Atypical hyperpachymorph Trypanosoma (Nannomonas) congolense forest-type in a dog returning from Senegal.

Trypanosoma congolense forest-type was identified by PCR in France, in a dog returning from Senegal. This paper describes the morphological features of the parasite on Giemsa-stained smears. Slender forms and "latent bodies" represent 30.4% and 20.4%, respectively. Some rosettes have been observed (0.8%). The predominant form (48.4%) is stumpy, close to "montgomeryi-form", but it is unusually broad, with a width/length ratio (WLr) of 0.40-0.55, while that of "montgomeryi-forms" is close to 0.3. To the best of our knowledge, this is the first description of such a form of T. (Nannomonas). Also unusual, the shape of the cytoplasm appears to be tightened by an "S-" or "C-" shaped flagellum. We propose naming this peculiar morphotype "hyperpachymorph", and adding its description to that of T. congolense forest-type. Thus T. (Nannomonas) forms would include: sphaeromorph or "latent body-form" (globular), hyperleptomorph (rodhaini-form, very long and slender, with a free flagellum); leptomorph (simiae-form, slender, with a free flagellum); isomorph (congolense-form, short, generally without a free flagellum); pachymorph (montgomeryi-form, short and stout; 0.25 < WLr < 0.34, without a free flagellum), and hyperpachymorph ("hyper montgomeryi-form", short and very stout; 0.35 < WLr < 0.7, without a free flagellum).

Costs and management of patients with hemophilia A in France: the Hemraude study.

OBJECTIVE: The Hemraude study was conducted to describe the profile of patients with HA, disease management, and economic burden in a collective perspective. METHODS: This retrospective study was conducted using the French administrative healthcare claims database SNIIRAM/SNDS. Male patients treated for hemophilia A with a long-term illness (ALD) status or invalidity were included in the study between January 1, 2016 and December 31, 2017. Patients were classified in six treatment groups: no treatment, on-demand FVIII, prophylactic FVIII, FVIII in immune tolerance induction (ITI) protocol, on-demand bypassing agents, and prophylactic bypassing agents. Patients treated with FVIII in ITI protocol and those treated with bypassing agents are deemed to have developed inhibitors. HA patients were compared to a control population without coagulation disorder and matched (ratio 1:3) on age and sex. RESULTS: A total of 4172 patients were included in the analysis, aged on average 35.2 years, 5.3% had HIV infection, and 8.8% had hepatitis B or C. In 2017, half of the patients received no treatment for HA, 46.7% were treated with FVIII (25% on demand, 20.6% with prophylaxis, and 1.1% ITI), 1.5% with bypassing agents. Patients treated with prophylactic treatments, either inhibitor or non-inhibitor, were less likely to be hospitalized for severe bleeding compared to patients receiving on-demand treatments. The average annual costs for HA management per patient were 72,209.60 €. The highest costs were observed in patients treated with FVIII in ITI protocol and those receiving prophylactic bypassing agents. CONCLUSION: Direct costs of HA treatments for HA may be very high especially in the small percentage of patients developing inhibitors or treated with ITI protocol.

Nanostructure of the fibrin clot.

The nanostructure of the fibrin fibers in fibrin clots is investigated by using spectrometry and small angle x-ray scattering measurements. First, an autocoherent analysis of the visible light spectra transmitted through formed clots is demonstrated to provide robust measurements of both the radius and density of the fibrin fibers. This method is validated via comparison with existing small-angle and dynamic light-scattering data. The complementary use of small angle x-ray scattering spectra and light spectrometry unambiguously shows the disjointed nature of the fibrin fibers. Indeed, under quasiphysiological conditions, the fibers are approximately one-half as dense as their crystalline fiber counterparts. Further, although the fibers are locally crystalline, they appear to possess a lateral fractal structure.

High-yield production of secreted active proteins by the Pseudomonas aeruginosa type III secretion system.

