BACKGROUND

Sepsis and endotoxemia are associated with concurrent activation of inflammation and the hemostatic mechanism, which both contribute to organ dysfunction and death. Electrical vagus nerve stimulation (VNS) has been found to inhibit tumor necrosis factor (TNF)-alpha release during endotoxemia in rodents.

OBJECTIVE

To determine the effect of VNS on activation of coagulation and fibrinolysis.

METHODS

Rats received a sublethal i.v. dose of lipopolysaccharide (LPS) after electrical VNS or sham stimulation. Activation of coagulation and fibrinolysis, as well as cytokine release, was measured before LPS injection and <em>2</em>, 4 and 6 h thereafter.

RESULTS

LPS induced activation of the coagulation system (increases in the plasma concentrations of thrombin-antithrombin complexes and D-dimer, and a decrease in antithrombin) and biphasic changes in the fibrinolytic system [early rises of plasminogen activator activity and tissue-type plasminogen activator, followed by a delayed increase in plasminogen activator inhibitor type 1 (PAI-1)]. VNS strongly inhibited all LPS-induced procoagulant responses and more modestly attenuated the fibrinolytic response. In addition, VNS attenuated the LPS-induced increases in plasma and splenic concentrations of the proinflammatory cytokines TNF-alpha and interleukin-6 (IL-6), while not influencing the release of the anti-inflammatory cytokine IL-10.

CONCLUSIONS

These data illustrate a thus far unrecognized effect of VNS and suggest that the cholinergic anti-inflammatory pathway not only impacts on inflammation but also on the coagulant-anticoagulant balance.

<em>D</em>ysregulated production of IL-6 and its receptor (IL-6R) are implicated in the pathogenesis of multiple myeloma, autoimmune diseases and prostate cancer. The IL-6R complex comprises two molecules each of IL-6, IL-6R, and the signaling molecule, gp130. Here, we report the x-ray structure (<em>2</em>.4 A) of the IL-6R ectodomains. The N-terminal strand of the Ig-like domain (<em>D</em>(1)) is disulfide-bonded to domain <em>D</em>(<em>2</em>), and domains <em>D</em>(<em>2</em>) and <em>D</em>(3), the cytokine-binding domain, are structurally similar to known cytokine-binding domains. The head-to-tail packing of two closely associated IL-6R molecules observed in the crystal may be representative of the configuration of the physiological <em>dimer</em> of IL-6R and provides new insight into the architecture of the IL-6R complex.

Background: Laboratory testing is commonly performed in patients with COVID-19. Each of the laboratory parameters has potential value for risk stratification and prediction of COVID-19 outcomes. This systematic review and meta-analysis aimed to evaluate the difference between these parameters in severe and nonsevere disease and to provide the optimal cutoff value for predicting severe disease.

Method: We performed a systematic literature search through electronic databases. The variables of interest were serum procalcitonin, albumin, C-reactive protein (CRP), D-dimer, and lactate dehydrogenase (LDH) levels in each group of severity outcomes from COVID-19.

Results: There were a total of 4848 patients from 23 studies. Our meta-analysis suggest that patients with severe COVID-19 infections have higher procalcitonin, (mean difference 0.07; 95% CI 0.05-0.10; p < 0.00001), CRP (mean difference 36.88; 95% CI 29.10-44.65; p < 0.00001), D-Dimer (mean difference 0.43; 95% CI 0.31-0.56; p < 0.00001), and LDH (mean difference 102.79; 95% CI 79.10-126.49; p < 0.00001) but lower levels of albumin (mean difference -4.58; 95% CI -5.76 to -3.39; p < 0.00001) than those with nonsevere COVID-19 infections. The cutoff values for the parameters were 0.065 ng/mL for procalcitonin, 38.85 g/L for albumin, 33.55 mg/L for CRP, 0.635 μ/L for D-dimer, and 263.5 U/L for LDH, each with high sensitivity and specificity.

Conclusion: This meta-analysis suggests elevated procalcitonin, CRP, D-dimer, and LDH and decreased albumin can be used for predicting severe outcomes in COVID-19.

Keywords: Biomarker; COVID-19; Coronavirus; Laboratory; SARS-CoV-2.

OBJECTIVE

Our aim was to evaluate the long-term effects of transplanted islets on diabetic macro-/microangiopathy in type 1 diabetic kidney-transplanted patients.

METHODS

A total of 34 type 1 diabetic kidney-transplanted patients underwent islet transplantation and were divided into two groups: successful islet-kidney transplantation (SI-K; 21 patients, fasting C-peptide serum concentration >0.5 ng/ml for >1 year) and unsuccessful islet-kidney transplantation (UI-K; 13 patients, fasting C-peptide serum concentration <0.5 ng/ml). Patients cumulative survival, cardiovascular death rate, and atherosclerosis progression were compared in the two groups. Skin biopsies, endothelial dependent dilation (EDD), nitric oxide (NO) levels, and atherothrombotic risk factors [von Willebrand factor (vWF) and D-dimer fragment (DDF)] were studied cross-sectionally.

