Thromboembolic events in patients with severe inherited fibrinogen deficiency

Inherited afibrinogenemia and hypofibrinogenemia are rare bleeding disorders characterized by markedly reduced levels of fibrinogen in blood. Thrombotic complications in these disorders have been rarely described. We performed a multicenter retrospective study and reviewed the occurrence of thrombotic complications among patients with inherited fibrinogen deficiency. Cases were identified during a review of medical records of all patients with inherited fibrinogen deficiency followed at three different university hospitals in Israel. Nine patients were included in this study: five were afibrinogenemic and four hypofibrinogenemic. There were seven thrombotic events, mostly venous, that occurred in four out of nine patients (44 %). All thrombotic events occurred in afibrinogenemic patients. Mean age at the time of thrombosis was 45 (range 28–61) years. Thrombophilic evaluation performed was negative in all cases. At the time of thrombosis in five out of seven (71.4 %) events, fibrinogen replacement therapy was concurrently given. Therapeutic approach was different among patients ranging from supportive therapy alone, antiplatelet agents and anticoagulant therapy with the concurrent administration of fibrinogen replacement therapy. This study discloses a high rate of thrombosis in patients with afibrinogenemia. Events were both venous and arterial and may be recurrent. Management is highly problematic due to the precarious balance between bleeding and thrombotic risk in these patients. Fibrinogen replacement therapy should be cautiously used in these patients as most thrombotic events followed the administration of fibrinogen replacement therapy. Larger cohorts are warranted to better characterize the best management strategy in these paradoxical events.     

The binding of fibrin sealant to collagen is influenced by the method of purification and the cross-linked fibrinogen-fibronectin (heteronectin) content of the 'fibrinogen' component.

Two fibrinogen preparations, each an intermediate in the manufacture of the 'fibrinogen' component of a commercial human tissue sealant, were made from a common cryoprecipitate source. The first preparation, prepared according to the process described by Schwartz et al. had a higher ratio of clottable to total protein than the second preparation, prepared according to that by Martinowitz and Bal but a much lower ratio of fibronectin to fibrinogen. After clotting with thrombin and solubilization and reduction of the clots, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a much higher content of high molecular weight polymers of fibrin(ogen) in the second preparation than in the first. The second preparation bound to collagen more strongly than did cryoprecipitate and much more strongly than did the first one. Experiments with highly purified proteins showed that fibronectin was essential in promoting progressive binding of fibrinogen to collagen under the action of activated factor XIII (transglutaminase). It was concluded that, because of their method of purification from cryoprecipitate, preparations of fibrinogen differ in their content of fibronectin and heteronectin. The binding of these proteins to collagen may improve the adhesion of tissue sealant clots to the extracellular matrix.     

Selective synthesis of bone morphogenetic proteins-1, -3, -4 and bone sialoprotein may be important for osteoinduction by Saos-2 cells

An ability to induce new bone formation at a required site would represent a considerable advance in bone repair and tissue engineering. It has been shown that the healing of critical-size bone defects in rats can be augmented by extracts of Saos-2 cells. These human osteosarcoma cells uniquely contain a bone-inducing activity, whereas other human osteosarcoma cells, e.g., U-2 OS cells, cannot replicate the osteoinductive capacity. To understand the necessary components of the Saos-2 bone-inducing activity, this study compared osteoinductive Saos-2 cells with non-osteoinductive U-2 OS cells with respect to the synthesis of bone morphogenetic proteins (BMPs)-1, -2, -3, -4, -5, -6, and -7 and the non-collagenous matrix proteins bone sialoprotein (BSP), osteonectin (ON), osteopontin (OPN), and osteocalcin (OC). The main differences were abundant synthesis of BMP-1/tolloid, BMP-3, -4, and BSP by Saos-2 cells, but absence or reduced synthesis in U-2 OS cells. BMP-2 and -7 were present in low amounts in both cell types, while BMP-5 and -6 were more abundant in U-2 OS cells, suggesting that these BMPs were of lesser importance for the osteoinductivity of Saos-2 cells. However, a relatively high expression of BMP-3 and -4, together with BMP-1/tolloid, may be important for the osteoinductive capacity of Saos-2 cells. The inability of U2-OS cells to induce bone, despite expressing most of the BMPs, may be due to an insufficiency of tolloid, BMP-3 or -4, BSP, and/or other unknown factors. A better understanding of the necessary components of the Saos-2 cell bone-inducing agent may, in future, lead to clinically useful Saos-2 cell products for bone repair and tissue engineering. Received: May 17, 2001 / Accepted: September 28, 2001     