The Escherichia coli system is the system of choice for recombinant protein production because it is possible to obtain a high protein yield in inexpensive media. The accumulation of protein in an insoluble form in inclusion bodies remains a major disadvantage. Use of the Pseudomonas aeruginosa type III secretion system can avoid this problem, allowing the production of soluble secreted proteins.

Impact of four direct oral anticoagulants on rotational thromboelastometry (ROTEM).

INTRODUCTION: Rotational Thromboelastometry (ROTEM) is a point of care method used to monitor coagulation during surgery and to guide transfusion strategies in patients presenting with severe bleeding. The aim of our study was to determine the impact of four direct oral anticoagulants (DOACs) on 3 commonly used ROTEM tests. METHODS: Whole blood samples from 20 healthy donors were spiked in vitro with apixaban, edoxaban, rivaroxaban or dabigatran at 5 different plasma concentrations (0-1000 ng/mL). EXTEM, INTEM and FIBTEM tests were systematically performed. RESULTS: There was a linear relationship between the increase in clotting time (CT) and plasma DOAC concentrations in both the EXTEM and INTEM tests. We found that the DOAC concentration required to double EXTEM CT was 1042 ± 225 ng/mL for apixaban, 134 ± 38 ng/mL for edoxaban, 176 ± 26 ng/mL for rivaroxaban and 284 ± 73 ng/mL for dabigatran. INTEM CT was less sensitive than EXTEM CT whatever the anticoagulant. EXTEM CT was above the normal range for 5 of 5 spiked samples when the plasma concentrations were ~1000 ng/mL for apixaban, ~100 ng/mL for edoxaban, ~200 ng/mL for rivaroxaban and ~200 ng/mL for dabigatran. Maximum Clot Firmness in EXTEM, INTEM and FIBTEM tests was not affected whatever the DOAC or its concentration. CONCLUSION: This study found a DOAC dose-dependent increase in ROTEM CTs. ROTEM tests were only poorly impacted by low levels of edoxaban, rivaroxaban or dabigatran. Apixaban had only a low effect even at high concentrations.

Effect of double-filtration plasmapheresis for antibody-mediated rejection on hemostasis parameters and thrombin generation.

INTRODUCTION: Donor-specific alloantibodies (DSAs) cause kidney-allograft loss in chronic antibody-mediated rejection (CAMR). Treatment relies on blocking antibody-producing cells and removing DSAs by apheresis: e.g., double-filtration plasmapheresis (DFPP). MATERIALS AND METHODS: To determine the impact of DFPP (6 or 8 sessions/patient) on clotting factors and natural anticoagulants, and on thrombin generation, we performed a prospective and observational study in five CAMR kidney-transplant patients who received DFPP plus rituximab therapy. Thrombin generation was performed in poor platelet plasma (PPP) with 5 pM tissue factor without and with 2 nM recombinant human thrombomodulin. RESULTS: After the first DFPP session, median levels of high molecular-weight proteins (fibrinogen, FV, FVIII, FXI, FXIII, von Willebrand factors and α2-MG) decreased significantly to <50% of baseline values, whereas levels of low molecular-weight factors (<100 kDa) were not significantly modified, except for protein S and TFPI. Of note, binding-protein (BP) S, i.e., C4BP, was significantly decreased. Over the course of successive DFPP sessions, both high and lower molecular-weight proteins (<100 kDa) with longer half-lives (>2 days, prothrombin and factor XII) were significantly decreased. DFPP also highly affected thrombin generation in the absence of thrombomodulin but not significantly in the presence of thrombomodulin. After the first DFPP session, mean endogenous thrombin potential (ETP) and peak thrombin (PH) significantly decreased when the thrombin generation assay was performed without thrombomodulin (respectively, 1084 nM·min for ETP and 210 nM for PH after the first DFPP session compared to 1616 nM·min and 264 nM at baseline). In the presence of thrombomodulin, there was only a slight decrease in ETP and PH (respectively 748 nM·min, and 172 nM after the first DFPP session compared to 822 nM·min and 179 nM at baseline). After the last session, median ETP and PH decreased respectively to 646 nM·min and 143 nM without thrombomodulin, and, to 490 nM·min and 117 nM with thrombomodulin. CONCLUSIONS: DFPP significantly removed high molecular-weight proteins from the haemostatic system and profoundly decreased levels of protein S and TFPI. Overall thrombin-generation balance was only moderately affected in the presence of thrombomodulin. Nevertheless, high depletion of fibrinogen, FXIII and Von Willebrand Factor may expose patients to an increased risk of bleeding.

