Background: Venous thromboembolism (VTE) is frequently observed in patients with coronavirus disease 2019 (COVID-19). However, reported VTE-rates differ substantially.

Objectives: We aimed at evaluating available data and estimating the prevalence of VTE in COVID-19 patients.

Methods: We conducted a systematic literature search (MEDLINE, EMBASE, WHO COVID-19 database) to identify studies reporting VTE-rates in COVID-19 patients. Studies with suspected high risk of bias were excluded from quantitative synthesis. Pooled outcome rates were obtained within a random effects meta-analysis. Subgroup analyses were performed for different settings (intensive care unit (ICU) vs. non-ICU hospitalization and screening vs. no screening) and the association of D-dimer levels and VTE-risk was explored.

Results: Eighty-six studies (33,970 patients) were identified and 66 (28,173 patients, mean age: 62.6 years, 60% men, 20% ICU-patients) were included in quantitative analysis. The overall VTE-prevalence estimate was 14.1% (95%CI 11.6-16.9), 40.3% (95%CI 27.0-54.3) with ultrasound-screening and 9.5% (95%CI 7.5-11.7) without screening. Subgroup analysis revealed high heterogeneity, with a VTE-prevalence of 7.9% (95%CI 5.1-11.2) in non-ICU and 22.7% (95%CI 18.1-27.6) in ICU patients. Prevalence of pulmonary embolism (PE) in non-ICU and ICU patients was 3.5% (95%CI 2.2-5.1) and 13.7% (95%CI 10.0-17.9). Patients developing VTE had higher D-dimer levels (weighted mean difference 3.26 µg/ml (95%CI 2.76-3.77) than non-VTE patients.

Conclusion: VTE occurs in 22.7% of patients with COVID-19 in the ICU, but VTE risk is also increased in non-ICU hospitalized patients. Patients developing VTE had higher D-dimer levels. Studies evaluating thromboprophylaxis strategies in patients with COVID-19 are needed to improve prevention of VTE.

Keywords: COVID‐19; Prevalence; Pulmonary Embolism; Severe Acute Respiratory Syndrome Coronavirus 2; Venous Thromboembolism.

<strong class="sub-title"> Background: </strong> New York City emerged as an epicenter of the coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) pandemic.

Objective: To describe the clinical characteristics and risk factors associated with mortality in a large patient population in the USA.

Design: Retrospective cohort study.

<strong class="sub-title"> Participants: </strong> 6493 patients who had laboratory-confirmed COVI<em>D</em>-19 with clinical outcomes between March 13 and April 17, <em>2</em>0<em>2</em>0, who were seen in one of the 8 hospitals and/or over 400 ambulatory practices in the New York City metropolitan area MAIN MEASURES: Clinical characteristics and risk factors associated with in-hospital mortality.

<strong class="sub-title"> Key results: </strong> A total of 858 of 6493 (13.<em>2</em>%) patients in our total cohort died: 5<em>2</em>/<em>2</em>785 (1.9%) ambulatory patients and 806/3708 (<em>2</em>1.7%) hospitalized patients. Cox proportional hazard regression modeling showed an increased risk of in-hospital mortality associated with age older than 50 years (hazard ratio [HR] <em>2</em>.34, CI 1.47-3.71), systolic blood pressure less than 90 mmHg (HR 1.38, CI 1.06-1.80), a respiratory rate greater than <em>2</em>4 per min (HR 1.43, CI 1.13-1.83), peripheral oxygen saturation less than 9<em>2</em>% (HR <em>2</em>.1<em>2</em>, CI 1.56-<em>2</em>.88), estimated glomerular filtration rate less than 60 mL/min/1.73m^<em>2</em> (HR 1.80, CI 1.60-<em>2</em>.0<em>2</em>), IL-6 greater than 100 pg/mL (HR 1.50, CI 1.1<em>2</em>-<em>2</em>.03), <em>D</em>-<em>dimer</em> greater than <em>2</em> mcg/mL (HR 1.19, CI 1.0<em>2</em>-1.39), and troponin greater than 0.03 ng/mL (HR 1.40, CI 1.<em>2</em>3-1.6<em>2</em>). <em>D</em>ecreased risk of in-hospital mortality was associated with female sex (HR 0.84, CI 0.77-0.90), African American race (HR 0.78 CI 0.65-0.95), and hydroxychloroquine use (HR 0.53, CI 0.41-0.67).

Conclusions: Among patients with COVID-19, older age, male sex, hypotension, tachypnea, hypoxia, impaired renal function, elevated D-dimer, and elevated troponin were associated with increased in-hospital mortality and hydroxychloroquine use was associated with decreased in-hospital mortality.

BACKGROUND

In patients with chronic urticaria (CU), plasma shows signs of thrombin generation and autologous plasma skin tests score positive in as many as 95% of cases.

OBJECTIVE

To evaluate the initiators of blood coagulation that lead to thrombin generation and fibrinolysis in CU.

METHODS

Activated factor VII, activated factor XII, fragment F(1+<em>2</em>), and <em>D</em>-<em>dimer</em> plasma levels were measured in 37 patients with CU and 37 controls. Skin specimens from 10 patients with CU and 10 controls were tested for tissue factor immunohistochemically.

RESULTS

Mean F(1+<em>2</em>) levels were higher in patients than controls (<em>2</em>.54 [S<em>D</em> <em>2</em>.57] nmol/L vs 0.87 [0.<em>2</em>6] nmol/L; P < .001); disease activity was moderate or severe in 9 of 11 (8<em>2</em>%) and 9 of <em>2</em>6 (35%) patients showing high or normal F(1+<em>2</em>) levels, respectively (P < .0<em>2</em>5). Mean <em>D</em>-<em>dimer</em> plasma levels were higher in patients than controls (3<em>2</em>9 [188] ng/mL vs <em>2</em>36 [81] ng/mL; P < .01); disease activity was moderate or severe in 6 of 8 (75%) and 11 of <em>2</em>9 (38%) showing elevated or normal plasma <em>D</em>-<em>dimer</em> levels (P = NS). Factor VIIa levels were higher in patients than controls (<em>2</em>.86 ng/mL [0.66] vs 1.97 ng/mL [0.65]; P < .001). Activated factor VII and F(1+<em>2</em>) levels were correlated (r = 0.5<em>2</em>9; P = .008). Tissue factor reactivity was observed only in CU skin specimens.

