

Conclusions

AEs are common and potentially amenable to prevention. GTT-identified AEs are seldom caught by commonly used AE-detection systems.     

Measuring insight through patient self-report: An in-depth analysis of the factor structure of the Birchwood Insight Scale

Little research has focused on item analysis and factor structure of the most commonly used measures of insight. We examined the factorial structure of the Birchwood Insight Scale (BIS), a brief, easy-to-administer, self-report measure. We studied the BIS in 327 first-episode psychosis patients, including a test sample (n=163) and a validation sample (n=164). We then used data from 100 patients with chronic serious mental illnesses as a second, external validation sample. Exploratory factor analysis was conducted with the test subsample, and confirmatory factor analyses with the two validation samples. Confirmatory factor analyses (in both the first-episode psychosis validation sample and the chronic serious mental illness sample) indicated that a single-factor solution, with seven items loading on a single factor—with item 1 (“Some of your symptoms are made by your mind”) eliminated—was the best-fitting model. Seven of the eight original BIS items loading on a single factor fit the data well in these samples. Researchers using this efficient measure of patient-reported insight should assess the item distributions and factor structure of the BIS in their samples, and potentially consider eliminating item 1.     

幽门螺杆菌cagⅡ对胃上皮细胞IL-8基因转录的影响及机制

目的探讨HpcagⅡ对胃上皮细胞IL-8基因转录的影响及信号传导机制。方法构建 cagⅡ基因位点缺失Hp突变株及带有IL-8报告基因的人胃癌细胞系L5F11,用液体闪烁计数仪测定荧光素酶(IL8转录)活性,用ELISA法测定IL8蛋白浓度。结果所有Hp突变株诱导荧光素酶活性与IL8蛋白浓度较亲代菌株26695均降低[(0．13±0．01)×cpm比(0．59±0．05)×(P<0．01);(0．73±0．13)ng／ml比(2．22±0．65)ng／ml,(P<0．05)]。PTK抑制剂herbimycinA不仅抑制Hp诱导的荧光素酶活性[(0．71±0．18)×cpm比(1．51±0．23)×cpm,(P<0．05)],而且抑制IL-8蛋白表达[(0．83±0．41)ng／ml比(3．22±0．59)ng／ml,(P<0．05)],但herbimycinA对TNFα诱导的荧光素酶活性及IL8蛋白表达均无影响(P均>0．05);PKA抑制剂H7抑制TNFα诱导的荧光素酶活性[(0．74±0．16)×cpm比(2．62±0．26)×cpm,(P<0．001)]及IL8蛋白表达[(1．45±0．38)ng／ml比(4．12±0．43)ng／ml,(P<0．01)],而对Hp诱导的荧光素酶活性无影响(P>0．05)。结论HpcagⅡ中的多基因能够调节胃上皮细胞IL-8基因转录,且这一作用主要经蛋白酪氨酸激酶途径。     

Tranexamic acid use decreases transfusion rate in children with cerebral palsy undergoing proximal femoral varus derotational osteotomy

Previous studies demonstrated the safety of tranexamic acid (TXA) use in cerebral palsy (CP) patients undergoing proximal femoral varus derotational osteotomy (VDRO), but were underpowered to determine if TXA alters transfusion rates or estimated blood loss (EBL). The purpose of this study was to investigate if intraoperative TXA administration alters transfusion rates or EBL in patients with CP undergoing VDRO surgery.We conducted a retrospective review of 390 patients with CP who underwent VDRO surgery between January 2004 and August 2019 at a single institution. Patients without sufficient clinical data and patients with preexisting bleeding or coagulation disorders were excluded. Patients were divided into 2 groups: those who received intraoperative TXA and those who did not.Out of 390 patients (mean age 9.4 ± 3.8 years), 80 received intravenous TXA (TXA group) and 310 did not (No-TXA group). There was no difference in mean weight at surgery (P = .25), Gross Motor Function Classification System level (P = .99), American Society of Anesthesiologist classification (P = .50), preoperative feeding status (P = .16), operative time (P = .91), or number of procedures performed (P = .12) between the groups. The overall transfusion rate was lower in the TXA group (13.8%; 11/80) than the No-TXA group (25.2%; 78/310) (P = .04), as was the postoperative transfusion rate (7.5%; 6/80 in the TXA group vs 18.4%; 57/310 in the No-TXA group) (P = .02). The intraoperative transfusion rate was similar for the 2 groups (TXA: 7.5%; 6/80 vs No-TXA: 10.3%; 32/310; P = .53). The EBL was slightly lower in the TXA group, although this was not significant (TXA: 142.9 ± 113.1 mL vs No-TXA: 177.4 ± 169.1 mL; P = .09). The standard deviation for EBL was greater in the No-TXA group due to more high EBL outliers. The percentage of blood loss based on weight was similar between the groups (TXA: 9.2% vs No-TXA: 10.1%; P = .40). The number needed to treat (NNT) with TXA to avoid one peri-operative blood transfusion in this series was 9.The use of intraoperative TXA in patients with CP undergoing VDRO surgery lowers overall and postoperative transfusion rates.Level of evidence: III, Retrospective Comparative Study.