Platelets modulate the proteolysis of factor VIII:C protein by plasmin

Factor VIII coagulant protein (VIII:C) functions as a critical cofactor with factor IXa, calcium ions, and phospholipid during the activation of factor X. In the course of this reaction, the activity of VIII:C is first increased and then is destroyed by one or more serine proteases that are part of the coagulation sequence. In this study, we have investigated the influence of platelets on the inactivation of VIII:C by plasmin. Platelets were separated from plasma proteins in the presence of granule release inhibitors and were incubated with plasmin and isolated VIII:C or the complex of purified VIII:C/von Willebrand factor (vWF); VIII:C activity and antigen levels were assessed over time. In the presence of platelets, the isolated VIII:C showed an initial increase in VIII:C activity that was not present when platelets were absent, and the VIII:C/vWF showed an increase in VIII:C activity over that seen when platelets were absent. In addition, platelets stabilized VIII:C activity over a one-hour time course when compared with buffer. The VIII:C antigen did not increase and decreased slowly whether platelets were present or absent. Preincubating the platelets with ristocetin, collagen, or plasmin did not alter the results, and experiments using platelets from a patient with severe von Willebrand's disease also showed a pattern similar to that seen with normal platelets. Experiments using fixed platelets or phospholipid vesicles showed that they did not support the activation reaction or delay the inactivation reaction. These studies demonstrate that platelets modulate the activation and inactivation of VIII:C by plasmin, apparently by a mechanism that is independent of the platelet release reaction.     

Lymphocyte recruitment to the lung

Lymphocyte recruitment in lymphoid tissues and inflammatory sites occurs in response to two events. The first is adherence of lymphocytes to specialized molecules expressed on the surface of appropriately stimulated vascular endothelial cells known as vascular addressins. The interaction occurs via specialized lymphocyte surface molecules known as homing receptors. There is considerable diversity among these molecules. At least three, and possibly four, different addressin-homing receptor pairs exist, regulating entry into peripheral lymph nodes, gut lymphoid tissue, BALT and intrathoracic lymphoid tissue, and inflamed synovium. Vascular addressins are expressed by specialized endothelial cells known as HEV. HEV are not found in normal lung parenchyma but may be induced to appear during an immune response. The mechanism for induction of HEV is unknown, although it may involve the action of inflammatory cytokines. It is not known whether separate endothelial cells exist with a propensity to develop into HEV or if any endothelial cells will develop into HEV if stimulated in the proper manner. Other accessory, lymphocyte-endothelium adhesion molecule pairs have been described, including LFA-1-ICAM-1 and CD4-HLA-DR. These molecules are induced by exposure of the endothelium to inflammatory cytokines, chiefly IFN-gamma. Thus, local humoral influences present during inflammation can alter the possibility of lymphocyte traffic through the endothelium by regulating the presence of lymphocyte adherence molecules. These processes have been documented to occur in the lung in normal homeostasis (e.g., BALT) and in disease (e.g., immunization with SRBC). After adherence, lymphocytes exit the circulation via amoeboid motility. This motility can be altered and enhanced through chemoattractant substances that act via surface receptors. The biochemical basis of cell motility is not entirely clear but appears to involve a link between the second messengers of receptor signaling and changes in the cytoskeleton, particularly actin filaments and microtubules. Like fibroblasts and smooth muscle cells, lymphocytes appear to respond to a number of "mitoattractants," substances that cause cell cycle entry and/or progression as well as enhanced motility. This relationship illustrates the integral relationship between cell motility and proliferation and suggests that the process of cell recruitment might also prime the recruitment cells to become activated to proliferate and perform effector function. Studies of lymphocyte-mediated lung disease confirm that antigen-specific as well as antigen-nonspecific lymphocytes are selectively recruited to the lung from the circulation during an inflammatory reaction in the lung.(ABSTRACT TRUNCATED AT 400 WORDS)     

Production of lymphocyte chemokinetic activity by stimulated alveolar macrophages