Non-conform diagnostic management of pulmonary embolism suspected patients is responsible for a higher risk of thrombotic event occurrence.

The aim of this 3-month follow-up prospective pragmatic study was to evaluate the implementation of a pulmonary embolism (PE) diagnostic strategy in clinical practice. One thousand and one hundred thirty-four consecutive in- and outpatients with clinically suspected PE were enrolled into a sequential diagnostic algorithm in which vascular medical unit plays a pivotal role in advising physicians and suggesting the most appropriate tests according to the diagnostic algorithm. In this observational study, patients that followed the proposed work-up were attributed to a so-called "conform group". Patients in whom diagnostic work-up was not according to protocol were attributed to a "non-conform group". Nine hundred and ninety-seven patients (87.9%) had a conform work-up, and 137 patients a non-conform work-up according to the proposed diagnostic algorithm. The non-conform work-up directly increased in relation to the age of the referred patients. PE was ruled out in 907 (80%) patients of whom 787 (86.8%) were in the conform group. Of the 797 patients who did not receive anticoagulant drugs, follow-up was obtained in 792 (99.4%). Among these patients, the incidence of acute thromboembolic events during the 3-month follow-up period was different in the group of patients that had a conform work-up (1%, [95% CI, 0.5-2.1%]) from the non-conform group patients (4.5%, [95% CI, 2-10.2%]. Therefore patients from the non-conform group have an independent increased risk to develop a thromboembolic event during the follow-up, adjusted odds ratio 3.3 [1.1-10, 95% CI]. Therefore we demonstrated that a non-conform diagnostic management strategy is associated with a higher risk of thrombotic event occurrence.

Influence of Breed Size, Age, Fecal Quality, and Enteropathogen Shedding on Fecal Calprotectin and Immunoglobulin A Concentrations in Puppies During the Weaning Period.

BACKGROUND: Fecal calprotectin and immunoglobulin A (IgA) are markers of intestinal inflammation and immunity in adult dogs. HYPOTHESIS: Fecal calprotectin and IgA concentrations in puppies are not influenced by fecal moisture in puppies but by enteropathogen shedding. ANIMALS: Three hundred and twenty-four puppies. METHODS: Fecal consistency was assessed by gross examination. Fecal moisture was evaluated before and after lyophilization. Canine parvovirus and coronavirus were detected in feces by qPCR and qRT-PCR respectively. Giardia intestinalis antigen was quantified by ELISA. The standard McMaster flotation technique was used to detect eggs and oocysts in feces. Fecal calprotectin and IgA concentrations were quantified by in-house radioimmunoassays. RESULTS: For each marker (IgA and calprotectin), a strong positive correlation was observed between concentration in fresh feces and concentration in fecal dry matter. 75.6% of the puppies were found to be infected by at ≥1 of the enteropathogens evaluated. Fecal calprotectin concentration was significantly influenced by age (P = .001), with higher concentrations in younger puppies, but not by viral (P = .863) or parasitic infection (P = .791). Fecal IgA concentration was significantly influenced by enteropathogen shedding (P = .01), with a lower fecal IgA concentration in puppies shedding at ≥1 enteropathogen compared to puppies without any enteropathogen shedding, but not by age. CONCLUSIONS: Fecal calprotectin and IgA are of no diagnostic value to detect presence of enteropathogens in clinically healthy puppies or puppies with abnormal feces, but could help to better understand the maturation of digestive tract.