CONCLUSIONS

The extrinsic pathway of clotting cascade is activated in CU. Disease severity is associated with the activation of the coagulation cascade.

CONCLUSIONS

The involvement of the coagulation pathway in CU opens new perspectives for a better understanding of the pathogenesis and, possibly, for the treatment of this disease.

Background: Identification of effective treatments in severe cases of COVID-19 requiring mechanical ventilation represents an unmet medical need. Our aim was to determine whether the administration of adipose-tissue derived mesenchymal stromal cells (AT-MSC) is safe and potentially useful in these patients.

Methods: Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine and/or tocilizumab, among others) were treated with allogeneic AT-MSC. Ten patients received two doses, with the second dose administered a median of 3 days (interquartile range-IQR- 1 day) after the first one. Two patients received a single dose and another patient received 3 doses. Median number of cells per dose was 0.98 × 10⁶ (IQR 0.50 × 10⁶) AT-MSC/kg of recipient's body weight. Potential adverse effects related to cell infusion and clinical outcome were assessed. Additional parameters analyzed included changes in imaging, analytical and inflammatory parameters.

Findings: First dose of AT-MSC was administered at a median of 7 days (IQR 12 days) after mechanical ventilation. No adverse events were related to cell therapy. With a median follow-up of 16 days (IQR 9 days) after the first dose, clinical improvement was observed in nine patients (70%). Seven patients were extubated and discharged from ICU while four patients remained intubated (two with an improvement in their ventilatory and radiological parameters and two in stable condition). Two patients died (one due to massive gastrointestinal bleeding unrelated to MSC therapy). Treatment with AT-MSC was followed by a decrease in inflammatory parameters (reduction in C-reactive protein, IL-6, ferritin, LDH and d-dimer) as well as an increase in lymphocytes, particularly in those patients with clinical improvement.

Interpretation: Treatment with intravenous administration of AT-MSC in 13 severe COVID-19 pneumonia under mechanical ventilation in a small case series did not induce significant adverse events and was followed by clinical and biological improvement in most subjects.

Funding: None.

Keywords: COVID-19; Case series; Cellular therapy; Mechanical ventilation; Mesenchymal stromal cells; Pneumonia; SARS-CoV-2.

BACKGROUND

Maraviroc treatment for HIV-1 infected patients results in larger C<em>D</em>4(+) T cell rises than are attributable to its antiviral activity alone. We investigated whether this is due to modulation of T cell activation and inflammation.

RESULTS

Thirty maraviroc-treated patients from the Maraviroc versus Efavirenz Regimens as Initial Therapy (MERIT) study were randomly selected from among those who had CCR5-tropic (R5) HIV on screening and achieved undetectable HIV RNA (<50 copies/mL) by Week 48. Efavirenz-treated controls were matched for baseline characteristics to the maraviroc-treated patients selected for this substudy. Changes in immune activation and inflammation markers were examined for associations with C<em>D</em>4(+) T cell changes. Maraviroc treatment tended to result in more rapid decreases in C<em>D</em>38 expression on C<em>D</em>4(+) T cells and in plasma <em>D</em>-<em>dimer</em> concentrations than did treatment with efavirenz. The proportion of patients with high-sensitivity C-reactive protein>><em>2</em> µg/mL increased from 45% to 66% in the efavirenz arm, but remained constant in the maraviroc arm (P = 0.033). <em>D</em>ecreases in C<em>D</em>38 expression on C<em>D</em>8(+) T cells were correlated with C<em>D</em>4(+) T cell rises for maraviroc treatment (r = -0.4, P = 0.048), but not for treatment with efavirenz.

CONCLUSIONS

Maraviroc-treated patients had earlier, modest decreases in certain markers of immune activation and inflammation, although in this small study, many of the differences were not statistically significant. Levels of high-sensitivity C-reactive protein remained constant in the maraviroc arm and increased in the efavirenz arm. <em>D</em>ecreases in immune activation correlated with increased C<em>D</em>4(+) T cell gains.

BACKGROUND

ClinicalTrials.gov NCT00098<em>2</em>93.

BACKGROUND

Previous studies have demonstrated increased markers of thrombogenesis in patients with atrial fibrillation (AF), suggesting the presence of a hypercoagulable or prothrombotic state. The objective of this study was to determine the effects of introducing ultra-low-dose warfarin (1 mg), conventional warfarin, and aspirin. (300 mg) therapy on thrombogenesis and platelet activation in AF.

RESULTS

We measured sequential changes in plasma fibrin <em>D</em>-<em>dimer</em> (an index of thrombogenesis) and beta-thromboglobulin (beta-TG, a measure of platelet activation) in 51 patients with chronic AF before and at <em>2</em> and 6 weeks after randomization to either 1 mg warfarin or 300 mg aspirin (phase 1). Then all patients were started on conventional warfarin therapy (phase <em>2</em>) with samples taken <em>2</em> and 6 weeks later. Pretreatment results were compared with those from <em>2</em>6 healthy control subjects in sinus rhythm. Baseline (pretreatment) beta-TG and <em>D</em>-<em>dimer</em> levels in patients with AF were elevated compared with those of control subjects (P < .001). In phase 1, there were no significant changes in median levels of fibrin <em>D</em>-<em>dimer</em> or beta-TG, despite warfarin 1 mg or aspirin 300 mg. With standard warfarin therapy (phase <em>2</em>), there was a reduction in median beta-TG at 6 weeks (P = .0<em>2</em>5) and a sequential reduction in median <em>D</em>-<em>dimer</em> levels at <em>2</em> (P = .001) and 6 (P < .001) weeks compared with baseline levels.

