Red cell microparticle enumeration: validation of a flow cytometric approach

Background and Objectives There is growing interest in the clinical application of red blood cell (RBC) microparticle (MP) enumeration as they have been postulated to be effectors of coagulation and inflammation following transfusion and in sickle cell disease. No uniform approach in MP enumeration exists and a key limitation is the lack of an internal validation process. We present and validate a flow cytometric approach where an internal standard is utilized. Materials and Methods Glycophorin A⁺ Annexin V⁺ events were enumerated using MPs isolated from RBC units or plasma samples obtained from volunteers. A mixture of absolute counting (7·6 μm) and calibration beads (0·5, 0·9 and 3 μm) at a fixed ratio was added to each sample. Results RBC MPs were initially selected based upon a fluorescence threshold, and the 0·5‐ and 0·9‐μm beads defined the upper and lower light scatter distribution of MPs. The ratio of 7·6:3‐μm bead events was used as an internal standard to validate the precision of MP enumeration across samples (coefficient of variation = 2·5–7·2%) and remained constant in both platelet‐rich plasma (PRP) and platelet‐free plasma (PFP). RBC MP counts increased in both PRP and PFP obtained from whole blood stimulated with ionophore and increasing calcium concentrations, with PRP showing higher MP counts than PFP at every concentration studied. Conclusion This method is a useful strategy to detect RBC MP counts across bio‐samples provided that the flow cytometer can reliably discriminate the size of the calibration beads.     

Factor VIII concentration is greater in female than male patients with HIV infection

The purpose of the present study was comparing plasma markers of coagulation between men and women in both HIV-infected patients and controls. Fifty-eight HIV-infected patients and 58 healthy participants who were individually matched with patients in age and sex were included in the study. We simultaneously collected blood samples for CD4+ T cell count, CD8+ T cell count, platelets count, hemoglobin, partial thromboplastin time (PTT), prothrombin time, international normalized ratio and blood type measurements. We used fresh plasma of the studied population to measure factor VIII, fibrinogen, antithrombin, protein C and protein S levels. CD4+ T cell count, CD8+ T cell count, platelet count, PTT, plasma fibrinogen, antithrombin, protein C and protein S levels were significantly lower, and plasma factor VIII levels were significantly higher in HIV patients. Factor VIII levels were significantly higher in HIV-infected women than HIV-infected men [200 (181–258) vs. 170 (150–194), p < 0.05]. This difference remained significant [219.7 (195.8–248.7) vs. 158 (136.5–180.3), p < 0.001] after multiple adjustments for age, CD8+ and CD4+ T cell count, using general linear model. Plasma factor VIII concentration was significantly higher in HIV-infected women after stratifying the patients into O, B, A and AB blood groups when there was not any gender difference in controls. We suggest that there is a sexual dimorphism in factor VIII concentration in HIV-infected patients.     

An intrinsic mechanism of secreted protein aging and turnover

The composition and functions of the secreted proteome are controlled by the life spans of different proteins. However, unlike intracellular protein fate, intrinsic factors determining secreted protein aging and turnover have not been identified and characterized. Almost all secreted proteins are posttranslationally modified with the covalent attachment of N-glycans. We have discovered an intrinsic mechanism of secreted protein aging and turnover linked to the stepwise elimination of saccharides attached to the termini of N-glycans. Endogenous glycosidases, including neuraminidase 1 (Neu1), neuraminidase 3 (Neu3), beta-galactosidase 1 (Glb1), and hexosaminidase B (HexB), possess hydrolytic activities that temporally remodel N-glycan structures, progressively exposing different saccharides with increased protein age. Subsequently, endocytic lectins with distinct binding specificities, including the Ashwell–Morell receptor, integrin αM, and macrophage mannose receptor, are engaged in N-glycan ligand recognition and the turnover of secreted proteins. Glycosidase inhibition and lectin deficiencies increased protein life spans and abundance, and the basal rate of N-glycan remodeling varied among distinct proteins, accounting for differences in their life spans. This intrinsic multifactorial mechanism of secreted protein aging and turnover contributes to health and the outcomes of disease.The secreted proteome is a diverse repertoire of proteins, each of which is maintained at concentrations appropriate to control various physiological processes. Although an intrinsic mechanism that determines the life span and turnover of secreted proteins has not been identified, its existence would explain how the composition of the secreted proteome is regulated and how it might differ in health and disease. Almost all secreted proteins are posttranslationally modified during their biosynthesis and transit through the secretory pathway with one or more N-glycans (1, 2). The N-glycans of mammalian proteins are multiantennary structures consisting of two or more oligosaccharide branches that typically include a characteristic terminal sequence of saccharides. This sequence includes sialic acid (Sia) as the distal linkage attached to underlying galactose (Gal), followed by N-acetylglucosamine (GlcNAc) and mannose (Man). Each is present in a multivalent context due to N-glycan branching emanating from the core structure. Distinct secreted proteins are therefore modified with similar N-glycans, each of which includes cryptic ligands of lectins that bind Gal, GlcNAc, or Man linkages.N-glycans have been primarily perceived as static posttranslational modifications that are eventually degraded along with the attached protein in the lysosome of the cell. Alterations of N-glycan structures thus far identified during a protein’s life span reflect the activity of exogenous glycosidases or targeted genetic mutations in N-glycan biosynthesis. For example, viral and bacterial neuraminidases cleave Sia linkages from the host glycans, thereby contributing to infection and modulating the severity of disease (3–6). Similarly, exogenous neuraminidase treatment or a genetic deficiency of the ST3Gal4 sialyltransferase was found to unmask Gal ligands of lectins known as asialoglycoprotein receptors in reducing the life spans of some secreted proteins (7, 8). It has been noted that blood plasma contains multiple unidentified glycosidases of unknown functions with activity levels that change in disease (9). We investigated whether the normal activities of circulating glycosidases may hydrolyze N-glycan linkages attached to secreted proteins, thereby generating multivalent ligands of endocytic lectin receptors in contributing to a mechanism of secreted protein aging and turnover.     

