Repertoire of microglial and macrophage responses after spinal cord injury

Macrophages from the peripheral circulation and those derived from resident microglia are among the main effector cells of the inflammatory response that follows spinal cord trauma. There has been considerable debate in the field as to whether the inflammatory response is good or bad for tissue protection and repair. Recent studies on macrophage polarization in non-neural tissues have shed much light on their changing functional states. In the context of this literature, we discuss the activation of macrophages and microglia following spinal cord injury, and their effects on repair. Harnessing their anti-inflammatory properties could pave the way for new therapeutic strategies for spinal cord trauma.     

Flagellin delivery by Pseudomonas aeruginosa rhamnolipids induces the antimicrobial protein psoriasin in human skin

The opportunistic pathogen Pseudomonas aeruginosa can cause severe infections in patients suffering from disruption or disorder of the skin barrier as in burns, chronic wounds, and after surgery. On healthy skin P. aeruginosa causes rarely infections. To gain insight into the interaction of the ubiquitous bacterium P. aeruginosa and healthy human skin, the induction of the antimicrobial protein psoriasin by P. aeruginosa grown on an ex vivo skin model was analyzed. We show that presence of the P. aeruginosa derived biosurfactant rhamnolipid was indispensable for flagellin-induced psoriasin expression in human skin, contrary to in vitro conditions. The importance of the bacterial virulence factor flagellin as the major inducing factor of psoriasin expression in skin was demonstrated by use of a flagellin-deficient mutant. Rhamnolipid mediated shuttle across the outer skin barrier was not restricted to flagellin since rhamnolipids enable psoriasin expression by the cytokines IL-17 and IL-22 after topical application on human skin. Rhamnolipid production was detected for several clinical strains and the formation of vesicles was observed under skin physiological conditions. In conclusion we demonstrate herein that rhamnolipids enable the induction of the antimicrobial protein psoriasin by flagellin in human skin without direct contact of bacteria and responding cells. Hereby, human skin might control the microflora to prevent colonization of unwanted microbes in the earliest steps before potential pathogens can develop strategies to subvert the immune response.     

Epidemiologic aspects of an emerging focus of visceral leishmaniasis in Tbilisi, Georgia

Background

Over the last 15 years, visceral leishmaniasis (VL) has emerged as a public health concern in Tbilisi, the capital of Georgia.

Methodology/Principal Findings

Seroepidemiological surveys were conducted to determine the prevalence and incidence of infection in children and dogs within the main focus of VL, and to identify risk factors associated with human infection. Of 4,250 children investigated, 7.3% were positive by direct agglutination test in a baseline survey; an apparent incidence rate of 6.0% was estimated by one year follow-up. None of the seropositive children progressed to VL during the survey. Increased seropositivity at one year was predicted by presence at baseline of clustered flying insects (OR = 1.49; P = 0.001), perceived satisfactory sanitation (OR = 1.65; P<0.001), stray dogs (OR = 1.33; P = 0.023), and by persistent fever during the 6 months prior to baseline survey (OR = 14.2; P<0.001). Overall, 18.2% (107/588) of domestic and 15.3% (110/718) of stray dogs were seropositive by the rk39 dipstick test. Clinical VL signs were found in 1.3% of domestic and 2.9% of stray, seropositive dogs. Parasites isolated from human and dog samples were identified by PCR and phylogenetic analysis of the Leishmania 70 kDa heat-shock protein (HSP70) gene as Leishmania infantum.

Conclusions/Significance

There is an active focus of L. infantum transmission in Tbilisi with a high prevalence of human and canine infections.     

Effect of a fixed-ratio (1:1:1) transfusion protocol versus laboratory-results–guided transfusion in patients with severe trauma: a randomized feasibility trial

Background:

Hemorrhage coupled with coagulopathy remains the leading cause of preventable in-hospital deaths among trauma patients. Use of a transfusion protocol with a predefined ratio of 1:1:1 (1 each of red blood cells [RBC], frozen plasma [FP] and platelets) has been associated with improved survival in retrospective studies in military and civilian settings, but such a protocol has its challenges and may increase the risk of respiratory complications. We conducted a randomized controlled trial to assess the feasibility of a 1:1:1 transfusion protocol and its effect on mortality and complications among patients with severe trauma.

Methods:

We included 78 patients seen in a tertiary trauma centre between July 2009 and October 2011 who had hypotension and bleeding and were expected to need massive transfusion (≥ 10 RBC units in 24 h). We randomly assigned them to either the fixed-ratio (1:1:1) transfusion protocol (n = 40) or to a laboratory-results–guided transfusion protocol (control; n = 38). The primary outcome, feasibility, was assessed in terms of blood product ratios and plasma wastage. Safety was measured based on 28-day mortality and survival free of acute respiratory distress syndrome.