Listeriolysin S: A bacteriocin from Listeria monocytogenes that induces membrane permeabilization in a contact-dependent manner

Listeriolysin S (LLS) is a thiazole/oxazole–modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes. LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

Listeria monocytogenes (Lm) is a gram-positive foodborne pathogen responsible for listeriosis, a disease characterized by meningitis in the newborn, bacteremia in immunocompromised or elderly individuals, and abortions in pregnant women (1, 2). To date, the most severe human listeriosis outbreaks have been associated with a subset of Lm lineage I strains (3, 4). These hypervirulent strains harbor a biosynthetic gene cluster that encodes for the small peptide Listeriolysin S (LLS) (3, 5). Though initially proposed to be a virulence factor via its hemolytic activity (3), it has since been shown that LLS is a weak hemolytic factor that does not display cytotoxic effects on eukaryotic cells, does not induce specific immune cell responses, and has no impact on cellular infection by Lm (6). Instead, LLS is a bacteriocin that targets several gram-positive bacterial species in vitro including Lactococcus lactis as well as hypovirulent Lm strains, and promotes intestinal colonization by Lm in vivo through modulation of the host gut microbiota composition (7, 8).Biosynthetic gene clusters similar to the LLS operon (shown in SI Appendix, Fig. S1A) are widely conserved in different bacterial phyla (9). They encode for 1) a prepeptide (unmodified peptide) (SI Appendix, Fig. S1B), 2) an ATP-binding cassette transporter (ABC transporter) that exports the toxin once it is posttranslationally modified, 3) an immunity protein, and 4) an enzymatic complex that allows the posttranslational modification (PTM) of the toxin with thiazole, oxazole, and/or methyl-oxazole heterocycles (10). This family of thiazole/oxazole–modified microcins (TOMMs) includes microcin B17 (MccB17) from Escherichia coli, streptolysin S (SLS) from Streptococcus pyogenes, and plantazolicin (PZN) from Bacillus methylotropicus (10–12). MccB17 is an antimicrobial peptide that targets E. coli and acts as a DNA gyrase inhibitor (13), while PZN displays narrow activity against Bacillus anthracis through bacterial membrane depolarization and association with cardiolipin microdomains (11). In contrast, SLS is a major cytotoxic and hemolytic virulence factor produced by group A S. pyogenes (14, 15), which targets erythrocytes, leukocytes, platelets, and subcellular organelles, and can display lytic activity against bacterial protoplasts (16–19).We have previously shown that LLS kills specific gram-positive bacteria (7) but its mechanism of action remains unknown. In the present study, we demonstrate that LLS remains localized to the bacterial cell membrane of LLS-producer bacteria and exerts its killing mechanism via direct contact between LLS-producer and -target bacteria, impairing membrane integrity of target bacteria and inducing membrane depolarization. Our previous findings identified a key role for LLS in the modulation of the host microbiota by Lm hypervirulent strains. Our present work suggests that hypervirulent Lm may outcompete bacteria by means of LLS in a contact-dependent manner.     

Identification of hypertension in hospitalized children prescribed as‐needed antihypertensive medication

Imperfect measurement conditions, transient blood pressure (BP) elevation due to pain or anxiety, and heavy clinical demands complicate hypertension (HTN) diagnosis in hospitalized children, and may prevent recognition of hypertensive episodes for children prescribed as‐needed (PRN) antihypertensive medication. The authors sought to describe the incidence and predictors of missed BP elevation among hospitalized children prescribed PRN antihypertensive medication at our hospital. BP data were retrospectively audited for children age 2‐18 admitted in 2018 to the general ward, and prescribed PRN oral nifedipine, intravenous [IV] or oral hydralazine, or IV labetalol. Appropriate recognition of BP elevation (exceeding the parameters in the medication order) was defined as administering medication within the ordered dosing interval, administering the medication earlier than planned, or physician documentation of why HTN treatment was withheld. Mixed‐effects logistic regression was used to identify factors associated with recognition of BP elevation. Fifty‐six hospitalizations including 616 BP measurements were analyzed. BP elevation was appropriately recognized in 230 (37%) instances, in most of which (n = 190) the antihypertensive medication was given after excessive BP was noted. On multivariable analysis, higher systolic BP and BP elevation occurring at night were associated with increased likelihood of appropriate recognition. BP elevations are frequently missed in hospitalized children prescribed PRN antihypertensive medication. Particularly, there was low recognition of diastolic BP elevation and of systolic BP elevation close to but still exceeding the ordered threshold. Further staff education may be needed to raise awareness of lower BP thresholds for HTN in younger and smaller children.     