RESULTS

The SI-K group showed a significant better patient survival rate (SI-K 100, 100, and 90% vs. UI-K 84, 74, and 51% at 1, 4, and 7 years, respectively, P = 0.04), lower cardiovascular death rate (SI-K 1/21 vs. UI-K 4/13, chi(2) = 3.9, P = 0.04), and lower intima-media thickness progression than the UI-K group (SI-K group: delta1-3 years -13 +/- 30 micro m vs. UI-K group: delta1-3 years 245 +/- 20 micro m, P = 0.03) with decreased signs of endothelial injuring at skin biopsy. Furthermore, the SI-K group showed a higher EDD than the UI-K group (EDD: SI-K 7.8 +/- 4.5% vs. UI-K 0.5 +/- 2.7%, P = 0.02), higher basal NO (SI-K 42.9 +/- 6.5 vs. UI-K 20.2 +/- 6.8 micro mol/l, P = 0.02), and lower levels of vWF (SI-K 138.6 +/- 15.3 vs. UI-K 180.6 +/- 7.0%, P = 0.02) and DDF (SI-K 0.61 +/- 0.22 vs. UI-K 3.07 +/- 0.68 micro g/ml, P < 0.01). C-peptide-to-creatinine ratio correlated positively with EDD and NO and negatively with vWF and DDF.

CONCLUSIONS

Successful islet transplantation improves survival, cardiovascular, and endothelial function in type 1 diabetic kidney-transplanted patients.

The pectate lyase (PL; EC 4.<em>2</em>.<em>2</em>.<em>2</em>) secreted by the plant pathogen Erwinia chrysanthemi is induced and catabolite repressed by different concentrations of its own product, digalacturonic acid 4,5-unsaturated at the nonreducing end [u(GalUA)(<em>2</em>)]. Both activities of u(GalUA)(<em>2</em>) depend on its cleavage by oligogalacturonide lyase (OGL; EC 4.<em>2</em>.<em>2</em>.6). This intracellular enzyme converts u(GalUA)(<em>2</em>) to the deoxyketuronic acid 4-deoxy-L-threo-5-hexosulose uronic acid, which is then isomerized to 3-deoxy-<em>D</em>-glycero-<em>2</em>,5-hexodiulosonic acid. An OGL-deficient mutant unable to grow on u(GalUA)(<em>2</em>) was poorly induced by u(GalUA)(<em>2</em>) or by <em>D</em>-galacturonan but produced wild-type levels of PL when supplied with 3-deoxy-<em>D</em>-glycero-<em>2</em>,5-hexodiulosonic acid. PL synthesis in the mutant could also be stimulated by 4,5-unsaturated trigalacturonic acid, from which deoxyketuronic acid is released by another intracellular enzyme. An OGL-deficient mutant that grew slowly on u(GalUA)(<em>2</em>) in comparison with the wild-type parent was hyperinduced by u(GalUA)(<em>2</em>) unless catabolite repression was relieved by cyclic AMP or imposed by logarithmic growth on glycerol. PL synthesis is also stimulated by saturated digalacturonic acid, which is released from <em>D</em>-galacturonan by another extracellular enzyme, exo-poly-alpha-<em>D</em>-galacturonosidase (EC 3.<em>2</em>.1.8<em>2</em>). Because these <em>dimers</em> stimulate PL synthesis at concentrations (wt/vol) 1/1000th of the concentration required by <em>D</em>-galacturonan, and because an OGL-deficient mutant uninducible by <em>dimers</em> was also uninducible by <em>D</em>-galacturonan, we postulate that PL induction by pectic polymers entails extracellular formation of <em>dimers</em> and subsequent intracellular conversion to deoxyketuronic acids, the apparent inducers of PL.

We compared the effects of oral estradiol (<em>2</em> mg), transdermal estradiol (50 microg), and placebo on measures of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in <em>2</em>7 postmenopausal women at baseline and after <em>2</em> and 1<em>2</em> weeks of treatment. Oral and transdermal estradiol induced similar increases in serum free estradiol concentrations. Oral therapy increased the plasma concentrations of factor VII antigen (FVIIag) and activated factor VII (FVIIa), and the plasma concentration of the prothrombin activation marker prothrombin fragment 1+<em>2</em> (F1+<em>2</em>). Oral but not transdermal estradiol therapy significantly lowered plasma plasminogen activator inhibitor-1 (PAI-1) antigen and tissue-type plasminogen activator (tPA) antigen concentrations and PAI-1 activity, and increased <em>D</em>-<em>dimer</em> concentrations, suggesting increased fibrinolysis. The concentration of soluble E-selectin decreased and serum C-reactive protein (CRP) increased significantly in the oral but not in the transdermal or placebo groups. In the oral but not in the transdermal or placebo estradiol groups low-density-lipoprotein (L<em>D</em>L) cholesterol, apolipoprotein B and lipoprotein (a) concentrations decreased while high-density-lipoprotein (H<em>D</em>L) cholesterol, apolipoprotein AI and apolipoprotein All concentrations increased significantly. L<em>D</em>L particle size remained unchanged. In summary, oral estradiol increased markers of fibrinolytic activity, decreased serum soluble E-selectin levels and induced potentially antiatherogenic changes in lipids and lipoproteins. In contrast to these beneficial effects, oral estradiol changed markers of coagulation towards hypercoagulability, and increased serum CRP concentrations. Transdermal estradiol or placebo had no effects on any of these parameters. These data demonstrate that oral estradiol does not have uniformly beneficial effects on cardiovascular risk markers and that the oral route of estradiol administration rather than the circulating free estradiol concentration is critical for any changes to be observed.