While the presence of a lymphocytic parenchymal infiltrate is characteristic of several lung diseases, the mechanisms responsible for the focal accumulation of lymphocytes within the lungs remain unclear. Since alveolar macrophages secrete several substances that affect lymphocyte function, we examined supernatants of stimulated, cultured guinea pig alveolar macrophages for their ability to alter lymphocyte motility. Guinea pigs were immunized by footpad injection of ovalbumin (OVA) emulsified in complete Freund's adjuvant. Fourteen days later, alveolar macrophages were obtained by bronchial lavage or teasing the lung parenchyma, enriched by adherence to plastic, and incubated for 3 and 24 hours in culture medium alone or medium containing either latex beads, OVA, or human serum albumin (HSA). Conditioned medium was harvested and assayed for chemoattractant activity against rat splenic lymphocytes in modified Boyden chambers. Regardless of stimulus, there was no evidence of enhanced lymphocyte motility above control values in supernatants harvested at 3 hours. At 24 hours, alveolar macrophages from OVA-sensitized guinea pigs stimulated with latex or OVA generated significant amounts of lymphocyte migration stimulating activity (LCA) (250 +/- 25 and 247 +/- 24 percent of control migration, respectively) compared to cells incubated in medium alone or with HSA (162 +/- 23 and 147 +/- 14 percent, respectively). Antigen recognition appears to be related to the presence of cytophilic anti-OVA antibody on the surfaces of alveolar macrophages of sensitized guinea pigs. LCA is resistant to neuraminidase, chymotrypsin, and heating to 56 degrees C, and was chemokinetic for T-lymphocytes. it elutes from Sephadex G-100 in two regions: one at approximately 67,000 d, and a second at approximately 15,000 d. These studies indicate that following systemic immunization, the guinea pig alveolar macrophage can react to specific antigen or phagocytosis of inert particulates by secreting a chemokinetic factor for T-lymphocytes, and may play a role in the pathogenesis of some types of antigen-induced lung disease.     

Hunger-promoting AgRP neurons trigger an astrocyte-mediated feed-forward autoactivation loop in mice

Hypothalamic feeding circuits have been identified as having innate synaptic plasticity, mediating adaption to the changing metabolic milieu by controlling responses to feeding and obesity. However, less is known about the regulatory principles underlying the dynamic changes in agouti-related protein (AgRP) perikarya, a region crucial for gating of neural excitation and, hence, feeding. Here we show that AgRP neurons activated by food deprivation, ghrelin administration, or chemogenetics decreased their own inhibitory tone while triggering mitochondrial adaptations in neighboring astrocytes. We found that it was the inhibitory neurotransmitter GABA released by AgRP neurons that evoked this astrocytic response; this in turn resulted in increased glial ensheetment of AgRP perikarya by glial processes and increased excitability of AgRP neurons. We also identified astrocyte-derived prostaglandin E₂, which directly activated — via EP2 receptors — AgRP neurons. Taken together, these observations unmasked a feed-forward, self-exciting loop in AgRP neuronal control mediated by astrocytes, a mechanism directly relevant for hunger, feeding, and overfeeding.     

The Type VI Secretion System Encoded in Salmonella Pathogenicity Island 19 Is Required for Salmonella enterica Serotype Gallinarum Survival within Infected Macrophages

Salmonella enterica serotype Gallinarum is the causative agent of fowl typhoid, a disease characterized by high morbidity and mortality that causes major economic losses in poultry production. We have reported that S. Gallinarum harbors a type VI secretion system (T6SS) encoded in Salmonella pathogenicity island 19 (SPI-19) that is required for efficient colonization of chicks. In the present study, we aimed to characterize the SPI-19 T6SS functionality and to investigate the mechanisms behind the phenotypes previously observed in vivo. Expression analyses revealed that SPI-19 T6SS core components are expressed and produced under in vitro bacterial growth conditions. However, secretion of the structural/secreted components Hcp1, Hcp2, and VgrG to the culture medium could not be determined, suggesting that additional signals are required for T6SS-dependent secretion of these proteins. In vitro bacterial competition assays failed to demonstrate a role for SPI-19 T6SS in interbacterial killing. In contrast, cell culture experiments with murine and avian macrophages (RAW264.7 and HD11, respectively) revealed production of a green fluorescent protein-tagged version of VgrG soon after Salmonella uptake. Furthermore, infection of RAW264.7 and HD11 macrophages with deletion mutants of SPI-19 or strains with genes encoding specific T6SS core components (clpV and vgrG) revealed that SPI-19 T6SS contributes to S. Gallinarum survival within macrophages at 20 h postuptake. SPI-19 T6SS function was not linked to Salmonella-induced cytotoxicity or cell death of infected macrophages, as has been described for other T6SS. Our data indicate that SPI-19 T6SS corresponds to a novel tool used by Salmonella to survive within host cells.     