CONCLUSIONS

Patients with AF have increased intravascular thrombogenesis and platelet activation compared with patients in sinus rhythm. Introduction of ultra-low-dose warfarin (1 mg) or aspirin 300 mg does not significantly alter these markers, although conventional warfarin therapy reduces beta-TG and fibrin D-dimer levels. This is consistent with the beneficial effect of full-dose warfarin in preventing stroke and thromboembolism in AF and suggests that ultra-low-dose warfarin and aspirin may not exert similar beneficial effects.

Tertiary and quaternary structural changes that occur during post-translational processing of the insulin proreceptor were examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S]methionine, labeled insulin receptor species, isolated by immuno- and insulin-affinity adsorption, were analyzed by sodium dodecyl sulfate (S<em>D</em>S)-polyacrylamide gel electrophoresis under conditions where intra- and intermolecular disulfide bonds remained intact or were cleaved by reduction. Reducing S<em>D</em>S-polyacrylamide gel electrophoresis confirmed that the insulin receptor is synthesized as a long-lived (t1/<em>2</em> = 3 h) proreceptor precursor of <em>2</em>10 k<em>D</em>a which undergoes proteolytic cleavage and carbohydrate maturation to form the alpha- and beta-subunits of the mature receptor. The proreceptor acquires insulin binding activity through a subtle structural change (t1/<em>2</em> = 45 min) detected only by an autoimmune antibody specific for an epitope of the active insulin binding site. Analysis of insulin receptor species by nonreducing S<em>D</em>S-polyacrylamide gel electrophoresis revealed that the proreceptor undergoes two additional structural changes not detected by reducing S<em>D</em>S-polyacrylamide gel electrophoresis. The proreceptor is synthesized as a monomer (M1) with an apparent molecular mass of 170 k<em>D</em>a that is converted by disulfide rearrangement to another monomeric form of 190-k<em>D</em>a apparent molecular mass (M<em>2</em>). N-Linked glycosylation is required for this transition, since aglycoproreceptor, synthesized in the presence of tunicamycin, does not undergo any detectable tertiary or quaternary structural changes. M<em>2</em> self-associates to form a disulfide-linked proreceptor <em>dimer</em> (<em>D</em>) which is subsequently proteolytically processed, forming the mature, disulfide-linked alpha <em>2</em> beta <em>2</em> receptor tetramer. The mature receptor was distinguished from the three proreceptor species (M1, M<em>2</em>, and <em>D</em>) by its cell surface location and its ability to bind tightly to wheat germ agglutinin-agarose, indicating the presence of complex oligosaccharide chains. Subcellular fractionation indicated that both the M1 to M<em>2</em> and M<em>2</em> to <em>D</em> conversions occur in the endoplasmic reticulum. Separation of the nonreduced proreceptor species into "active" and "inactive" forms by affinity chromatography on insulin-agarose revealed that neither the transition of M1 to M<em>2</em>, nor of M<em>2</em> to <em>D</em>, is correlated with the acquisition of insulin binding function. Rather, during its life-time, the M<em>2</em> species acquires insulin binding activity and an epitope recognized by a binding site specific autoimmune antibody through a subtle structural change not detected by reducing or nonreducing S<em>D</em>S-polyacrylamide gel electrophoresis.

T(4) levels are determinant of several components of the fibrinolytic system. However, relationships between hypothyroidism and alteration of fibrinolytic capacity are not well established, and published data remain conflicting. As the impact of hypothyroidism on both degradation and synthesis of proteins may vary according to the severity of the disease, we measured fibrinolytic activity across varying states of hypothyroidism. We measured fibrinogen, <em>D</em>-<em>dimers</em> (<em>D</em><em>D</em>I), alpha(<em>2</em>)-antiplasmin activity, tissue plasminogen activator antigen (t-PA Ag), plasminogen, plasminogen activator inhibitor antigen (PAI-1 Ag), and factor XII (FXII) of the coagulation. We prospectively included 76 middle-aged female subjects: <em>2</em>5 controls, <em>2</em>4 patients displaying moderate hypothyroidism (TSH, 10--50 mU/L), and <em>2</em>7 patients with severe hypothyroidism (TSH, >50 mU/L). Blood pressure, body mass index, smoking habits, total cholesterol as well as high and low density lipoprotein subfractions, triglyceride, fasting glycemia, and insulinemia were recorded. We found a different pattern of fibrinolytic abnormalities according to the severity of hypothyroidism. Compared with controls, patients with moderate hypothyroidism displayed a decreased fibrinolytic activity, as reflected by lower <em>D</em><em>D</em>I levels, higher alpha(<em>2</em>)-antiplasmin activities, and higher levels of t-PA and PAI-1 Ag. In sharp contrast, patients with severe hypothyroidism exhibited higher <em>D</em><em>D</em>I levels, lower alpha(<em>2</em>)-antiplasmin activities, and lower t-PA and PAI-1 Ag levels. These results were not accounted for by confounding factors such as age, smoking, and components of the insulin resistance syndrome. Free T(4) was significantly associated with fibrinogen, alpha(<em>2</em>)-antiplasmin, PAI-1 Ag, total cholesterol, and triglyceride and was negatively associated with <em>D</em><em>D</em>I. The main hypotheses underlying the mechanisms by which thyroid status may affect the fibrinolytic system remain to be established. In conclusion, patients with moderate hypothyroidism, who were consistently shown to be at high risk for cardiovascular disease, have decreased fibrinolytic activity. Subjects with severe hypothyroidism have a tendency toward increased fibrinolytic activity, and these modifications may participate to the bleeding tendency observed in such patients.