Quantitative high D-dimer value is predictive of pulmonary embolism occurrence independently of clinical score in a well-defined low risk factor population.

We performed a prospective study to assess whether positive quantitative D-dimer (DD) levels could be integrated for a selected population in a defined strategy to accurately diagnose pulmonary embolism (PE). For this purpose, 1528 in- or outpatients with clinically suspected PE were investigated according to our prescription rules. Clinical probability was defined as low, intermediate or high. Patients in whom DD levels were measured met criteria defined by our previously described decision-making algorithm: in- and outpatients, < 80 years, without surgery in the previous 30 days or active cancer. Nine hundred and twenty-three patients (60.4%) had quantitative DD measurement using automated latex DD assay (STA-Liatest D-Di). According to our decision-making algorithm, DD measurement was applied to 70.5% of out-, and 55.7% of inpatients, and PE diagnosis was ruled out in 49.5% of the 923 patients. This allowed us to confirm prospectively that our specific rules greatly improve the DD testing efficiency. PE was diagnosed in 115 (12.5%) patients. For a 0.5 mg L(-1) cut-off, the test sensitivity was 97.4%, but its specificity was only 56.7%. However, PE prevalence increased gradually with DD levels. The true observed PE prevalence, according to the quantitative assessment of DD levels, differed from that predicted with pretest clinical probability only. Moreover, in this well-defined patient group, a quantitative DD level > 2 mg L(-1) was predictive of PE occurrence independently of the clinical score (odds ratio 6.9, 95% confidence interval 3.7, 12.8). As part of a defined strategy, knowledge of positive DD quantitative value, together with the clinical probability score, improves the PE predictive model. A clinical validation of these results in a follow-up study would now be necessary before considering the implementation of this strategy into clinical practice.

Protein C level at birth.

The protein C level was determined, on cord blood, for 30 healthy newborns by electro-immuno assay using a monospecific antiserum. For the newborns the mean level of protein C related antigen is about one third of normal adults' mean level. There is a good correlation between Protein C related antigen and prothrombin related antigen. The low level of these vitamin-K-dependent proteins is probably a consequence of partial liver immaturity at birth. Using two-dimensional immuno-electrophoresis we were unable to detect subcarboxylated forms of protein C. However these abnormal forms could be seen in vitamin-K deficiencies of neonates.

Monocyte tissue factor expression induced by Plasmodium falciparum-infected erythrocytes.

Monocytes are active elements of the host response against Plasmodium falciparum. They are able to express tissue factor and trigger the extrinsic pathway of blood coagulation the activation of which remained unclear in malaria. Our aim was to assess the tissue factor expression of purified blood monocytes stimulated by cultured Plasmodium falciparum-infected erythrocytes. Malaria parasite induced an early generation of tissue factor with a peak between 8 and 12 h of stimulation. Maximum expression was observed for parasitemia ranging from 1 to 2%. Plasmodium falciparum culture supernatants had the same effect showing the existence of a soluble factor able to induce the tissue factor expression. These data, demonstrating an activation of the tissue factor pathway by the malaria parasite, emphasize thrombin generation. Therefore, thrombin could participate in malaria pathology either in the microcirculatory blockade via platelet and fibrinogen activation or as a mitotic.

Combined hereditary disorders of haemophilia B Leyden (-6 G-->A) and type 1 von Willebrand disease.