The cGMP-binding cGMP-specific phosphodiesterase (P<em>D</em>E5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding P<em>D</em>Es, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for <em>dimer</em>ization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human P<em>D</em>E5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-<em>dimer</em>ization occurs between GAF a modules (K(<em>D</em>) < 30 nM) and between GAF b modules (K(<em>D</em>) = 1-<em>2</em>0 pM), and the sequence between the GAFs (Thr3<em>2</em><em>2</em>-Asp403) contributes to <em>dimer</em> stability; <em>2</em>) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(<em>D</em>) < 40 nM) than does the isolated R domain (K(<em>D</em>) = 110 nM) or holoenzyme (K(<em>D</em>) = <em>2</em>00 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu3<em>2</em>1) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser10<em>2</em> phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into P<em>D</em>E5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to <em>dimer</em>ization.

Fatal multiple organ failure after severe infection may be related to an early activation of protease cascade systems. This study aimed to relate changes in coagulation, fibrinolysis, and kallikrein to shock and outcome. Of 53 patients with severe infection, 30 did not develop shock, 1<em>2</em> survived septic shock, and 11 died from organ failure after septic shock. No patient had overt disseminated intravascular coagulation. We measured 17 components of the coagulation/fibrinolysis/kallikrein pathways on admission and on the next <em>2</em> days. High values for fibrinogen, factor VIII:C, von Willebrand factor antigen, and <em>D</em>-<em>dimer</em> were seen in all patients; factor XII, prekallikrein, factor VII, antithrombin, protein C, and fibronectin were low. The patients thus appeared to be hypercoagulable. These disturbances were more pronounced in septic shock survivors, who also had low plasminogen and antiplasmin, indicating ongoing fibrinolysis. Nonsurvivors of sepsis were distinguished mainly by high plasminogen activator inhibitor values; this suggests an impaired functional fibrinolysis in fatal sepsis, with possible therapeutic implications. Cryoprecipitate infusion increased the fibronectin concentration, but did not influence the other factors studied.

The enzymatic synthesis of the complete l-alanyl(1)-l-alanine(<em>2</em>) si<em>d</em>e chain of the pepti<em>d</em>oglycan precursors of Enterococcus faecalis was obtaine<em>d</em> in vitro using purifie<em>d</em> enzymes. The pathway involve<em>d</em> alanyl-tRNA synthetase an<em>d</em> two ligases, BppA1 an<em>d</em> BppA<em>2</em>, that specifically transfer alanine from Ala-tRNA to the first an<em>d</em> secon<em>d</em> positions of the si<em>d</em>e chain, respectively. The structure of the UDP-N-acetylmuramoyl-l-Ala-gamma-<em>d</em>-Glu-l-Lys(N(epsilon)-l-Ala(1)-l-Ala(<em>2</em>))-<em>d</em>-Ala-<em>d</em>-Ala pro<em>d</em>uct of BppA1 an<em>d</em> BppA<em>2</em> was confirme<em>d</em> by mass spectrometry (MS) an<em>d</em> MS/MS analyses. The pepti<em>d</em>oglycan structure of the wil<em>d</em>-type E. faecalis strain JH<em>2</em>-<em>2</em> was <em>d</em>etermine<em>d</em> by tan<em>d</em>em reverse-phase high-pressure liqui<em>d</em> chromatography-MS revealing that most muropepti<em>d</em>es containe<em>d</em> two l-alanyl resi<em>d</em>ues in the cross-bri<em>d</em>ges an<em>d</em> in the free N-terminal en<em>d</em>s. Deletion of the bppA<em>2</em> gene was associate<em>d</em> with pro<em>d</em>uction of muropepti<em>d</em>es containing a single alanyl resi<em>d</em>ue at these positions. The relative abun<em>d</em>ance of monomers, <em>dimers</em>, trimers, an<em>d</em> tetramers in the pepti<em>d</em>oglycan of the bppA<em>2</em> mutant in<em>d</em>icate<em>d</em> that precursors containing an incomplete si<em>d</em>e chain were efficiently use<em>d</em> by the <em>d</em><em>d</em>-transpepti<em>d</em>ases in the cross-linking reaction. However, the bppA<em>2</em> <em>d</em>eletion impaire<em>d</em> expression of intrinsic beta-lactam resistance suggesting that the low affinity penicillin-bin<em>d</em>ing protein 5 <em>d</em>i<em>d</em> not function optimally with precursors substitute<em>d</em> by a single alanine.