Catabolism of Asn-linked glycoproteins to monosaccharides and amino acids occurs in lysosomes. Break-down must be complete to avoid lysosomal storage diseases that occur when fragments as small as <em>dimers</em> are left undigested. Recent results have clarified several aspects of Asn-linked glycoprotein catabolism in mammals. First, degradation of the oligosaccharide portion is accomplished by exo-glycosidases, which act only from the nonreducing end of chains to release sugar monomers as products. In contrast, proteolysis can proceed from both end and internal points along the polypeptide to eventually yield free amino acids. A second important feature of the glycoprotein disassembly pathway is that the hydrolytic steps can be grouped into two sets of ordered reactions: I) stepwise hydrolysis of the major portion of the oligosaccharide chains by a set of exoglycosidases, and II) ordered disassembly of the protein and the oligosaccharide-to-protein linkage region. Process II can vary at a single reaction step depending on the species in which degradation takes place. Thus, the last step of reaction sequence II can be either: 1) hydrolysis of the actual peptide-to-carbohydrate linkage, or <em>2</em>) removal of the reducing-end GlcNAc from a previously freed oligosaccharide. The latter cleavage is catalyzed by the lysosomal glycosidase chitobiase. Chitobiase has been found only in humans and rats and not in other mammals (dogs, cats, goats, sheep, cats, or cattle). The hydrolytic mechanism of this enzyme is unique as it appears to be a reducing-end glycosidase and can be viewed as an accessory step in the human and rat digestive pathways. The species that lack this enzyme likely rely on exo-beta-<em>D</em>-glucosaminidase to cleave GlcNAc from both outer chain residues and the chitobiose moiety at the protein-to-carbohydrate linkage.

1. Cell walls were isolate<em>d</em> from Bacillus licheniformis N.C.T.C. 6346 an<em>d</em> Bacillus subtilis Marburg strain 168 trp grown on casein hy<em>d</em>rolysate into exponential phase. Autolysis was carrie<em>d</em> out an<em>d</em> the soluble pro<em>d</em>ucts, separate<em>d</em> by chromatography on DEAE-cellulose, from the two wall preparations are broa<em>d</em>ly similar in composition an<em>d</em> are in agreement with autolysis procee<em>d</em>ing with hy<em>d</em>rolysis of ami<em>d</em>e bon<em>d</em>s between l-alanine an<em>d</em> N-acetylmuramic aci<em>d</em> resi<em>d</em>ues in the mucopepti<em>d</em>e components. <em>2</em>. Pepti<em>d</em>es originating from the mucopepti<em>d</em>e components were isolate<em>d</em> an<em>d</em> shown to be a monomer pepti<em>d</em>e, l-alanyl-<em>d</em>-glutamyl-meso-<em>d</em>iaminopimelic aci<em>d</em> an<em>d</em> a <em>dimer</em> pepti<em>d</em>e containing two monomer pepti<em>d</em>es linke<em>d</em> through a resi<em>d</em>ue of <em>d</em>-alanine. Approximately one ami<em>d</em>e group is present for each equivalent tripepti<em>d</em>e unit an<em>d</em> is probably substitute<em>d</em> on <em>d</em>iaminopimelic aci<em>d</em> resi<em>d</em>ues. 3. Oligosacchari<em>d</em>es originating from the mucopepti<em>d</em>e components were isolate<em>d</em> an<em>d</em> after hy<em>d</em>rolysis containe<em>d</em> almost equimolar amounts of glucosamine an<em>d</em> muramic aci<em>d</em> an<em>d</em> only very small amounts of amino aci<em>d</em>s. The number-average chain length, estimate<em>d</em> by the release of non-re<em>d</em>ucing en<em>d</em> groups of N-acetylglucosamine with exo-beta-N-acetylglucosamini<em>d</em>ase, is approximately ten hexosamine resi<em>d</em>ues for oligosacchari<em>d</em>es isolate<em>d</em> from either organism. The oligosacchari<em>d</em>es are poly<em>d</em>isperse. 4. N-Acetylglucosamine resi<em>d</em>ues are the only re<em>d</em>ucing terminals <em>d</em>etectable in the oligosacchari<em>d</em>es isolate<em>d</em> from B. subtilis or B. licheniformis cell-wall autolysates. The number-average chain lengths of the oligosacchari<em>d</em>es were <em>d</em>etermine<em>d</em> by estimation of the content of these resi<em>d</em>ues an<em>d</em> are higher than those foun<em>d</em> by enzymic assay. Possible reasons for the <em>d</em>iscrepancy are <em>d</em>iscusse<em>d</em>.

Osteocalcin (OC) is a small (6 k<em>D</em>a) polypeptide whose expression was thought to be limited to mature osteoblasts. The discovery of OC expression in prostate cancer specimens led us to study the regulation of OC gene in androgen-independent metastatic human prostate PC3 cells. An 800-bp human OC (hOC) promoter-luciferase construct exhibited strong basal and vitamin <em>D</em>-induced activity in OC-positive human prostate and osteosarcoma cell lines. Through deletion analysis of the hOC promoter, the functional hierarchy of the cis-acting elements, OSE1, OSE<em>2</em>, and AP-1/V<em>D</em>RE, was established in PC3 cells (OSE1>> AP-1/V<em>D</em>RE>> OSE<em>2</em>). By juxtaposing <em>dimers</em> of these 3 cis-elements, we produced a minimal hOC promoter capable of displaying high tissue specific activity in prostate cancer cells. Our study demonstrated three groups of transcription factors, Runx<em>2</em>, Jun<em>D</em>/Fra-<em>2</em>, and Sp1, responsible for the high hOC promoter activity in PC3 cells by binding to the OSE<em>2</em>, AP-1/V<em>D</em>RE, and OSE1 elements, respectively. Among the three groups of transcription factors, the expression levels of Runx<em>2</em> and Fra-<em>2</em> are higher in the OC-positive PC3 cells and osteoblasts, compared with the OC-negative LNCaP cells. Interestingly, unlike the mouse OC promoter, the OSE1 site in hOC promoter is regulated by members of Sp1 family instead of the osteoblast-specific factor Osf1. The molecular basis for androgen-independent prostate cancer cells behaving like mature osteoblasts may be explained by the interplay and coordination of these transcription factors under the tight regulation of autocrine and paracrine mediators.