Results:

Overall, a transfusion ratio of 1:1:1 was achieved in 57% (21/37) of patients in the fixed-ratio group, as compared with 6% (2/32) in the control group. A ratio of 1:1 (RBC:FP) was achieved in 73% (27/37) in the fixed-ratio group and 22% (7/32) in the control group. Plasma wastage was higher with the intervention protocol (22% [86/390] of FP units v. 10% [30/289] in the control group). The 28-day mortality and number of days free of acute respiratory distress syndrome were statistically similar between the groups.

Interpretation:

The fixed-ratio transfusion protocol was feasible in our study, but it was associated with increased plasma wastage. Larger randomized trials are needed to evaluate the efficacy of such a protocol in trauma care. Trial registration: ClinicalTrials.gov, no. {"type":"clinical-trial","attrs":{"text":"NCT00945542","term_id":"NCT00945542"}}NCT00945542A fixed-ratio (1:1:1) transfusion strategy is a resuscitation strategy for trauma patients that promotes the transfusion of red blood cells (RBC), plasma and platelets (PLT) at a 1:1:1 ratio while minimizing crystalloid infusion.¹ This balanced transfusion strategy aims to correct both the early coagulopathy of trauma and the volume status of patients in hemorrhagic shock, thus targeting preventable hemorrhage-related deaths.^2,3 Retrospective studies of the 1:1:1 transfusion protocol reported marked reductions in mortality based on retrospectively calculated ratios of plasma:PLT:RBC.^4–6 Methodologic limitations, particularly survivorship bias (where higher mortality was associated with low ratios of plasma and PLT to RBC in unsalvageable patients who died before 1:1:1 transfusion could be achieved), preclude any definitive conclusion on the potential benefit of a 1:1:1 transfusion strategy in terms of efficacy and safety.^7–10The 1:1:1 transfusion strategy has been widely adopted by trauma centres worldwide^11,12 and is being increasingly used in prehospital care and in the care of patients without traumatic injuries.^13–15 Widespread adoption of the strategy has significant resource and safety implications. Its full implementation requires access to thawed type AB plasma, which is chronically in short supply.¹⁶ In addition, because of the difficulty in predicting the need for massive transfusion (commonly defined as ≥ 10 RBC units in 24 h), the 1:1:1 transfusion protocol may lead to unnecessary exposure to blood components and an increased risk of acute respiratory distress syndrome, sepsis and multiple organ dysfunction.¹⁷We conducted a pilot randomized controlled trial comparing a 1:1:1 transfusion strategy with the standard of care at our institution (laboratory-results–guided transfusion; laboratory results are available for transfusion decisions throughout resuscitation) in trauma patients predicted to need massive transfusion. Our primary objective was to assess the feasibility and safety of the fixed-ratio protocol in patients with severe trauma.     

Aggregation and structural changes of α(S1)-, β- and κ-caseins induced by homocysteinylation

Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. α(S1)-, β- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one β-, α(S1)-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1-3μm. Homocysteinylation of α(S1)- and β-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400-800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features.     

The Structure of Microbial Community and Degradation of Diatoms in the Deep Near-Bottom Layer of Lake Baikal

Insight into the role of bacteria in degradation of diatoms is important for understanding the factors and components of silica turnover in aquatic ecosystems. Using microscopic methods, it has been shown that the degree of diatom preservation and the numbers of diatom-associated bacteria in the surface layer of bottom sediments decrease with depth; in the near-bottom water layer, the majority of bacteria are associated with diatom cells, being located either on the cell surface or within the cell. The structure of microbial community in the near-bottom water layer has been characterized by pyrosequencing of the 16S rRNA gene, which has revealed 149 208 unique sequences. According to the results of metagenomic analysis, the community is dominated by representatives of Proteobacteria (41.9%), Actinobacteria (16%); then follow Acidobacteria (6.9%), Cyanobacteria (5%), Bacteroidetes (4.7%), Firmicutes (2.8%), Nitrospira (1.6%), and Verrucomicrobia (1%); other phylotypes account for less than 1% each. For 18.7% of the sequences, taxonomic identification has been possible only to the Bacteria domain level. Many bacteria identified to the genus level have close relatives occurring in other aquatic ecosystems and soils. The metagenome of the bacterial community from the near-bottom water layer also contains 16S rRNA gene sequences found in previously isolated bacterial strains possessing hydrolytic enzyme activity. These data show that potential degraders of diatoms occur among the vast variety of microorganisms in the near-bottom water of Lake Baikal.     