The effects on thrombosis and hemostasis of thrombin-induced activation of endogenous protein C (PC) were evaluated in baboons. Thrombosis was induced by placing into arteriovenous shunts a segment of <em>D</em>acron vascular graft, which generated arterial platelet-rich thrombus, followed by an expansion region of low-shear blood flow, which in turn accumulated fibrin-rich venous-type thrombus. Thrombosis was quantified by 111In-platelet imaging and 1<em>2</em>5I-fibrinogen accumulation. Intravenous infusion of alpha-thrombin, 1-<em>2</em> U/kg-min for 1 h, increased baseline activated PC levels (approximately 5 ng/ml) to <em>2</em>50-500 ng/ml (P < 0.01). The lower thrombin dose, which did not deplete circulating platelets, fibrinogen, or PC, reduced arterial graft platelet deposition by 48% (P < 0.05), and platelet and fibrin incorporation into venous-type thrombus by>> 85% (P < 0.01). Thrombin infusion prolonged the activated partial thromboplastin clotting time, elevated fibrinopeptide A (FPA), thrombin-antithrombin III complex (T:AT III), and fibrin <em>D</em>-<em>dimer</em> plasma levels (P < 0.01), but did not affect bleeding times. Thrombin's antithrombotic effects were blocked by infusing a monoclonal antibody (HPC-4) which prevented PC activation in vivo, caused shunt occlusion, increased the consumption of platelets and fibrinogen, elevated plasma FPA and T:AT III levels, and reduced factor VIII (but not factor V) procoagulant activity (P < 0.05). We conclude that activated PC is a physiologic inhibitor of thrombosis, and that activation of endogenous PC may represent a novel and effective antithrombotic strategy.

BACKGROUND

The stability of hybrids of <em>2</em>'-O-methyl-ribonucleotides with complementary RNA is considerably higher than that of the corresponding DNA.RNA duplexes. The <em>2</em>'-O-modified ribonucleotides are thus an attractive class of compounds for antisense applications. Understanding how these substituents stabilize the structure of the hybrid duplex may be important in the design of ribonucleotides with novel properties.

RESULTS

The crystal structure of a dimer of the self-complementary DNA strand d(GCGT)O<em>2</em>'mer(A)d(TACGC), which has a <em>2</em>'-O-methylated ribonucleotide incorporated at position 5, was determined at <em>2</em>.1 A resolution. This strand forms a duplex with an overall A-type conformation; the methyl groups of the two modified adenosines point into the relatively wide minor groove. Both <em>2</em>'-methoxy groups are hydrogen-bonded to solvent molecules. These results allowed us to build a model of a fully <em>2</em>'-O-methylated RNA double helix.

CONCLUSIONS

Insertion of <em>2</em>'-O-modified RNA residues into a stretch of DNA can nucleate a local A-type conformation, in part because modification with a bulky residue at this position stabilizes a C3'-endo type sugar pucker. The increased stability of fully <em>2</em>'-O-methylated RNA may result from hydrophobic interactions between substituents in the minor groove. As the <em>2</em>'-O-methyl groups are directed into the minor groove, it may be worthwhile to introduce tailor-made <em>2</em>'-O-substituents into RNA; it might be possible to design groups that both stabilize the hybrid duplexes and carry a nuclease function, further improving the efficacy of these modified RNAs in antisense applications.

BACKGROUND

Paroxysmal nocturnal hemoglobinuria (PNH) is associated with an increased risk of thrombosis through unknown mechanisms.

METHODS

We studied <em>2</em>3 patients with PNH, before and after five and 11 weeks of treatment with eculizumab. We examined markers of thrombin generation and reactional fibrinolysis (prothrombin fragment 1+<em>2</em> (F1+<em>2</em>), <em>D</em>-<em>dimers</em>, and plasmin antiplasmin complexes (P-AP), and endothelial dysfunction tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), soluble thrombomodulin (sTM), intercellular adhesion molecule 1 (sICAM-1), vascular cell adhesion molecule (sVCAM-1), endothelial microparticles (EMPs), and tissue factor pathway inhibitor (TFPI).

RESULTS

At baseline, vWF, sVCAM-1, the EMP count, and F1+<em>2</em> and <em>D</em>-dimer levels were significantly elevated in the patients, including those with no history of clinical thrombosis. Treatment with eculizumab was associated with significant decreases in plasma markers of coagulation activation (F1+<em>2</em>, P=0.01<em>2</em>, and <em>D</em>-<em>dimers</em>, P=0.01), and reactional fibrinolysis (P-AP, P=0.000<em>2</em>). Eculizumab treatment also significantly reduced plasma markers of endothelial cell activation (t-PA, P=0.0005, sVCAM-1, P<0.0001, and vWF, P=0.0047) and total (P=0.0008) and free (P=0.0013) TFPI plasma levels.

CONCLUSIONS

Our results suggest a new understanding of the contribution of endothelial cell activation to the pathogenesis of thrombosis in PNH. The terminal complement inhibitor, eculizumab, induced a significant and sustained decrease in the activation of both the plasma hemostatic system and the vascular endothelium, likely contributing to the protective effect of eculizumab on thrombosis in this setting.

BACKGROUND

Despite its benefits, it is uncommon to apply the nested case-control design in diagnostic research. We aim to show advantages of this design for diagnostic accuracy studies.

METHODS

We used data from a full cross-sectional diagnostic study comprising a cohort of 1<em>2</em>95 consecutive patients who were selected on their suspicion of having deep vein thrombosis (DVT). We draw nested case-control samples from the full study population with case:control ratios of 1:1, 1:<em>2</em>, 1:3 and 1:4 (per ratio 100 samples were taken). We calculated diagnostic accuracy estimates for two tests that are used to detect DVT in clinical practice.