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. <em>D</em>ue to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv <em>dimer</em>) and the T84.66 minibody (scFv-CH3 <em>dimer</em>), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (<em>D</em>OTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 k<em>D</em>a) exhibited very rapid tumor targeting with 1<em>2</em>.5 +/- 0.4% injected dose per gram (% I<em>D</em> g(-1) +/- standard error) at <em>2</em> h and reached a maximum of 13.3 +/- 0.9% I<em>D</em> g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- <em>2</em>1.0% I<em>D</em> g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of <em>D</em>-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.<em>2</em> +/- 1.<em>2</em>% I<em>D</em> g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 k<em>D</em>a), displayed rapid tumor targeting of 14.<em>2</em> +/- 6.1% I<em>D</em> g(-1) at <em>2</em> h, and reached a maximum activity of <em>2</em>4.5 +/- 6.1% I<em>D</em> g(-1) by 1<em>2</em> h. Renal uptake achieved a plateau of 1<em>2</em>-13% I<em>D</em> g(-1) which cleared to 7.<em>2</em>% I<em>D</em> g(-1) at 7<em>2</em> h. However, hepatic uptake was elevated and reached a maximum of <em>2</em>6.0 +/- 1.0% I<em>D</em> g(-1) at 1<em>2</em> h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 1<em>2</em> h to 16.6 +/- 1.5% I<em>D</em> g(-1), indicative of an intrinsic hepatic accumulation of the [111In]<em>D</em>OTA-T84.66 minibody or metabolites. While the anti-CEA [111In]<em>D</em>OTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]<em>D</em>OTA forms appeared problematic for RIS and RIT applications. <em>D</em>evelopment of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.

Tumor necrosis factor (TNF) may be involved in the disturbance of the procoagulant-fibrinolytic balance in septicemia, leading to microvascular thrombosis. To assess the dynamics of the fibrinolytic response to TNF in humans, we performed a crossover saline-controlled study in six healthy men, investigating the effects of a bolus intravenous injection of recombinant human TNF (50 micrograms/m<em>2</em>) on the stimulation and inhibition of plasminogen activation as well as on plasmin activity and inhibition. TNF induced a brief fourfold increase in the overall plasma plasminogen activator (PA) activity peaking after 1 h (p less than 0.0001), which was associated with rises in the antigenic levels of urokinase-type plasminogen activator (p less than 0.0001) and tissue-type plasminogen activator (p less than 0.0001). Plasminogen activator inhibitor type I antigen remained unchanged in the first hour, but showed a rapid eightfold increase thereafter (p less than 0.0001), which coincided with the decrease in PA activity. Generation of plasmin activity in the first hour was signified by an 11-fold rise in <em>D</em>-<em>dimer</em> levels (p less than 0.0001); inhibition of plasmin was reflected by a 36-fold rise in plasmin-alpha <em>2</em> antiplasmin complexes (p less than 0.0001), as well as by a transient 16% decrease in alpha <em>2</em>-antiplasmin activity (p less than 0.01). In conclusion, TNF induced an early activation of the fibrinolytic system becoming maximal in 1 h, with a rapid inhibition thereafter. Earlier observations in the same subjects showed sustained coagulation activation for 6-1<em>2</em> h. The observed disbalance between the procoagulant and fibrinolytic mechanisms after TNF injection confirms the in vivo relevance of the effects of TNF on vascular endothelium in vitro and may explain the tendency towards microvascular thrombosis in septicemia.

BACKGROUND

Animal studies have demonstrated that hemostatic disorders occurring after cardiac arrest affect outcome. We investigated hemostatic changes during and after cardiopulmonary resuscitation (CPR) in humans.

RESULTS

The prospective study included <em>2</em>3 patients (<em>2</em>9 to 86 years) who underwent out-of-hospital CPR for nontraumatic causes. Blood samples were drawn immediately and 15 and 30 minutes after initiation of CPR. In the case of restoration of spontaneous circulation (ROSC; n = 7), additional blood samples were taken immediately, 30 minutes, and <em>2</em>, 8, <em>2</em>4, 48, and 7<em>2</em> hours after ROSC. A marked activation of blood coagulation was found in all patients. The specific markers of activated blood coagulation and fibrin formation, thrombin-antithrombin complex (TAT; median during CPR, <em>2</em>60 micrograms/L; median after ROSC, 57 micrograms/L; normal range, 1.0 to 4.1 micrograms/L), and fibrin monomers (FM; median during CPR, 34.3 micrograms/mL; median after ROSC, 65.4 micrograms/mL; normal range, 0 to 3.6 micrograms/mL) were markedly increased during and in the early phase after CPR. When patients survived for 48 hours, TAT and FM values returned to the normal range. In most patients, the plasma levels of <em>D</em>-<em>dimer</em>, an indicator of endogenous fibrinolytic activity, were not markedly increased during CPR (median, < 0.<em>2</em>5 microgram/mL; normal range, < 0.<em>2</em>5 microgram/mL) but increased moderately after ROSC (median, 0.56 microgram/mL). Levels of plasminogen activator inhibitor type 1 (normal range, 0.3 to 3.5 U/mL), a marker for endogenous inhibition of fibrinolytic activity, were moderately increased in most patients (median during CPR, 4.<em>2</em><em>2</em> U/mL; median after ROSC, 8.08 U/mL).

CONCLUSIONS

Our data clearly demonstrate that there is a marked activation of blood coagulation and fibrin formation after prolonged cardiac arrest and CPR in humans that is not balanced adequately by concomitant activation of endogenous fibrinolysis. These changes may contribute to reperfusion disorders, such as the cerebral "no-reflow" phenomenon, by inducing fibrin deposition and formation of microthrombi.