Procoagulant microparticles are increased in patients with Behçet’s disease but do not define a specific subset of clinical manifestations

Microparticles (MP) are considered a key component in the haemostatic response. Beyond their in vitro procoagulant properties, a number of pieces of evidence points to procoagulant MP as efficient effectors in the haemostatic response and as pathogenic markers of thrombotic disorders and vascular damage. The aim of the present study was to analyze the procoagulant activity of MP and its correlation with clinical manifestations focusing on vascular involvement in patients with Behçet’s disease (BD). We analyzed 55 BD patients in inactive phase of the disease (26 men; mean age, 35?±?15 years) of which 19 had previously suffered from thrombosis (deep venous thrombosis in 17 and ischemic stroke in 2), and 73 healthy controls matched for age and sex. Procoagulant MP were assessed by a functional assay. BD patients showed higher procoagulant MP values than controls (22.89?±?15.74 nM versus 14.47?±?7.34 nM; p?<?0.0001). Conversely, we did not find differences in the levels of procoagulant MP according to the gender of patients (22.22?±?16.23 nM for men versus 21.46?±?16.47 for women; p?=?0.846) or to previous and current treatments. Moreover, the plasmatic concentration of MP does not define any clinical phenotype and it was not related to the time of evolution of the disease. Although inactive BD patients had high values of procoagulant MP, they did not differentiate between BD patients with or without thrombosis.     

Lipoprotein subfraction composition in non-insulin-dependent diabetes treated by diet, sulphonylurea, and insulin

Lipoprotein abnormalities may predispose to an increased risk of coronary heart disease in type II (non-insulin-dependent) diabetes mellitus. To investigate the effects of different treatment modalities, the composition and concentrations of fasting plasma lipoproteins were determined in a cross-sectional study of patients with type II diabetes at diagnosis, treated by diet alone, treated by diet + glibenclamide (2.5 to 15 mg/d for 6 to 48 months), and treated by diet + insulin (25 to 65 U/d for 8 to 144 months). Compared with normal subjects matched for sex, age, body mass index, exercise, alcohol consumption and smoking, type II patients at diagnosis showed increased concentrations of nonesterified and esterified cholesterol, triglyceride, phospholipid, and protein in the very low density lipoprotein (VLDL) fraction. However, the only alteration in VLDL composition was a small decrease in the relative proportion of phospholipid. Apolipoprotein-B and low density lipoprotein (LDL) cholesterol concentrations were also raised in type II patients at diagnosis. Plasma concentrations of high density lipoprotein (HDL) nonesterified and esterified cholesterol, phospholipid, and apo-AI were lower in type II patients at diagnosis. This was largely accounted for by reduced concentrations of these components in the HDL2 subfraction, which retained a normal composition. Type II patients treated by diet alone and diet + glibenclamide exhibited similar abnormalities of plasma lipoprotein concentrations, which are associated with premature coronary disease, to the type II patients at diagnosis. However, in type II patients treated with insulin, plasma lipoprotein concentrations and composition were normal, except LDL cholesterol, which was lower than normal in insulin-treated patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasma fibronectin in sickle cell disease