Multiple coagulation disorders are unusual. We report here a combination of haemophilia B Leyden with type 1 von Willebrand disease (vWD) affecting different members of the same family. Haemophilia B Leyden was due to a -6 G-->A mutation within the promoter of the factor IX gene and was responsible for a mild haemophilia in the father of the proband. The proband and her sister (age 4 and 6) exhibited a twofold lower level of factor IX activity (0.4 IU/ml) than the paternal grandmother (0.95 IU/ml). The differences in F IX levels in the three carriers of the same -6 G-->A mutation suggest the implication of an age-related mechanism responsible for the increase in factor IX plasma level. Haemophilia B Leyden patient and carriers suffered also from a mild von Willebrand disease. The diagnosis of this associated type 1 vWD was performed by assaying plasma von Willebrand factor together with multimer electrophoretic studies and DDAVP test. The inheritance of this vWD was investigated by haplotype analysis of the vWF gene. Individuals affected by such an association are actually asymptomatic, but per- and post-operative bleeding risk remains to be evaluated.

Retinoids induced t-PA synthesis by C6 glioma cells--role in tumoral haemorrhagic necrosis.

Treatment of rat C6 glioma with high doses of 13 cis-retinoic acid (cRA) was responsible for death related to haemorrhagic necrosis localized to the tumor. Our aim was to explore this adverse effect of retinoid treatment. We show that cRA-treated C6 glioma at 25 mg/kg/day for 18 days exhibits in vivo an increase T-PA activity, which is responsible for a localized tumor fibrinolytic activity. Production of t-PA is supported by specific enhancement of gene expression, as was shown by the increase in t-PA mRNA (x 2.3). This production is a direct effect of cRA when treating the tumor, since tumor cells themselves do not produce enough t-PA and treatment of control rats does not increase the t-PA level. T-PA production by rat C6 glioma is in vivo related to the specific synthesis of t-PA by the C6 cell-line. The stimulation of C6 cell-line by cRA in vitro is dose-dependent and reached a maximum for 3 and 30 microM at the 72nd h. So cRA-treated C6 glioma cells produce t-PA which appears to be the major species associated with the fibrinolytic activity-induced intra-tumoral haemorrhage after exposure to retinoid treatment.

Measurement of low molecular weight heparin ex vivo activities in clinical laboratories using various anti-Xa assays: interlaboratory variability and requirement for an agreed low molecular weight heparin standard.

The only sensitive and convenient assay to assess the biological activity of low molecular weight heparins (LMWHs) is based on the potentiation of activated factor Xa inhibition. Several procedures for measuring the socalled anti Xa activity have been proposed. In this collaborative study including eight laboratories, we have used four different assays (three amidolytic and one clotting based methods) for measuring the anti Xa activity of ex vivo samples obtained after injecting three different LMWHs. The dispersion of the results obtained by calibration against standard heparin could be reduced by using any of the three LMWHs for calibration. A coefficient of variation less than 0.20 between values obtained in different laboratories using a variety of methods seems acceptable. However it is necessary to refer to a common international standard for expressing the results in units and to define, for each of the three products, the therapeutic range.

Effects of two sex steroids (17beta estradiol and testosterone) on proliferation and clonal growth of the human monoblastic leukemia cell line, U937.

We investigated the effects of two sex steroids (17beta estradiol and testosterone) on five human leukemia cell lines. We observed a statistically significant inhibition of proliferation, dose and time dependent, of the human monoblastic leukemia cell line U937. This inhibition was associated with a dose dependent decrease in the number of CFU-blasts in clonogenic cultures. Cytostatic effect was obtained with doses of 5 microM for estrogen and 10 microM for androgen and was not due to a non-specific cytotoxic effect, some cell viability remained high (> 90%) even after 6 days of incubation. More accurately, we demonstrated that growth inhibition was associated with a cell cycle arrest, U937 cells accumulating in G2/M phase. This blockade was dose related with a maximum number of cells accumulating at day 4. Sensitivity of these cells to an S-phase specific agent (hydroxyurea) was not increased, suggesting that these cells were blocked in G2/M and did not undergo mitosis. Expression in U937 cells of high affinity nuclear receptors for estrogen and androgen was negative which was in favour of a type II estrogen binding site, mediated mechanism. Moreover, a small fraction of these cells underwent apoptosis or differentiation with about 12% apoptotic cells and a significant increase (more than 30%) of two myelomonocytic markers (CD13 and CD64). These results demonstrate that the proliferation of some leukemic cells may be inhibited by micromolar concentrations of sex steroids, independently of nuclear receptor expression. The main mechanism seems to be a block in cell cycle associated with modulation of apoptosis and differentiation. It provided additional evidence for the potential value of sex steroids and their analogues in the treatment of leukemias.