Background The association of pulmonary embolism (PE) with deep vein thrombosis (<em>D</em>VT) in patients with coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) remains unclear, and the diagnostic accuracy of <em>D</em>-<em>dimer</em> tests for PE is unknown. Purpose To conduct meta-analysis of the study-level incidence of PE and <em>D</em>VT and to evaluate the diagnostic accuracy of <em>D</em>-<em>dimer</em> tests for PE from multicenter individual patient data. Materials and Methods A systematic literature search identified studies evaluating the incidence of PE or <em>D</em>VT in patients with COVI<em>D</em>-19 from January 1, <em>2</em>0<em>2</em>0, to June 15, <em>2</em>0<em>2</em>0. These outcomes were pooled using a random-effects model and were further evaluated using metaregression analysis. The diagnostic accuracy of <em>D</em>-<em>dimer</em> tests for PE was estimated on the basis of individual patient data using the summary receiver operating characteristic curve. Results Twenty-seven studies with 334<em>2</em> patients with COVI<em>D</em>-19 were included in the analysis. The pooled incidence rates of PE and <em>D</em>VT were 16.5% (95% CI: 11.6, <em>2</em><em>2</em>.9; <i>I</i>^<em>2</em> = 0.93) and 14.8% (95% CI: 8.5, <em>2</em>4.5; <i>I</i>^<em>2</em> = 0.94), respectively. PE was more frequently found in patients who were admitted to the intensive care unit (ICU) (<em>2</em>4.7% [95% CI: 18.6, 3<em>2</em>.1] vs 10.5% [95% CI: 5.1, <em>2</em>0.<em>2</em>] in those not admitted to the ICU) and in studies with universal screening using CT pulmonary angiography. <em>D</em>VT was present in 4<em>2</em>.4% of patients with PE. <em>D</em>-<em>dimer</em> tests had an area under the receiver operating characteristic curve of 0.737 for PE, and <em>D</em>-<em>dimer</em> levels of 500 and 1000 μg/L showed high sensitivity (96% and 91%, respectively) but low specificity (10% and <em>2</em>4%, respectively). Conclusion Pulmonary embolism (PE) and deep vein thrombosis (<em>D</em>VT) occurred in 16.5% and 14.8% of patients with coronavirus disease <em>2</em>019 (COVI<em>D</em>-19), respectively, and more than half of patients with PE lacked <em>D</em>VT. The cutoffs of <em>D</em>-<em>dimer</em> levels used to exclude PE in preexisting guidelines seem applicable to patients with COVI<em>D</em>-19. © RSNA, <em>2</em>0<em>2</em>0 <i>Supplemental material is available for this article.</i> See also the editorial by Woodard in this issue.

Three forms of the glycolytic enzyme, enolase [<em>2</em>-phospho-<em>D</em>-glycerate hydrolase (E.C. No. 4.<em>2</em>.1.11)] have been prepared from rat whole brain extract. The most acidic enolase form is neuron specific enolase (NSE) which had previously been designated neuron specific protein (NSP). The least acidic form designated non-neuronal enolase (NNE) has been purified and compared structurally, immunologically and functionally to NSE. NNE is a <em>dimer</em> of 86,500 M.W. consistint of two very similar subunits. The data establish that NNE is larger than NSE which has been shown to be composed of two apparently identical 39,000 molecular weight subunits (78,000). NNE is less acidic than NSE having a pI of 5.9 compared to the value of 4.7 for NSE. Structural and immunological analysis establishes that the NNE subunit is distinct from the NSE subunit, and are therfore products of two separate genes. The structural designation of NSE is (gammagamma) and that of NNE (alpha' alpha'). NSE is strictly localized in neurons indicating that the gene coding for the gamma subunit is only expressed in neuronal cells. The intermediate brain enolase form has been partially purified; structural and immunological evidence indicate that it is a hybrid molecule consisting of one NNE subunit and one NSE subunit (alpha'gamma).

<strong class="sub-title"> Background: </strong> Coronavirus disease <em>2</em>019 (COVI<em>D</em>-19) has rapidly become a global pandemic. Since the severity of the disease is highly variable, predictive models to stratify patients according to their mortality risk are needed.

Objective: To develop a model able to predict the risk of fatal outcome in COVID-19 patients, which could be used easily upon arrival of patients to the hospital.

<strong class="sub-title"> Methods: </strong> We constructed a prospective cohort with 611 adult patients diagnosed with COVI<em>D</em>-19 between March 10 and April 1<em>2</em>, <em>2</em>0<em>2</em>0, in a tertiary hospital in Madrid, Spain. We included in the analysis 501 patients who had been discharged or had died by April <em>2</em>0, <em>2</em>0<em>2</em>0. The capacity to predict mortality of several biomarkers, measured at the beginning of hospitalisation, was assessed individually. Those biomarkers that independently contributed to improve mortality prediction were included in a multivariable risk model.

<strong class="sub-title"> Results: </strong> High interleukin-6 (IL-6), C-reactive protein, lactate dehydrogenase (L<em>D</em>H), ferritin, <em>D</em>-<em>dimer</em>, neutrophil count, neutrophil-to-lymphocyte (N/L) ratio, and low albumin, lymphocyte count, monocyte count and peripheral blood oxygen saturation/fraction of inspired oxygen ratio (SpO_<em>2</em>/FiO_<em>2</em>), were all predictive of mortality (area under the curve (AUC)>0.70). A multivariable mortality risk model including SpO_<em>2</em>/FiO_<em>2</em>, N/L ratio, L<em>D</em>H, IL-6, and age, was developed and showed high accuracy for the prediction of fatal outcome (AUC=0.94). The optimal cut-off reliably classified patients into survivor and non-survivor, including patients with no initial respiratory distress, with 0.88 sensitivity and 0.89 specificity.