Immunoturbidimetric assay technique was used to determine plasma fibronectin concentration in healthy Nigerian children (age 2-14 years), patients with sickle cell disease in steady state and patients with sickle cell disease in crises. Compared with controls, the plasma levels of fibronectin were greatly reduced in patients with sickle cell crises. Values within the normal reference range were seen in the group of patients with sickle cell disease in steady state. The data suggest that the significantly (p less than 0.001) reduced plasma fibronectin in patients with sickle cell crisis may be due to the consumption of this plasma protein in the process of erythrocyte endothelial adhesion.     

Binding of primitive hematopoietic progenitor cells to marrow stromal cells involves heparan sulfate.

Blast colony-forming cells (BI-CFC) and pre-colony-forming unit-granulocyte, monocyte (CFU-GM) in human bone marrow bind to marrow-derived stromal layers grown in the presence of methylprednisolone (MP+), but do not bind to stroma grown without MP (MP-). The BI-CFC bind to stroma and form colonies when overlaid with agar; the pre-CFU-GM bind to stroma and release CFU-GM into the supernatant culture medium (delta assay). These two classes of progenitor may represent similar stages of hematopoietic cell development. Their binding to stroma depends on the presence of heparan sulfate proteoglycan (HS-PG) in the extracellular matrix secreted by the stromal cells. Here, we have analyzed the functional and biochemical properties of HS-PG isolated from MP+ and MP- stromal cultures. HS-PG or isolated HS glycosaminoglycan (GAG) side chains partially blocked progenitor cell binding when they were added to the 2-hour binding phase of the BI-CFC or delta assays. Gel electrophoresis of HS-PG resolved more bands in matrix preparations from MP+ cultures than in preparations from MP- cultures. The blocking activity of the eluted MP+ HS-PG bands depended partly on the amount of GAG attached to the protein core and presumably partly on the structure of the core itself. Time course studies demonstrated that the HS-dependent phase of the binding interaction was limited to the first 30 to 60 minutes of the 2-hour binding phase. The different blocking effects of MP+ and MP- HS indicate that they have different biochemical properties. The HS-GAG in MP+ stroma has a higher degree of sulfation and a greater negative charge to mass ratio compared with MP- HS-GAG. Variations in HS may determine specific binding by hematopoietic progenitor cells and a heparan sulfate receptor is envisaged as acting in concert with further cell adhesion molecules (CAMs) on the progenitor cell surface.     

Coat protein regulates formation of replication complexes during tobacco mosaic virus infection

The genome of tobacco mosaic virus (TMV) encodes replicase protein(s), movement protein (MP), and capsid protein (CP). On infection, one or more viral proteins direct the assembly of virus replication complexes (VRCs), in association with host-derived membranes. The impact of CP-mediated resistance on the structures of the replication complexes was examined in nontransgenic and transgenic BY-2 cell lines that produce wild-type CP, mutant CP(T42W), and Ds-Red, which was targeted to endoplasmic reticulum by using immunofluorescence and 3D microscopy. We developed a model of VRCs that shows a clear association of MP with and surrounding the endoplasmic reticulum. Replicase is located within the MP bodies, as well as isolated sites throughout the cell. CP surrounds the VRCs. CP enhances the production of MP and increases the size of the VRC; however, the mutant CP(T42W) reduces the amount of MP and interferes with the formation of VRCs. We propose a regulatory role of the CP in the establishment of the VRC. We suggest that the lack of formation of VRCs restricts the efficiency of virus replication and the formation of virus movement complexes, resulting in restriction of cell-cell spread of infection. This results in higher levels of plant CP-mediated protection provided by CP(T42W).     

Low plasma levels of dehydroepiandrosterone sulphate in HIV-positive patients coinfected with hepatitis C virus

Objectives

To evaluate plasma levels of dehydroepiandrosterone sulphate (DHEAS) in a cohort of HIV‐infected patients and to analyse factors associated with DHEAS levels.

Methods

We conducted a cross‐sectional survey in the Nîmes University Hospital cohort of HIV‐infected patients in south‐eastern France. All HIV‐infected patients with at least one outpatient visit between 1 January and 1 September 2002 were included in the study. Sociodemographic, clinical, therapeutic, immuno‐virological and plasma DHEAS level data were collected during this period. Hepatitis C virus (HCV) coinfection was defined as the presence of HCV antibody with positive RNA. To identify factors associated with plasma DHEAS levels, Spearman's rank correlation and univariate and multivariate linear regression analyses were used.