RESULTS

Estimates of diagnostic accuracy in the nested case-control samples were very similar to those in the full study population. For example, for each case:control ratio, the positive predictive value of the D-dimer test was 0.30 in the full study population and 0.30 in the nested case-control samples (median of the 100 samples). As expected, variability of the estimates decreased with increasing sample size.

CONCLUSIONS

Our findings support the view that the nested case-control study is a valid and efficient design for diagnostic studies and should also be (re)appraised in current guidelines on diagnostic accuracy research.

Background: Spain has been one of the countries most affected by the COVID-19 pandemic.

Objective: To create a registry of patients with COVID-19 hospitalized in Spain, in order to improve our knowledge of the clinical, diagnostic, therapeutic, and prognostic aspects of this disease.

Methods: A multicentre retrospective cohort study, including consecutive patients hospitalized with confirmed COVID-19 throughout Spain. Epidemiological and clinical data, additional tests at admission and at seven days, treatments administered, and progress at 30 days of hospitalization were collected from electronic medical records.

Results: Up to June 30th 2020, 15,111 patients from 150 hospitals were included. Their median age was 69.4 years (range: 18-102 years) and 57.2% were male. Prevalences of hypertension, dyslipidemia, and diabetes mellitus were 50.9%, 39.7%, and 19.4%, respectively. The most frequent symptoms were fever (84.2%) and cough (73.5%). High values of ferritin (73.5%), lactate dehydrogenase (73.9%), and D-dimer (63.8%), as well as lymphopenia (52.8%), were frequent. The most used antiviral drugs were hydroxychloroquine (85.6%) and lopinavir/ritonavir (61.4%); 33.1% developed respiratory distress. Overall mortality rate was 21.0%, with a marked increase with age (50-59 years: 4.7%, 60-69 years: 10.5%, 70-79 years: 26.9%, ≥80 years: 46.0%).

Conclusions: The SEMI-COVID-19 Network provides data on the clinical characteristics of patients with COVID-19 hospitalized in Spain. Patients with COVID-19 hospitalized in Spain are mostly severe cases, as one in three patients developed respiratory distress and one in five patients died. These findings confirm a close relationship between advanced age and mortality.

Keywords: 2019-nCoV; COVID-19; Coronavirus; España; SARS-CoV-2; Spain.

Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homo<em>dimer</em>ization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concentration dependence of the relative distribution of monomer HL and <em>dimer</em> (HL)(<em>2</em>) species, the apparent dissociation constant (K(<em>D</em>)) for this interaction was determined. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding K(<em>D</em>) values quantified over a range of 10(-10)-10(-4) M. The critical importance of this noncovalent interaction in maintaining the intact <em>dimer</em>ic structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quantitative view of local structural features that define biological properties of IgG4.

BACKGROUND

Plasma levels of D-dimer, the primary degradation product of cross-linked fibrin, are elevated in several acute thrombotic disorders. However, whether elevated D-dimer levels among healthy individuals are associated with future coronary thrombosis is unknown.

RESULTS

To evaluate whether levels of D-dimer are associated with the occurrence of future myocardial infarction (MI) among apparently healthy men, levels were measured in plasma samples collected at baseline from 296 participants in the Physicians' Health Study who later developed a first MI and from an equal number of age- and smoking status-matched control subjects who remained free of vascular disease during a mean follow-up period of 60.2 months. In univariate analyses, baseline plasma concentrations of D-dimer in the upper ranges of normal were associated with elevated risks of MI. Specifically, the relative risk of future MI for individuals with baseline D-dimer concentration exceeding the 95th percentile of the control distribution was two times higher than that of individuals with lower levels (relative risk [RR], 2.02; 95% confidence interval [CI], 1.04 to 4.02; P = .04). This association persisted in multivariate analyses controlling for nonlipid cardiovascular risk factors (RR, 2.12; 95% CI, 1.05 to 4.28; P = .04) and for lipoprotein(a) (RR, 2.02; 95% CI, 1.04 to 3.94; P = .03). In contrast, this association was attenuated and no longer statistically significant in analyses that controlled for total and high-density lipoprotein cholesterol (RR, 1.74; 95% CI, 0.78 to 3.91; P = .2) or for endogenous tissue-type plasminogen activator and its primary inhibitor, plasminogen activator inhibitor type 1 (RR, 1.58; 95% CI, 0.67 to 3.77; P = .3).

CONCLUSIONS

Elevated levels of D-dimer are associated with increased risks of future MI, although they do not appear to be an independent predictor when other risk factors are considered. As the presence of D-dimer in plasma reflects ongoing fibrin degradation, these data support the hypothesis that activation of the endogenous fibrinolytic system occurs many years in advance of coronary arterial occlusion.