Conclusion: This mortality risk model allows early risk stratification of COVID-19 hospitalised patients, before the appearance of obvious signs of clinical deterioration, and can be used as a tool to guide clinical decision-making.

Keywords: COVID-19; interleukin-6; mortality risk; predictive model.

BACKGROUND

The strength and direction of the associations between inflammation and coagulation biomarkers with kidney disease onset and progression remain unclear, especially in a population-based setting.

METHODS

Prospective observational study.

METHODS

4,966 participants from the Multi-Ethnic Study of Atherosclerosis (MESA) with a cystatin C-based estimate of glomerular filtration rate (eGFR(cys)) >60 mL/min/1.73 m(<em>2</em>) and at least one follow-up measurement of kidney function. All participants were free of cardiovascular disease at entry.

METHODS

We evaluated the associations of C-reactive protein (CRP), interleukin 6 (IL-6), fibrinogen, factor VIII, and d-dimer levels with kidney function decrease.

METHODS

Kidney function decrease was assessed primarily by repeated measurements of eGFR(cys) over 5 years. Rapid decrease in kidney function was defined as eGFR decrease >3 mL/min/1.73 m(<em>2</em>) per year. Incident low eGFR was defined as the onset of eGFR(cys) <60 mL/min/1.73 m(<em>2</em>) at any follow-up examination and eGFR(cys) decrease ≥1 mL/min/1.73 m(<em>2</em>) per year.

RESULTS

Mean age was 60 years, 39% were white, 5<em>2</em>% were women, and 11% had diabetes. Mean eGFR(cys) was 96 mL/min/1.73 m(<em>2</em>) and 7% had albuminuria. Median follow-up was 4.77 years. Higher factor VIII levels (per 1 standard deviation [SD] of biomarker) had the strongest association with kidney function decrease (β = -0.<em>2</em>5; 95% CI, -0.38 to -0.1<em>2</em>; P < 0.001), followed by IL-6 (β = -0.16; 95% CI, -0.<em>2</em>9 to -0.03; P = 0.01), CRP (β = -0.09; 95% CI, -0.<em>2</em><em>2</em> to 0.03; P = 0.1), and fibrinogen levels (β = -0.09; 95% CI, -0.<em>2</em><em>2</em> to 0.04; P = 0.<em>2</em>). Each 1-SD higher concentration of IL-6 (OR, 1.15; 95% CI, 1.07-1.<em>2</em>3), factor VIII (OR, 1.11; 95% CI, 1.03-1.18), and CRP (OR, 1.09; 95% CI, 1.0<em>2</em>-1.16) at baseline was associated significantly with rapid kidney function decrease. Only IL-6 level was associated significantly with incident low eGFR (OR, 1.09; 95% CI, 1.00-1.19).

CONCLUSIONS

Observational study design and absence of measured GFR.

CONCLUSIONS

Inflammation and coagulation biomarkers are associated with decreasing kidney function in ambulatory adults without established cardiovascular disease or chronic kidney disease.

OBJECTIVE

Intestinal ischaemia is a life-threatening condition with high mortality, and the lack of accurate and readily available diagnostic methods often results in delay in diagnosis and treatment. The aim of this study was to investigate the accuracy of different plasma biomarkers in diagnosing intestinal ischaemia.

METHODS

Prospective inclusion of patients older than 50 years with acute abdomen admitted to hospital in Karlskrona, Sweden, between 2001 and 2003. Venous blood was sampled prior to any surgery and within 24 h from onset of pain. D-lactate, alpha glutathione S-transferase, intestinal fatty acid binding protein, creatine kinase B, isoenzymes of lactate dehydrogenase (LD) and alkaline liver phosphatase (ALP) were analysed. D-dimer was analysed using four different commercially available test kits.

RESULTS

In-hospital mortalities among patients with (n = 10) and without (n = 61) intestinal ischaemia were 40 % and 3 %, respectively (p = 0.003). D-dimer was associated with intestinal ischaemia (p = 0.001) independently of which assay was used. No patient presenting with a normal D-dimer had intestinal ischaemia. D-dimer >0.9 mg/L had a specificity, sensitivity and accuracy of 82 %, 60 % and 79 %, respectively. Total LD, isoenzymes of LD 1-4 and liver isoenzyme of ALP (ALP liver) were significantly higher in patients with intestinal ischaemia, and accuracies for LD 2 (cut-off 2.3 microkat/L) and ALP liver (cut-off 0.7 microkat/L) were 69 % and 66 %, respectively.

CONCLUSIONS

D-dimer may be used as an exclusion test for intestinal ischaemia, but lacks specificity. The other plasma biomarkers studied had insufficient accuracy for this group of patients. Further studies are needed.

In the present study, we have characterized the reactivity of two mAbs that are directed at the human TCR-gamma/<em>delta</em>. These reagents, designated anti-A13 and anti-TiV <em>delta</em> <em>2</em>, were found to recognize antigenic determinants encoded by the TCR V <em>delta</em> 1 and V <em>delta</em> <em>2</em> gene segments, respectively. Immunofluorescence analyses performed with the antibodies confirmed that, in the TCR-gamma/<em>delta</em>+ cell subpopulation, the expression of V <em>delta</em> <em>2</em>+ <em>delta</em> chains is largely predominant, as compared with the V <em>delta</em> 1+ counterparts. However, these experiments led to an apparently discrepant finding. Indeed, the total number of cells recognized by the anti-A13 plus the anti-TiV <em>delta</em> <em>2</em> antibodies was often greater than that detected with anti-TCR-<em>delta</em> 1, a reagent specific for a constant epitope of the human <em>delta</em> chain. Further investigation showed that the presence of a sizeable peripheral lymphocyte subset coexpressing the BMA031 and the A13 epitopes. Because the former antibody is known to recognize an invariant antigenic determinant of the TCR-alpha/beta <em>dimer</em>, these results suggested that the V <em>delta</em> 1 gene segment may be expressed with either C <em>delta</em> or C alpha. This hypothesis was confirmed using T<em>2</em>, an IL-<em>2</em>-dependent BMA031+ A13+ polyclonal cell line developed from peripheral blood of a healthy adult donor. Indeed, T<em>2</em> cells were found to have productively rearranged the V <em>delta</em> 1 gene. Together, results of Northern blot analysis and cDNA cloning indicated that V <em>delta</em> 1 was expressed in these cells as part of a 1.6-kb full-length message including J alpha-C alpha segments.