Results

The DHEAS plasma level was measured in 137 patients (104 men and 33 women), 37 (27.0%) of whom were HCV coinfected. The median age of the patients was 39.1 years [interquartile range (IQR): 34.9–48.7] for women and 41.8 years (36.5–47.7) for men. The median DHEAS level was 5.5 μmol/L (IQR: 2.3–8.8) for the whole sample of 137 patients, and was lower in women (2.4 μmol/L; 1.5–6.6) than in men (6.1 μmol/L; 2.5–9.0) (P<0.01), and lower in patients coinfected with HCV (2.1 μmol/L; 0.6–6.7) than in those not coinfected (6.6 μmol/L; 3.0–9.1) (P<0.01). Of all prognostic factors studied in the variance covariance analysis, three factors were associated with DHEAS: age, gender and HCV coinfection. Subgroup analysis revealed that the age‐adjusted mean of the DHEAS level was lower in HCV coinfected patients for both women (1.3±1.1 μmol/L) and men (4.0±0.7 μmol/L), compared with patients not HCV coinfected (women, 5.3±0.7 μmol/L; men, 7.2±0.4 μmol/L) (P<0.01).

Conclusions

This is the first report of the determination of DHEAS plasma levels in HIV/HCV coinfected patients. When age and sex were taken into account, the DHEAS plasma level was found to be significantly lower in HCV coinfected patients. To date, the pathophysiology of such findings is unknown.

     

Bradycardia after high-dose intravenous methylprednisolone therapy

In 5 consecutive patients with rheumatoid arthritis who received intravenous high-dose methylprednisolone (MP) therapy (1 g daily for 2 or 3 consecutive days), a decline in pulse rate was observed, most pronounced on day 4. In one of the 5 patients the bradycardia was associated with complaints of substernal pressure. Reversal to normal heart rate was found on day 7. Electrocardiographic registrations showed sinus bradycardia in all cases. No significant changes in plasma concentrations of electrolytes were found. Careful observation of patients receiving high-dose MP is recommended. High-dose MP may be contraindicated in patients with known heart disease.     

White Blood Cell Count Predicts All-Cause Mortality in Patients with Suspected Peripheral Arterial Disease

Objective

We investigated whether markers of inflammation—white blood cell (WBC) count, C-reactive protein (CRP), and lipoprotein-associated phospholipase A2—are associated with mortality in patients referred for noninvasive lower-extremity arterial evaluation.

Methods

Participants (n = 242, mean age 68 years, 54% men) were followed for a median of 71 months. Ankle-brachial index (ABI), WBC count, plasma CRP, and lipoprotein-associated phospholipase A2 were measured at the start of the study. Factors associated with all-cause mortality were identified using Cox proportional hazards.

Results

During the follow-up period, 56 patients (25%) died. Factors associated with higher mortality were greater age, history of coronary artery disease/cerebrovascular disease, lower ABI, higher serum creatinine, and higher WBC count/plasma CRP. In stepwise multivariable regression analysis, ABI, serum creatinine, WBC count, and CRP were associated significantly with mortality. Patients in the top tertile of WBC count and CRP level had a relative risk of mortality of 3.37 (confidence interval [CI], 1.56-7.27) and 2.12 (CI, 0.97-4.62), respectively. However, only the WBC count contributed incrementally to prediction of mortality. Inferences were similar when analyses were limited to patients with peripheral arterial disease (ABI < 0.9, n = 114).

Conclusion

WBC count, but not plasma CRP level, provides incremental information about the risk of death in patients referred for lower-extremity arterial evaluation and in the subset of these patients with peripheral arterial disease.     

Arabidopsis synaptotagmin SYTA regulates endocytosis and virus movement protein cell-to-cell transport