Pentraxin 3 (PTX3) inhibits plasma cell/stromal cell cross‐talk in the bone marrow of multiple myeloma patients

Pentraxin 3 (PTX3) is a soluble pattern recognition receptor that binds with high affinity and selectivity to fibroblast growth factor‐2 (FGF2), thus inhibiting its pro‐angiogenic activity. Here we investigated the effects of PTX3 on monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patient‐derived bone marrow (BM) plasma cells (PCs), endothelial cells (ECs), and fibroblasts (FBs), and assessed whether PTX3 can modulate the cross‐talk between PCs and those microenvironment cells. PTX3 and FGF2 expression was evaluated by ELISA. Functional studies, including cell viability, wound healing, chemotaxis, and Matrigel^® assays, were performed on MGUS and MM ECs and FBs upon the PTX3 treatment. Through western blot PTX3‐induced modulation in FGF2/FGF receptor signalling pathways was evaluated in MGUS and MM ECs and FBs through western blot. Co‐cultures between MM ECs/FBs and human PC lines were used to evaluate possible PTX3 indirect effects on MM PCs. Adhesion molecules were studied by flow cytometry. PTX3 provides a direct time‐ and dose‐dependent apoptotic effect on MM ECs and FBs, but not on either MM primary PCs or human PC lines. PTX3 inhibits migration of MM ECs and FBs in a dose‐dependent manner, and impacts in vitro and in vivo FGF2‐mediated MM angiogenesis. Co‐cultures of PCs and ECs/FBs show that PTX3 treatment indirectly impairs PC viability and adhesion. We conclude that PTX3 is an anti‐angiogenic factor in MM and behaves as a cytotoxic molecule on MM cells by inhibiting the cross‐talk between PCs and ECs/FBs. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Relationship between clinical outcomes measures and personal and social performance functioning in a prospective,interventional study in schizophrenia

ObjectivesTo explore clinical and demographic characteristics impacting patient functioning by determining extent of overlap in factors driving change in Personal and Social Performance (PSP) and other clinical outcomes.MethodsPost‐hoc analysis from a single‐arm trial of paliperidone extended release in adult patients with nonacute symptomatic schizophrenia. Psychosocial functioning measures: PSP, Clinical Global Impression–Severity (CGI‐S), Positive and Negative Syndrome Scale (PANSS), Short‐Form 36 (SF‐36), treatment satisfaction, sleep quality/daytime drowsiness, and Extrapyramidal Symptoms Rating Scale.ResultsHighest correlations with PSP total score change included PANSS total score change (Spearman''s r = 0.607), PANSS general psychopathology change (r = 0.579), and CGI‐S change (r = 0.569). A PSP score change of −32 predicted 90% probability of deterioration in CGI‐S (score change of ≥1). The power of PSP change to predict PANSS total score change was lower. Linear stepwise regression demonstrated independent relationships for PSP change and: PANSS total change; CGI‐S change; SF‐36 Mental Component change; treatment satisfaction at endpoint; PSP at baseline; previous psychiatric hospitalizations. R ² = 0.55 meant that 45% of PSP variation could not be explained by other clinical outcome measures.ConclusionsPsychosocial functioning improvement is important in schizophrenia. PSP may be valuable for assessing functioning; it encompasses psychosocial and clinical factors not measured by other established assessments.     

Recommendations for Managing Endodontic Emergencies during Coronavirus Disease 2019 Outbreak

IntroductionThe management of endodontic emergencies has been particularly challenging during the coronavirus disease 2019 (COVID-19) outbreak because of the possible generation of airborne particles and aerosols. The aim of this report was to contribute to the practice of endodontics by proposing a general protocol for the management of emergencies showing the rationale for remote diagnosis, clinical procedures, and the use of personal protective equipment and barriers at the dental office during the COVID-19 outbreak.MethodsA review of the literature was conducted up to May 2020 on relevant institutional sites, aiming to retrieve the best updated evidence. The reporting considered the Reporting Tool for Practice Guidelines in Health Care statement.ResultsRecommendations from Cochrane Oral Health, the American Dental Association, and the Centers for Disease Control and Prevention were included along with the American Association of Endodontists resources and scientific articles that addressed the issue.ConclusionsThe proposed protocol could contribute to the management of endodontic emergencies at the dental office during the COVID-19 outbreak.