Catechins (flavan-3-ols), the most important secondary metabolites in the tea plant, have positive effects on human health and are crucial in defense against pathogens of the tea plant. The aim of this study was to elucidate the biosynthetic pathway of galloylated catechins in the tea plant. The results suggested that galloylated catechins were biosynthesized via 1-O-glucose ester-dependent two-step reactions by acyltransferases, which involved two enzymes, U<em>D</em>P-glucose:galloyl-1-O-β-<em>D</em>-glucosyltransferase (UGGT) and a newly discovered enzyme, epicatechin:1-O-galloyl-β-<em>D</em>-glucose O-galloyltransferase (ECGT). In the first reaction, the galloylated acyl donor β-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphosphate glucose. In the second reaction, galloylated catechins were produced by ECGT catalysis from β-glucogallin and <em>2</em>,3-cis-flavan-3-ol. <em>2</em>,3-cis-Flavan-3-ol and 1-O-galloyl-β-<em>D</em>-glucose were appropriate substrates of ECGT rather than <em>2</em>,3-trans-flavan-3-ol and 1,<em>2</em>,3,4,6-pentagalloylglucose. Purification by more than 1641-fold to apparent homogeneity yielded ECGT with an estimated molecular mass of <em>2</em>41 to 1<em>2</em>1 k<em>D</em>a by gel filtration. Enzyme activity and S<em>D</em>S-PAGE analysis indicated that the native ECGT might be a <em>dimer</em>, trimer, or tetramer of 60- and/or 58-k<em>D</em>a monomers, and these monomers represent a hetero<em>dimer</em> consisting of pairs of 36- or 34- of and <em>2</em>8-k<em>D</em>a subunits. MAL<em>D</em>I-TOF-TOF MS showed that the protein SCPL1199 was identified. Epigallocatechin and epicatechin exhibited higher substrate affinities than β-glucogallin. ECGT had an optimum temperature of 30 °C and maximal reaction rates between pH 4.0 and 6.0. The enzyme reaction was inhibited dramatically by phenylmethylsulfonyl fluoride, HgCl(<em>2</em>), and sodium deoxycholate.

<strong class="sub-title"> Background: </strong> Coronavirus disease <em>2</em>019 (COVID-19) is a novel infectious disease caused by the severe acute respiratory syndrome coronavirus <em>2</em> (SARS-CoV-<em>2</em>) emerged in Wuhan and has quickly spread across the world. The mortality rate in critically ill patients with COVID-19 is high. This study analyzed clinical and biochemical parameters between mild and severe patients, helping to identify severe or critical patients early.

Methods: In this single center, cross-sectional study, 143 patients were included and divided to mild/moderate and sever/critical groups. Correlation between the disease criticality and clinical features and peripheral blood biochemical markers was analyzed. Cut-off values for critically ill patients were speculated through the ROC curve.

<strong class="sub-title"> Results: </strong> Significantly, disease severity was associated with age (r = 0.458, P < 0.001), comorbidities (r = 0.445, P < 0.001), white cell count (r = 0.<em>2</em><em>2</em>9, P = 0.006), neutrophil count (r = 0.<em>2</em>38, P = 0.004), lymphocyte count (r = - 0.<em>2</em>95, P < 0.001), albumin (r = - 0.603, P < 0.001), high-density lipoprotein cholesterol (r = - 0.36<em>2</em>, P < 0.001), serum potassium (r = - 0.<em>2</em>37, P = 0.004), plasma glucose (r = 0.383, P < 0.001), total bilirubin (r = 0.340, P < 0.001), serum amyloid A (r = 0.58, P < 0.001), procalcitonin (r = 0.345, P < 0.001), C-reactive protein (r = 0.477, P < 0.001), lactate dehydrogenase (r = 0.548, P < 0.001), aspartate aminotransferase (r = 0.34<em>2</em>, P < 0.001), alanine aminotransferase (r = 0.<em>2</em>64, P = 0.001), erythrocyte sedimentation rate (r = 0.<em>2</em>84, P = 0.001) and D-dimer (r = 0.477, P < 0.001) .

<strong class="sub-title"> Conclusions: </strong> With the following parameters such as age > 5<em>2</em> years, C-reactive protein > 64.79 mg/L, lactate dehydrogenase > <em>2</em>45 U/L, D-dimer > 0.96 μg/mL, serum amyloid A > 100.0<em>2</em> mg/L, or albumin < 36 g/L, the progress of COVID-19 to critical stage should be closely observed and possibly prevented. Lymphocyte count, serum potassium, high-density lipoprotein cholesterol and procalcitonin may also be a prognostic indicator.

<strong class="sub-title"> Keywords: </strong> COVID-19; Correlation analysis; Prognostic indicator; SARS-CoV-<em>2</em>.