Serologic evidence for bovine immunodeficiency virus infection in France.

We report herein on the first serologic detection of antibodies to bovine immunodeficiency virus (BIV) in France. Serum samples from dairy and beef cattle from southwestern and western France (Landes and Vendée) were tested using a western blot assay with a recombinant 53 kDa gag precursor derived from the Louisiana BIV R29 isolate. We performed our study on the oldest animals from 37 different herds that were under serologic follow up for previous bovine leukemia virus infection. Overall, 398 selected bovine sera were assayed and 15 serum samples from 8 herds reacted with the recombinant 53 kDa BIV R29 gag. Interestingly, reactions obtained with French sera were weaker than with positive Louisiana sera, a finding that may indicate the occurrence of distinct French and Louisiana BIV variants.

[Biological activity of enoxaprine (PK 10169) in hemodialysis. A dose study]. / Activités biologiques de l'éjnoxaparine (PK 10,169) en hémodialyse. Etude de dose.

Anti-Xa and anti-IIa activity were evaluated in 42 patients with chronic renal insufficiency, in an open randomized trial, to determine optimal dose of PK 10169 for prevention of coagulation in extracorporeal circuit during hemodialysis sessions. PK 10169 was given as single doses of 0.75, 1 and 1.25 mg/kg at start of dialysis, into the arterial line. All dialysis sessions were continued over the 4 hours provided for without the need for further injections. A linear relation existed between anti-Xa and anti-IIa activity measured at end of dialysis and the dose injected (p less than 0.001). Efficacy and tolerance were assessed clinically and biologically and were rated excellent at the 3 dose levels, the best tolerance/efficacy ratio being at the 1 mg/kg dosage.

[The use of enoxaparine for anticoagulation in extracorporeal circulation in hemodialysis at high risk for hemorrhage]. / Utilisation de l'énoxaparine pour l'anticoagulation du circuit extracorporel des hémodialyses à haut risque hémorragique.

After evaluation of efficacy of a low molecular weight heparin (LMWH), enoxaparine (Lovenox), in patients on continuous hemodialysis without a particular known hemorrhagic risk, this same LMWH was administered during 493 dialysis sessions to 46 patients presenting various degrees of risk of hemorrhage. Lower doses of 0.5 mg/kg or 0.75 mg/kg as bolus injections were administered at the start of the 4 or 5 hourly session. Clotting in the extracorporeal circulation (ECC) was noted in 0.6% treatments, the product being effective in all other sessions. Only one case of bleeding can be imputed to the LMWH injected during hemodialysis (0.2% of sessions). Although an open trial, the superiority of enoxaparine both for antithrombotic activity in ECC, and its simple management, as well as the small number of hemorrhages noted, has led to the routine use of this method in all patients at hemorrhagic risk.

[Thrombin: a multifunctional enzyme]. / La thrombine: enzyme multifonctionnelle.

Thrombin is the final enzyme of blood coagulation cascade. It belongs to the trypsin family of serine proteases. Its two primary actions are to cleave fibrinogen to release fibrin and to activate platelets through a limited proteolysis of a specific receptor. In addition, thrombin is the major regulator of blood coagulation. It is both a procoagulant enzyme in the activation of factors V and VIII, and an anticoagulant enzyme through the activation of protein C and TAFI. This multi-functionality of thrombin depends upon the conformation of its active site: depth for high specificity and shape for a finely tuned selection of substrates. Since new anticoagulant molecules, some with anti-thrombin activity, are emerging, it is important to understand the mechanisms allowing thrombin to be so specifically multifunctional.