Exploring of Primate Models of Tick-Borne Flaviviruses Infection for Evaluation of Vaccines and Drugs Efficacy

Tick-borne encephalitis virus (TBEV) is one of the most prevalent and medically important tick-borne arboviruses in Eurasia. There are overlapping foci of two flaviviruses: TBEV and Omsk hemorrhagic fever virus (OHFV) in Russia. Inactivated vaccines exist only against TBE. There are no antiviral drugs for treatment of both diseases. Optimal animal models are necessary to study efficacy of novel vaccines and treatment preparations against TBE and relative flaviviruses. The models for TBE and OHF using subcutaneous inoculation were tested in Cercopithecus aethiops and Macaca fascicularis monkeys with or without prior immunization with inactivated TBE vaccine. No visible clinical signs or severe pathomorphological lesions were observed in any monkey infected with TBEV or OHFV. C. aethiops challenged with OHFV showed massive hemolytic syndrome and thrombocytopenia. Infectious virus or viral RNA was revealed in visceral organs and CNS of C. aethiops infected with both viruses; however, viremia was low. Inactivated TBE vaccines induced high antibody titers against both viruses and expressed booster after challenge. The protective efficacy against TBE was shown by the absence of virus in spleen, lymph nodes and CNS of immunized animals after challenge. Despite the absence of expressed hemolytic syndrome in immunized C. aethiops TBE vaccine did not prevent the reproduction of OHFV in CNS and visceral organs. Subcutaneous inoculation of M. fascicularis with two TBEV strains led to a febrile disease with well expressed viremia, fever, and virus reproduction in spleen, lymph nodes and CNS. The optimal terms for estimation of the viral titers in CNS were defined as 8–16 days post infection. We characterized two animal models similar to humans in their susceptibility to tick-borne flaviviruses and found the most optimal scheme for evaluation of efficacy of preventive and therapeutic preparations. We also identified M. fascicularis to be more susceptible to TBEV than C. aethiops.     

Changes in the anatomical structure of tree rings of the rootstock and scion in the heterografts of Siberian pine

The analysis of the anatomical characteristics of tree rings of the Pinus sibirica scions and Pinus sylvestris rootstocks is presented. The main anatomical features maintain the seasonal dynamics characteristic of the reference (ungrafted) trees of scion and rootstock, pointing to the stability of the genetic control of the xylem differentiation. However, various anatomical characteristics are reduced for rootstocks and increased for scions relative to the reference trees. A mechanism consistent with our results is that the graft union zone creates a barrier for the ascending and descending transport of substances, including phytohormones. The reaction of the scion and rootstock to climatic factors is weakened relative to the reaction of the reference trees. Thus, the presumed shift towards phytohormone disbalance reduces the influence of external factors on xylem differentiation. The interannual variation of the individual characteristics of the scions and rootstocks is increased relative to the reference trees, reflecting either the influence of the graft partners on each other or the non-uniform distribution of the growth regulators across the graft union. Thus, heterografts can be used to evaluate the interaction of the internal and external mechanisms of xylem differentiation.     

Disulfide linked pyrazole derivatives inhibit phagocytosis of opsonized blood cells

Immune thrombocytopenia (ITP) is caused by production of an autoantibody to autologous platelets. ITP can be treated either by reducing platelet destruction or by increasing platelet production. Fcγ receptor mediated phagocytosis of the opsonized blood cells is a well-accepted mechanism for the underlying pathogenesis of ITP and inhibition of this phagocytosis process with small molecules is a potential strategy for the development of drugs against ITP. A broad screen indicated that 4-methyl-1-phenyl-pyrazole derivative (1) could inhibit the phagocytosis of opsonized blood cells with weak potency. We reveal here the discovery of the polysulfide products, synthesis of various 1-phenyl-pyrazole derivatives, and the biological evaluation of pyrazole derivatives as inhibitors of phagocytosis for potential use as therapeutics for ITP. Substitution at C4 of the pyrazole moiety in the disulfide-bridged dimers influenced the potency in the increasing order of 10 ? 11 ? 16 < 19 < 20. A novel scaffold, 20 with an IC₅₀ of 100 nM inhibiting opsonized blood cell phagocytosis was identified as a potential candidate for further studies. Confirmation of the disulfide bridge additionally provides clues for the non-thiol or non-disulfide bridge carrying ligands targeting ITP and other similar disorders.     

Fulminant ectopic Cushing syndrome in a patient with metastatic neuroendocrine carcinoma and Crohn's disease

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) have been rarely reported in patients with Crohn's disease, being usually small and incidentally detected in areas uninvolved by the inflammatory process. We describe the case of a young female patient with Crohn's disease and a fulminant Cushing's syndrome induced by the ectopic secretion of adrenocorticotropic hormone (ACTH) by an aggressive gastrointestinal neuroendocrine carcinoma (NEC). Despite a multi-therapeutic approach, including the administration of multiple courses of chemotherapy, hypo-cortisolemic agents, somatostatin analogues, as well as the performance of bilateral adrenal vein embolization followed by bilateral adrenalectomy, patient's condition progressively deteriorated and she died nine months after the diagnosis of NEC due to liver failure. The available literature addressing the possible connection between Crohn's disease and NEC is discussed in detail.