The amino acid modification, gel filtration chromatographic, and electrophoretic characteristics of bovine and human serum albumins irreversibly modified by methylglyoxal (MG-SA) and by glucose-derived advanced glycation endproducts (AGE-SA) were investigated. Methylglyoxal selectively modified arginine residues at low concentration (1 mM); at high methylglyoxal concentration (100 mM), the extent of arginine modification increased and lysine residues were also modified. Both arginine and lysine residues were modified in AGE-SA. Analytical gel filtration HPLC of serum albumin derivatives suggested that the proportion of <em>dimers</em> and oligomers increased with modification in both low and highly modified MG-SA and AGE-SA derivatives relative to unmodified serum albumins. In SDS-PAGE analysis, <em>dimers</em> and oligomers of low-modified MG-SA were dissociated into monomers, but not in highly modified MG-SA. MG-SA had increased anodic electrophoretic mobility under nondenaturing conditions at pH 8.6, indicating an increased net negative charge, which increased with extent of modification; highly modified MG-SA and AGE-SA had similar high electrophoretic mobilities. MG-SA derivatives were fluorescent: the fluorescence was characteristic of the arginine-derived imidazolone N <em>delta</em>-(5-methyl-4-imidazolon-<em>2</em>-yl)ornithine, but other fluorophores were also present. AGE-SA had similar fluorescence, attributed, in part, to glucose-derived imidazolones. AGE formed from glucose-modified proteins and AGE-like compounds formed from methylglyoxal-modified proteins may both be signals for recognition and degradation of senescent macromolecules.

The crystal structures of complexes of isolectins 1 and <em>2</em> of wheat germ agglutinin (WGA1 and WGA<em>2</em>) with N-acetylneuraminyl-lactose (NeuNAc-alpha(<em>2</em>-3)-Gal-beta(1-4)-Glc) have been refined on the basis of data in the 8 to <em>2</em>.<em>2</em> A resolution range to final crystallographic R-factors of 17.<em>2</em>% and 15.3% (Fo greater than 1 sigma), respectively. Specific binding interactions and water association, as well as changes in conformation and mobility of the structure upon ligand binding, were compared in the two complexes. The temperature factors (B = 16.3 A<em>2</em> and 18.4 A<em>2</em>) were found to be much lower compared with those of their respective native structures (19 to <em>2</em><em>2</em> A<em>2</em>). Residues involved in sugar binding, <em>dimer</em>ization and in lattice contacts exhibit the largest decreases in B-value, suggesting that sugar binding reduces the overall mobility of the protein molecules in the crystal lattice. The binding mode of this sialyl-trisaccharide, an important cell receptor analogue, has been compared in the two isolectins. Only one of the two unique binding sites (4 per <em>dimer</em>), located in the subunit/subunit interface, is occupied in the crystals. This site, termed the "primary" binding site, contains one of the five amino acid substitutions that differentiate WGA1 and WGA<em>2</em>. Superposition of the refined models in each of the independent crystallographic environments indicates a close match only of the terminal non-reducing NeuNAc residue (root-mean-square <em>delta</em> r of 0.5 to 0.6 A). The Gal-Glc portion was found to superimpose poorly, lack electron density, and possess high atomic thermal factors. In both complexes NeuNAc is stabilized through contact with six amino acid side-chains (Ser114 and Glu115 of subunit 1 and Ser6<em>2</em>, Tyr64, Tyr(His)66 and Tyr73 of subunit <em>2</em>), involving all NeuNAc ring substituents. Refinement has allowed accurate assessment of the contact distances for four hydrogen bonds, a strong buried non-polar contact with the acetamido CH3 group and a large number of van der Waals' interactions with the three aromatic side-chains. The higher affinity of N-acetylneuraminyl-lactose observed by nuclear magnetic resonance studies for WGA1 can be explained by the more favorable binding interactions that occur when residue 66 is a Tyr. The tyrosyl side-chain provides a larger surface for van der Waals' stacking against the NeuNAc pyranose ring than His66 and a hydrogen bond contact with Gal (C<em>2</em>-OH), not possible in WGA<em>2</em>.(ABSTRACT TRUNCATED AT 400 WORDS)

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in <em>D</em>NA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homo<em>dimer</em> of the beta subunit, at a single site on the <em>dimer</em>. The numerous beta(<em>2</em>)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(<em>2</em>) site, synthetic peptides derived from the putative beta(<em>2</em>)-binding motifs were assessed for their abilities to inhibit protein-beta(<em>2</em>) interactions, to bind directly to beta(<em>2</em>), and to inhibit <em>D</em>NA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/<em>D</em>)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(<em>2</em>) in various stages or circumstances of <em>D</em>NA replication and repair.

Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. <em>D</em>ioxolane-linked gA <em>dimers</em> formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked <em>dimer</em> in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of <em>dimer</em>ization reaction. The most commonly observed and most stable gramicidin A <em>dimer</em> is the main object of this study. This gramicidin <em>dimer</em> remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA <em>dimers</em>. g(max) and K(<em>D</em>), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its <em>dimer</em>. In GMO bilayers, however, proton affinities of gA and the dioxolane-<em>dimer</em> were significantly lower (K(<em>D</em>) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and <em>2</em>150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-<em>2</em> M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only <em>2</em>5%. <em>2</em>) <em>D</em>ifferences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the <em>dimer</em> channel. However, other factors can also influence closing flickers.