Clinical significance of lifetime mood and panic-agoraphobic spectrum symptoms on quality of life of patients with rheumatoid arthritis

BACKGROUND: Previous studies suggested that rheumatoid arthritis (RA) is associated with depressive and anxiety symptomatology. The well-being and functioning of patients with RA may be significantly influenced by subthreshold psychiatric comorbidity. Health-related quality of life (HRQoL) of patients with RA, compared with the Italian norms and patients with diabetes, was assessed by the influence of lifetime mood and panic-agoraphobic spectrum symptoms and demographic and clinical variables. METHODS: Ninety-two patients were consecutively recruited at the Department of Rheumatology at the University Hospital of Pisa, Italy. All patients met diagnostic criteria of RA according to the American College of Rheumatology. Health-related quality of life was measured using the Medical Outcomes Study 36-Item Short-Form Health Survey questionnaire (MOS SF-36). Mood and panic-agoraphobic spectra were assessed by two different structured self-report instruments: the Mood Spectrum (MOODS-SR) and the Panic-Agoraphobic Spectrum (PAS-SR), respectively. RESULTS: Patients with RA were compared, as regards the MOS SF-36 scale scores, with the Italian normative population and patients with diabetes. Compared with the Italian population, patients with RA showed significantly lower MOS SF-36 scale scores, except for role emotional. Moreover, patients with RA scored significantly lower on the role physical, bodily pain, and social functioning scales compared with patients with diabetes and higher on role emotional and mental health. A significant worsening of all MOS SF-36 scale scores was related to higher scores of the depressive domains of MOODS-SR, except for social functioning and bodily pain. A statistically significant negative association was also found between PAS-SR total score and the MOS SF-36 scales physical functioning, vitality, role emotional, and mental health. There were no statistically significant correlations between MOS SF-36 scales and the manic MOODS spectrum. In the multivariate models, the negative correlations between depressive MOODS, role emotional, and mental health were confirmed and the severity of arthritis showed a significant impact on all MOS SF-36 areas with the exception for social functioning; moreover, manic MOODS was associated with better general health. CONCLUSIONS: The present report shows that lifetime depressive spectrum symptoms negatively affects HRQoL of patients with RA and subthreshold mania improves the perception of general health. Diagnosis and appropriate clinical management of depression, including subthreshold symptoms, might enhance HRQoL in these patients.     

The prediction of small airway dimensions using computed tomography

Chronic obstructive pulmonary disease is characterized by destruction of the lung parenchyma and/or small airway narrowing. To determine whether the dimensions of relatively large airways assessed using computed tomography (CT) reflect small airway dimensions measured histologically, we assessed these variables in nonobstructed or mild to moderately obstructed patients having lobar resection for a peripheral tumor. For both CT and histology, the square root of the airway wall area (Aaw) was plotted versus lumen perimeter to estimate wall thickness. The wall area percentage was calculated as wall area/lumen area + wall area x 100. Although CT overestimated Aaw, the slopes of the relationships between the square root of Aaw and internal perimeter (Pi) measured with both techniques were related (CT slope = 0.2059 histology slope + 0.1701, R2 = 0.32, p < 0.01). The mean wall area percentage measured by CT for airways with a Pi of greater than 0.75 cm predicted the mean dimensions of the small airways with an internal diameter of 1.27 mm (R2 = 0.57, p < 0.01). We conclude that CT measurements of airways with a Pi of 0.75 cm or more could be used to estimate the dimensions of the small conducting airways, which are the site of airway obstruction in chronic obstructive pulmonary disease.     