Cancer-associate<em>d</em> point mutations in isocitrate <em>d</em>ehy<em>d</em>rogenase 1 an<em>d</em> <em>2</em> (IDH1 an<em>d</em> IDH<em>2</em>) confer a neomorphic enzymatic activity: the re<em>d</em>uction of α-ketoglutarate to <em>d</em>-<em>2</em>-hy<em>d</em>roxyglutaric aci<em>d</em>, which is propose<em>d</em> to act as an oncogenic metabolite by in<em>d</em>ucing hypermethylation of histones an<em>d</em> DNA. Although selective inhibitors of mutant IDH1 an<em>d</em> IDH<em>2</em> have been i<em>d</em>entifie<em>d</em> an<em>d</em> are currently un<em>d</em>er investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well un<em>d</em>erstoo<em>d</em>. A high throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R13<em>2</em>H i<em>d</em>entifie<em>d</em> compoun<em>d</em> 1, a bis-imi<em>d</em>azole phenol that inhibits <em>d</em>-<em>2</em>-hy<em>d</em>roxyglutaric aci<em>d</em> pro<em>d</em>uction in cells. We investigate<em>d</em> the mo<em>d</em>e of inhibition of compoun<em>d</em> 1 an<em>d</em> a previously publishe<em>d</em> IDH1 mutant inhibitor with a <em>d</em>ifferent chemical scaffol<em>d</em>. Stea<em>d</em>y-state kinetics an<em>d</em> biophysical stu<em>d</em>ies show that both of these compoun<em>d</em>s selectively inhibit mutant IDH1 by bin<em>d</em>ing to an allosteric site an<em>d</em> that inhibition is competitive with respect to Mg(<em>2</em>+). A crystal structure of compoun<em>d</em> 1 complexe<em>d</em> with R13<em>2</em>H IDH1 in<em>d</em>icates that the inhibitor bin<em>d</em>s at the <em>dimer</em> interface an<em>d</em> makes <em>d</em>irect contact with a resi<em>d</em>ue involve<em>d</em> in bin<em>d</em>ing of the catalytically essential <em>d</em>ivalent cation. These results show that targeting a <em>d</em>ivalent cation bin<em>d</em>ing resi<em>d</em>ue can enable selective inhibition of mutant IDH1 an<em>d</em> suggest that <em>d</em>ifferences in magnesium bin<em>d</em>ing between wil<em>d</em>-type an<em>d</em> mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.

OBJECTIVE

Increased cardiovascular (CV) risk is a rheumatoid arthritis (RA) hallmark and it has been mainly related to chronic systemic inflammation. Since inflammation is linked to coagulation perturbation, both may play a role in increasing CV risk. Treatment with tumor necrosis factor (TNF)-alpha blocking agents is effective in RA and reduces local and systemic inflammation but there is little information on its effect on coagulation. We therefore investigated inflammation and coagulation plasma biomarkers before and after infliximab treatment in RA patients.

METHODS

We studied <em>2</em>0 patients with active RA and 40 healthy controls. Patients were treated with: a stable dose of methotrexate (10mg/week), and infliximab (3mg/kg) at weeks 0, <em>2</em>, 6 and 14. At baseline and week 14, we determined: disease activity score (<em>D</em>AS-<em>2</em>8), visual analogue scale pain, erythrocyte sedimentation rate (ESR), and plasma levels of C-reactive protein (CRP), TNF-alpha, interleukin (IL)-6, prothrombin fragment 1+<em>2</em> (F1+<em>2</em>) and <em>D</em>-<em>dimer</em>. The same inflammation and coagulation parameters were evaluated 1h after infliximab infusion in 10 patients.

RESULTS

At baseline, ESR, CRP, TNF-alpha, IL-6, F1+<em>2</em> and <em>D</em>-<em>dimer</em> levels were significantly higher in RA patients than in controls (P=0.0001). After 14weeks of infliximab treatment, there was a significant clinical improvement and ESR and CRP, IL-6, F1+<em>2</em> and <em>D</em>-<em>dimer</em> level decrease (P=0.001-P=0.008). The levels of TNF-alpha, IL-6, F1+<em>2</em> and <em>D</em>-<em>dimer</em> significantly decreased 1h after infliximab infusion (P=0.005).

CONCLUSIONS

Infliximab decreases inflammation and coagulation biomarkers in RA patients. Such a combined effect may be pivotal in reducing the whole thrombotic risk in these patients.

Background: Obesity is a risk factor for pneumonia and acute respiratory distress syndrome.

<strong class="sub-title"> Objective: </strong> To determine whether obesity is associated with intubation or death, inflammation, cardiac injury, or fibrinolysis in coronavirus disease <em>2</em>019 (COVID-19).

Design: Retrospective cohort study.

Setting: A quaternary academic medical center and community hospital in New York City.

<strong class="sub-title"> Participants: </strong> <em>2</em>466 adults hospitalized with laboratory-confirmed severe acute respiratory syndrome coronavirus <em>2</em> infection over a 45-day period with at least 47 days of in-hospital observation.

Measurements: Body mass index (BMI), admission biomarkers of inflammation (C-reactive protein [CRP] level and erythrocyte sedimentation rate [ESR]), cardiac injury (troponin level), and fibrinolysis (D-dimer level). The primary end point was a composite of intubation or death in time-to-event analysis.

<strong class="sub-title"> Results: </strong> Over a median hospital length of stay of 7 days (interquartile range, 3 to 14) days, 533 patients (<em>2</em><em>2</em>%) were intubated, 6<em>2</em>7 (<em>2</em>5%) died, and 59 (<em>2</em>%) remained hospitalized. Compared with overweight patients, patients with obesity had higher risk for intubation or death, with the highest risk among those with class 3 obesity (hazard ratio, 1.6 [95% CI, 1.1 to <em>2</em>.1]). This association was primarily observed among patients younger than 65 years and not in older patients (<i>P</i> for interaction by age = 0.04<em>2</em>). Body mass index was not associated with admission levels of biomarkers of inflammation, cardiac injury, or fibrinolysis.