Synaptotagmins are calcium sensors that regulate synaptic vesicle exo/endocytosis. Thought to be exclusive to animals, they have recently been characterized in plants. We show that Arabidopsis synaptotagmin SYTA regulates endosome recycling and movement protein (MP)-mediated trafficking of plant virus genomes through plasmodesmata. SYTA localizes to endosomes in plant cells and directly binds the distinct Cabbage leaf curl virus (CaLCuV) and Tobacco mosaic virus (TMV) cell-to-cell movement proteins. In a SYTA knockdown line, CaLCuV systemic infection is delayed, and cell-to-cell spread of TMV and CaLCuV movement proteins is inhibited. A dominant-negative SYTA mutant causes depletion of plasma membrane-derived endosomes, produces large intracellular vesicles attached to plasma membrane, and inhibits cell-to-cell trafficking of TMV and CaLCuV movement proteins, when tested in an Agrobacterium-based leaf expression assay. Our studies show that SYTA regulates endocytosis, and suggest that distinct virus movement proteins transport their cargos to plasmodesmata for cell-to-cell spread via an endocytic recycling pathway.Synaptotagmins (Syts) are a large family of Ca²⁺/lipid binding proteins widely studied in animals due to their role in neurotransmitter release. They are also found in Drosophila and Caenorhabditis elegans and were recently described in plants (1, 2). Syts have a conserved domain structure: a short uncleaved N-terminal signal peptide that overlaps a transmembrane (TM) domain, followed by a cytosolic variable region and two C-terminal C₂ domains, C₂A and C₂B. Whereas C₂A and C₂B each bind phospholipids in a Ca²⁺-dependent manner, fold independently and act synergistically, C₂B is essential for activity (1). SytI, the best studied Syt, is proposed to act as a Ca²⁺ sensor to regulate rapid and synchronous synaptic vesicle exocytosis (1). Whether it regulates SNARE complex formation in a temporal and spatial manner, or is itself fusogenic, is unclear. Studies in PC12 cells, and of mouse and Drosophila sytI mutants, suggest that the SNARE complex VAMP1/SNAP25/syntaxin-1 targets the synaptic vesicle to the plasma membrane to create a metastable fusion intermediate. SytI on the vesicle membrane, and perhaps a distinct partner Syt on the plasma membrane, would then interact with phospholipids and the SNARE complex to accelerate SNARE-mediated fusion pore dilation. Liposome studies suggest a direct fusogenic role for SytI, in which shallow insertion of the C₂ region into target membranes induces curvature to destabilize the lipid bilayer and form the fusion pore opening (1, 3). Studies in mice, Drosophila, and C. elegans show that SytI also regulates the kinetics of endocytosis at nerve terminals, apparently in a clathrin-mediated manner (1, 4).Plant virus movement proteins (MPs) mediate the transport of progeny genomes across the cell wall for local and systemic infection. Despite diverse strategies for cell-to-cell movement, two common features have emerged: movement proteins alter plasmodesmata (PD), transwall pores that connect adjacent plant cells; and protein localization and interaction studies implicate the endoplasmic reticulum (ER) and membrane trafficking in this process (5 –8). This is typified by the Begomoviruses Cabbage leaf curl virus (CaLCuV) and Squash leaf curl virus (SqLCV) and Tobamovirus Tobacco mosaic virus (TMV), with their respective single strand DNA (ssDNA) or RNA (ssRNA) genomes. CaLCuV and SqLCV encode two movement proteins: the nuclear shuttle protein NSP and the cell-to-cell movement protein MP. NSP binds replicated viral ssDNA in the nucleus and shuttles it to the cytoplasm, where MP traps these complexes to direct them to and across the cell wall via ER-derived transwall tubules. NSP then targets the viral genome to the nucleus for new cycles of replication (7). The ER-derived tubules are proposed to be the analog of the desmotubule, the PD axial membrane component that is first derived from cortical ER “trapped” by the wall during cell division (6). TMV genomes replicate at ER-derived membrane sites in the cytosol. TMV encodes a single 30-kDa movement protein (30K), which binds and targets progeny genomes to cortical ER sites and PD. The 30K protein increases PD size exclusion limits to allow viral ssRNA to move cell to cell (5, 7). Mutational and antisense suppression studies show that interaction of 30K with a cell wall pectin methylesterase (PME) is required for TMV cell-to-cell movement and infection. Hence, PME may direct 30K, complexed with TMV genomes, to PD and/or act to alter PD gating (9). These studies, and those of other movement proteins, link vesicular traffic to virus movement and lead to speculation that viral genomes and other macromolecules may target to and through PD by “grabbing” a receptor or exo/endocytosis (10).We report here the functional analysis of a plant synaptotagmin, Arabidopsis SYTA, which we identified in a yeast interactive screen using CaLCuV MP (MP_CaLCuV) as bait. SYTA directly binds to MP_CaLCuV in vitro, and to the related SqLCV MP (MP_SqLCV) and the distinct TMV 30K. We found that SYTA localizes to endosomes, using FM4-64 and compartment-specific markers. To establish the functions of SYTA in virus movement and in plant cells, we showed that CaLCuV infection is delayed, and TMV 30K and MP_CaLCuV cell-to-cell spread are inhibited, in an SYTA knockdown line; and a dominant-negative form of SYTA inhibited endocytosis and the recycling of an endosome marker at the plasma membrane, and the cell-to-cell trafficking of TMV 30K and MP_CaLCuV in an Agrobacterium tumefaciens-based leaf transient expression assay. We conclude that SYTA regulates both endosome recycling and the activities of the diverse MP_CaLCuV and TMV 30K in virus cell-to-cell movement, and suggest that distinct virus movement proteins transport their cargos to PD for cell-to-cell spread via an endocytic recycling pathway.     