Quality assurance in gerontological and geriatric training programs: the European case

Quality assurance (QA) in gerontological and geriatric education programs is regarded as essential to maintain standards, strengthen accountability, improve readability of qualifications, and facilitate professional mobility. In this article the authors present a summary of international developments in QA and elaborate four international trends, including the pros and cons of QA. Furthermore, the authors focus on accreditation and credit transfer opportunities in vocational and academic education programs for primary care practitioners, including nurses, home care workers, social workers, physiotherapists, and family doctors involved in the care of older people in nine European countries and highlight changes that have occurred over the last decade. Vocational education and professional training in elderly care at the basic and postgraduate specialization level remains extremely diversified, reflecting the lack of standardization for programs outside the higher education sector. The situation is ripe for the implementation of the European Qualifications Framework, which is intended to promote transparency, comparability and portability of qualifications at different levels and the introduction of a credit transfer system for vocational education to be established in 2012.     

Genetic deficit of KCa3.1 channels protects against pulmonary circulatory collapse induced by TRPV4 channel activation

Background and Purpose

The intermediate conductance calcium/calmodulin-regulated K⁺ channel K_Ca3.1 produces hyperpolarizing K⁺ currents that counteract depolarizing currents carried by transient receptor potential (TRP) channels, and provide the electrochemical driving force for Cl⁻ and fluid movements. We investigated whether a deficiency in K_Ca3.1 (K_Ca3.1^−/−) protects against fatal pulmonary circulatory collapse in mice after pharmacological activation of the calcium-permeable TRP subfamily vanilloid type 4 (TRPV4) channels.

Experimental Approach

An opener of TRPV4 channels, GSK1016790A, was infused in wild-type (wt) and K_Ca3.1^−/− mice; haemodynamic parameters, histology and pulmonary vascular reactivity were measured; and patch clamp was performed on pulmonary arterial endothelial cells (PAEC).

Key Results

In wt mice, GSK1016790A decreased right ventricular and systemic pressure leading to a fatal circulatory collapse that was accompanied by increased protein permeability, lung haemorrhage and fluid extravasation. In contrast, K_Ca3.1^−/− mice exhibited a significantly smaller drop in pressure to GSK1016790A infusion, no haemorrhage and fluid water extravasation, and the mice survived. Moreover, the GSK1016790A-induced relaxation of pulmonary arteries of K_Ca3.1^−/− mice was significantly less than that of wt mice. GSK1016790A induced TRPV4 currents in PAEC from wt and K_Ca3.1^−/− mice, which co-activated K_Ca3.1 and disrupted membrane resistance in wt PAEC, but not in K_Ca3.1^−/− PAEC.

Conclusions and Implications

Our findings show that a genetic deficiency of K_Ca3.1 channels prevented fatal pulmonary circulatory collapse and reduced lung damage caused by pharmacological activation of calcium-permeable TRPV4 channels. Therefore, inhibition of K_Ca3.1channels may have therapeutic potential in conditions characterized by abnormal high endothelial calcium signalling, barrier disruption, lung oedema and pulmonary circulatory collapse.     

The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells

Recent studies have described malignant stem cells as central to the initiation, growth, and potential relapse of acute and chronic myelogenous leukemia (AML and CML). Because of their important role in pathogenesis, rare and biologically distinct leukemia stem cells (LSCs) represent a critical target for therapeutic intervention. However, to date, very few agents have been shown to directly target the LSC population. The present studies demonstrate that parthenolide (PTL), a naturally occurring small molecule, induces robust apoptosis in primary human AML cells and blast crisis CML (bcCML) cells while sparing normal hematopoietic cells. Furthermore, analysis of progenitor cells using in vitro colony assays, as well as stem cells using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenograft model, show that PTL also preferentially targets AML progenitor and stem cell populations. Notably, in comparison to the standard chemotherapy drug cytosine arabinoside (Ara-C), PTL is much more specific to leukemia cells. The molecular mechanism of PTL-mediated apoptosis is strongly associated with inhibition of nuclear factor kappa B (NF-kappaB), proapoptotic activation of p53, and increased reactive oxygen species (ROS). On the basis of these findings, we propose that the activity of PTL triggers LSC-specific apoptosis and as such represents a potentially important new class of drugs for LSC-targeted therapy.