<strong class="sub-title"> Limitations: </strong> Body mass index was missing for <em>2</em>8% of patients. The primary analyses were conducted with multiple imputation for missing BMI. Upper bounding factor analysis suggested that the results are robust to possible selection bias.

Conclusion: Obesity is associated with increased risk for intubation or death from COVID-19 in adults younger than 65 years, but not in adults aged 65 years or older.

Primary funding source: National Institutes of Health.

Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RG<em>D</em> motif. In addition, the RG<em>D</em> peptide (RG<em>D</em>-4C) was covalently linked to a proapoptotic heptapeptide <em>dimer</em>, <em>D</em>(KLAKLAK)<em>2</em>, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RG<em>D</em>-containing cyclic peptide (RG<em>D</em>-4C) that binds selectively to the alpha(v)beta3 and alpha(v)beta5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RG<em>D</em>-4C phage to inflamed synovium was inhibited by co-administration of soluble RG<em>D</em>-4C. Intravenous injections of the RG<em>D</em>-4C-<em>D</em>(KLAKLAK)<em>2</em> chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RG<em>D</em>-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis.

A quantitative method for the analysis of EPR spectra from dinuclear Mn(II) complexes is presented. The complex [(Me(3)TACN)(<em>2</em>)Mn(II)(<em>2</em>)(mu-OAc)(3)]BPh(4) (1) (Me(3)TACN=N, N('),N(")-trimethyl-1,4,7-triazacyclononane; OAc=acetate(1-); BPh(4)=tetraphenylborate(1-)) was studied with EPR spectroscopy at X- and Q-band frequencies, for both perpendicular and parallel polarizations of the microwave field, and with variable temperature (<em>2</em>-50K). Complex 1 is an antiferromagnetically coupled <em>dimer</em> which shows signals from all excited spin manifolds, S=1 to 5. The spectra were simulated with diagonalization of the full spin Hamiltonian which includes the Zeeman and zero-field splittings of the individual manganese sites within the <em>dimer</em>, the exchange and dipolar coupling between the two manganese sites of the <em>dimer</em>, and the nuclear hyperfine coupling for each manganese ion. All possible transitions for all spin manifolds were simulated, with the intensities determined from the calculated probability of each transition. In addition, the non-uniform broadening of all resonances was quantitatively predicted using a lineshape model based on <em>D</em>- and r-strain. As the temperature is increased from <em>2</em>K, an 11-line hyperfine pattern characteristic of dinuclear Mn(II) is first observed from the S=3 manifold. <em>D</em>- and r-strain are the dominate broadening effects that determine where the hyperfine pattern will be resolved. A single unique parameter set was found to simulate all spectra arising for all temperatures, microwave frequencies, and microwave modes. The simulations are quantitative, allowing for the first time the determination of species concentrations directly from EPR spectra. Thus, this work describes the first method for the quantitative characterization of EPR spectra of dinuclear manganese centers in model complexes and proteins. The exchange coupling parameter J for complex 1 was determined (J=-1.5+/-0.3 cm(-1); H(ex)=-<em>2</em>JS(1).S(<em>2</em>)) and found to be in agreement with a previous determination from magnetization. The phenomenon of exchange striction was found to be insignificant for 1.

This study was designed to evaluate major fibrinolytic parameters in relation to parameters of inflammation associated with different kinds of pleural effusion. Sixty patients with pleural effusion were studied. The underlying aetiology was empyema in 10 cases, tuberculosis in 9, cancer in 31, cardiac failure in 7, and undetermined in 3. Plasminogen, plasminogen activator inhibitor 1 (PAI-1) and <em>2</em> (PAI-<em>2</em>), tissue type plasminogen activator (t-PA), urokinase (u-PA) and <em>D</em>-<em>dimers</em> (<em>D</em>-<em>D</em>) were quantified in plasma samples and pleural effusion specimens. These data were then correlated with inflammatory or infectious parameters, i.e. fibrinogen, von Willebrand factor (vWF), erythrocyte sedimentation rate (ESR), protein concentration, and white blood cell count. <em>D</em>-<em>D</em> levels were higher in pleural fluid than in plasma. <em>D</em>-<em>D</em> levels were not correlated with either plasminogen activator or plasminogen activator inhibitor levels, suggesting the presence of other fibrinolytic pathways. PAI levels (PAI activity, PAI-1 antigenicity, PAI-<em>2</em> antigenicity) and vWF levels were significantly higher in patients with tuberculosis and empyema than in patients with cancer or cardiac failure. Regression analysis between inflammatory and fibrinolytic parameters showed that pleural PAI levels were significantly correlated with pleural neutrophil count, vWF levels, and plasma fibrinogen levels. <em>D</em>-<em>D</em> levels were correlated with blood ESR. No significant difference in pleural t-PA, u-PA and <em>D</em>-<em>D</em> levels was observed between aetiologies. The highest pleural t-PA and u-PA values were noted in patients with cancer, especially lymphoma. Plasma t-PA levels were higher inpatients with pleural effusion secondary to congestive heart failure, but this difference did not reach statistical significance.(ABSTRACT TRUNCATE<em>D</em> AT <em>2</em>50 WOR<em>D</em>S)