Potential novel biomarkers of disease activity in rheumatoid arthritis patients: CXCL13, CCL23, transforming growth factor alpha, tumor necrosis factor receptor superfamily member 9, and macrophage colony-stimulating factor

OBJECTIVE: To determine whether the plasma levels of a range of inflammatory proteins have utility as biomarkers of disease activity in rheumatoid arthritis (RA) patients. METHODS: Plasma proteins (n = 163) were profiled in 44 patients with RA diagnosed according to the American College of Rheumatology 1987 criteria (22 with active and 22 with quiescent disease) and in 16 age- and sex-matched healthy controls. The utility of a subset of differentially expressed proteins as predictors of RA disease activity was investigated using partial least-squares discriminant analysis, and their response to therapeutic intervention was evaluated in plasma from an additional cohort of 16 patients with active RA treated with anti-tumor necrosis factor alpha (anti-TNFalpha). RESULTS: The protein profiling study identified 25 proteins that were differentially expressed in plasma samples from patients with active RA (P for the false discovery rate < or = 0.01) compared with those with quiescent RA, including the previously described interleukin-6 (IL-6), oncostatin M, and IL-2, and the 5 less-established markers macrophage colony-stimulating factor (M-CSF), tumor necrosis factor receptor superfamily member 9, CCL23, transforming growth factor alpha, and CXCL13. Systemic levels of these 5 markers correlated with the C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor level, tender joint count in 68 joints, and Disease Activity Score in 28 joints (DAS28), and their combined plasma levels were shown to be good predictors of disease activity (kappa = 0.64). In anti-TNFalpha-treated RA patients, plasma levels of CXCL13 were reduced after 1 and 7 days of therapy, and levels of CCL23, M-CSF, and CXCL13 showed a statistically significant positive correlation with the DAS28 score. CONCLUSION: This exploratory study for biomarker discovery led to the identification of several proteins predictive of RA disease activity that may be useful in the definition of disease subphenotypes and in the measurement of response to therapy in clinical studies.     

Clinical severity of <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis>(MP) infection is associated with bacterial load in oropharyngeal secretions but not with MP genotype

Background  

Disease severity in Mycoplasma pneumoniae (MP) infection could potentially be related to bacterial factors such as MP genotype (MP1 or MP2; distinguished by different adhesions proteins) or bacterial load in airway secretions. We have compared these parameters in patients who were hospitalized for MP pneumonia, with outpatients with mild MP disease.     

Alternative Pathway of Complement in Children with Diarrhea-Associated Hemolytic Uremic Syndrome

Background and objectives: Diarrhea-associated hemolytic uremic syndrome (D+HUS) is a common cause of acute kidney injury in children. Mutations in alternative pathway (AP) complement regulatory proteins have been identified in severe cases of thrombotic microangiopathy, but the role of the AP in D+HUS has not been studied. Therefore, we determined whether plasma levels of markers of activation of the AP are increased in D+HUS and are biomarkers of the severity of renal injury that predict the need for dialysis.Design, setting, participants, & measurements: Patients were randomly selected from among participants in the HUS-SYNSORB Pk trial. Plasma samples were collected on days 1, 4, 7, and 10 after enrollment and day 28 after discharge from the hospital. Levels of two complement pathway products, Bb and SC5b-9, were determined by ELISA.Results: Seventeen children (6 boys and 11 girls; age, 5.4 ± 3.5 yr) were studied. Eight (47%) required dialysis support, and two had serious extrarenal events. On the day of enrollment, plasma levels of Bb and SC5b-9 were significantly increased in all patients compared with healthy controls (P < 0.01). The elevated concentrations normalized by day 28 after discharge. Circulating levels of complement pathway fragments did not correlate with severity of renal injury or occurrence of complications.Conclusions: Patients with acute-onset D+HUS manifest activation of the AP of complement that is temporally related to the onset of disease and that resolves within 1 mo. Therapies to inhibit the AP of complement may be useful in attenuating the severity of renal injury and extrarenal complications.Diarrhea-associated hemolytic uremic syndrome (D+HUS) is one of most common causes of acute kidney injury in previously healthy children (1). It is caused by antecedent infection with Shiga toxin-producing strains of Escherichia coli (STEC). These organisms elaborate Shiga toxins (Stx) 1 and/or 2 that bind to the globotriaosylceramide (Gb3) receptor on the surface of endothelial cells, especially in the glomerular microcirculation. After internalization of the toxin, there is retrograde transport to the ribosome, inhibition of protein synthesis, endothelial cell death, and organ hypoperfusion and dysfunction (1,2). In addition, there is activation of numerous inflammatory cytokines and chemokines that have the potential to cause vascular injury and mediate tissue damage (3).D+HUS is one manifestation of thrombotic microangiopathy (TMA), a histopathologic phenotype characterized by endothelial cell swelling and detachment from the basement membrane and deposition of fibrin-platelet thrombi in the vascular lumen (4). In addition to D+HUS, TMA can occur sporadically in response to various medications, infectious agents, pregnancy, malignancies, rheumatological disorders, and in patients with thrombotic thrombocytopenic purpura. Finally, there is a rare group of patients who develop TMA as a consequence of genetic abnormalities in complement activation and regulatory proteins that lead to uncontrolled activation of the alternative pathway (AP). Recent reviews of TMA have proposed that the disease occurs due to disturbances in one of two distinct pathways—either dysregulation of complement activation or a relative loss of function of ADAMTS13, a protease that modulates the interaction between von Willebrand factor and endothelial cells. Although endothelial damage is the primary step in D+HUS, it has not been definitively attributed to abnormalities in the function of the complement pathway or ADAMTS13.There are anecdotal reports of low serum C3 levels and C3 deposition in the kidney of children with D+HUS (5). However, there has been no consistent evidence of activation of the AP of complement in children with D+HUS. We hypothesized that the AP is activated by Shiga toxin-induced endothelial damage in D+HUS. To test this hypothesis, we performed the following study using stored plasma samples from patients who were enrolled in the HUS-SYNSORB Pk multicenter clinical trial to determine whether there was evidence of activation of the AP of complement in this disease and to assess whether it correlated with disease activity or outcome.     

Lack of Correlation Between Activation of Hemostatic Mechanism and Inflammation in Unstable Angina Pectoris

In the acute phase of unstable angina, activation of the hemostatic mechanism is demonstrated by an increase in the plasma levels of markers of thrombin generation (prothrombin fragment 1+2) and thrombin activity (fibrinopeptide A). Increased concentrations of plasma C-reactive protein, an acute-phase reactant, have also been reported in patients with unstable angina. However, whether there is a correlation between the activation of the hemostatic mechanism and the acute-phase reaction of inflammation remains unclear. We measured the plasma levels of prothrombin fragment 1+2, fibrinopeptide A, and C-reactive protein in 91 patients consecutively hospitalized with recent-onset rest angina (Class IIIB Braunwald's classification), finding that they were above the normal limits in 48 (53%), 45 (49%), and 30 (33%) patients, respectively. There was no correlation between prothrombin fragment 1+2 and fibrinopeptide A (P = 0.34), prothrombin fragment 1+2 and C-reactive protein (P = 0.10), or fibrinopeptide A and C-reactive protein (P = 0.75). Plasma levels of prothrombin fragment 1+2 and fibrinopeptide A were both above normal levels in 32% of patients; 19% had both prothrombin fragment 1+2 and C-reactive protein, and 18% both fibrinopeptide A and C-reactive protein levels above the upper normal limits. All three markers were abnormally high in 11% of patients. According to the kappa cofficient test, the agreement between the elevation of the plasma concentrations of the markers was "random." In approximately half of the patients with acute unstable angina, there was an increase in the markers of the activation of the hemostatic mechanism and, in a smaller proportion, an increase in plasma C-reactive protein levels. The activation of the coagulation cascade and the acute-phase reaction of inflammation were infrequently associated in individual patients.     

Protein balance during very-low-calorie diets for the treatment of severe obesity

Very-low-calorie diets (less than 500 kcal/day; VLCD) are widely used for the treatment of severe obesity. We report the effects of such diets, consisting of proteins only or proteins and carbohydrates (CH), on nitrogen balance and protein nutritional status of morbidly obese patients. Cumulative nitrogen loss, serum albumin, transferrin, prealbumin (PA) and retinol-binding protein (RBP) concentrations, and plasma amino acid profile were determined in two groups of obese patients: 5 subjects (3 women, 2 men: BMI 55.3 +/- 2.2 kg/m2) subjected for 4 weeks to a protein VLCD (40 g protein + 2 g fat) and 7 others (4 women, 3 men: BMI 45.6 +/- 2.8 kg/m2) received for the same length of time a protein + CH VLCD (34 g protein + 26 g CH). Nitrogen balance was determined weekly whilst plasma and serum variables were measured on days 0, 3, 5, 10, 20 and 28 of treatment. Nitrogen balance did not significantly differ between the two groups of patients throughout the treatment. Serum PA and RBP concentrations decreased from day 5 and day 10, respectively, in both groups. Plasma amino acids showed a similar pattern in the protein and protein + CH groups. Alanine gradually decreased below baseline values; after a peak value on day 5, branched-chain amino acids (valine, leucine, isoleucine) returned to baseline values in both groups. In conclusion, in severely obese patients subjected to VLCD, nitrogen balance, labile protein concentrations and plasma amino acid profile are not significantly affected by adding CH to proteins.     

Alteration of haemostasis in non-metastatic gastric cancer

BACKGROUND: Cancer is one of the most common acquired causes of venous thromboembolism. AIM: To evaluate haemostasis disorders in patients with non-metastatic gastric cancer. PATIENTS AND METHODS: We studied 11 patients with non-metastatic gastric cancer (9 males and 2 females, median age 54 years) and 20 healthy subjects (15 males and 5 females, median age 48 years) control. We measured prothrombin time, activated partial thromboplastin time, coagulation time, clot lysis time, fibrinogen, clotting factors (II, VII, VIII, IX, X), C protein, S protein, AT III, activated protein C resistance, prothrombin 1+2 fragment, tissue plasminogen activator and D-Dimer in all subjects. RESULTS: Fibrinogen plasma levels were significantly higher in patients with non-metastatic gastric cancer than in control group (505+/-24 mg/dl vs 336+/-30 mg/dl, p<0.001). We also found a significant increase in prothrombin 1+2 fragment plasma concentration compared with controls (3.8+/-0.6 nM vs 0.83+/-0.09 nM, p<0.001). Plasma D-dimer levels were 20-fold higher in patients with non-metastatic gastric cancer compared with controls (9.57+/-0.4 ng/dl vs 0.4+/-0.05 ng/dl, p<0.001). Also tissue plasminogen activator was significantly higher in gastric cancer patients than in controls (20.8+/-2.32 ng/ml vs 9.1+/-1.37 ng/ml, p<0.01). Finally clot lysis time was significantly accelerated in gastric cancer patients compared with control subjects (81+/-37 min vs 233+/-74 min, p<0.01). CONCLUSIONS: Patients with non-metastatic gastric cancer are at risk for thrombotic events due to the combined increase in fibrinogen plasma levels and thrombin formation.     

Identification of CML-modified proteins in hemofiltrate of diabetic patients by proteome analysis.

The posttranslational modification of extra- and intracellular proteins by non-enzymatic glycation results in the formation of advanced glycation end products (AGEs) in physiological systems and is associated with the loss of protein structure and function. Modification by N (epsilon)-carboxymethyl lysine (CML) correlates with the risk for retinopathy in diabetes mellitus and has been discussed as a marker for the prediction of mortality in hemodialysis patients. AGEing of proteins is particularly increased under hyperglycemia associated with different late complications of diabetes mellitus. Modification of proteins to form AGE residues is significantly more enhanced in patients suffering from chronic renal disease than in hyperglycemia and is associated with increased risk for cardiovascular complications and inflammation in patients with chronic renal insuffiency. In order to identify and define the protein "substrates" for non-enzymatic glycation we used a proteome approach combining two-dimensional gel electrophoresis and immunoblotting with Edman protein sequencing to identify specific CML-modified proteins in human hemofiltrate, which essentially resembles plasma with respect to protein composition. Albumin, Ig kappa chain, prostaglandin D2 synthase, lysozyme C, plasma retinol binding protein and beta-2-microglobulin were identified as the major CML-modified proteins. CML-modified fragments of these proteins were also found in hemofiltrate. All identified proteins have in common that they appeared in hemofiltrate predominantly in their CML-modified form(s). Further studies of the functional roles of proteins identified by this new experimental approach could lead to the development of diagnostic tools to follow the progression of diabetes and contribute to the understanding of the pathogenesis of AGE-related diseases.