Self-reactivity in the dimeric intravenous immunoglobulin fraction

Therapeutic intravenous immunoglobulin (IVIg) preparations contain antibodies reflecting the cumulative antigen experience of the donor population. IVIg contains variable amounts of monomeric and dimeric IgG, but there is little information available on their comparative antibody specificities. We have isolated highly purified fractions of monomeric and dimeric IgG by size-exclusion chromatography. Following treatment of all fractions at pH4, analyses by immunodot and immunocytology on human cell lines showed a preferential recognition of autoantigens in the dimeric IgG fraction. Investigation of the HEp-2 cytoplasmic proteome by 2D-PAGE, Western blot, and subsequent identification of IVIg reactive spots by mass spectrometry (LC-MS/MS) showed that IVIg recognized only a restricted set of the total proteins. Similar experiments showed that more antigens were recognized by the dimeric IgG fraction, especially when the dissociated dimer fraction was used, as compared to its monomeric counterpart. These observations are consistent with idiotype-anti-idiotype masking of auto-specific Abs in the dimeric fraction of IVIg.

Transcriptional silencing of nonsense codon-containing immunoglobulin micro genes requires translation of its mRNA

Eukaryotes have evolved quality control mechanisms that prevent the expression of genes in which the protein coding potential is crippled by the presence of a premature translation-termination codon (PTC). In addition to nonsense-mediated mRNA decay (NMD), a well documented posttranscriptional consequence of the presence of a PTC in an mRNA, we recently reported the transcriptional silencing of PTC-containing immunoglobulin (Ig) mu and gamma minigenes when they are stably integrated into the genome of HeLa cells. Here we demonstrate that this transcriptional silencing of PTC-containing Ig-mu constructs requires active translation of the cognate mRNA, as it is not observed under conditions where translation of the PTC-containing mRNA is inhibited through an iron-responsive element in the 5'-untranslated region. Furthermore, RNA interference-mediated depletion of the essential NMD factor Upf1 not only abolishes NMD but also reduces the extent of nonsense-mediated transcriptional gene silencing (NMTGS). Collectively, our data indicate that NMTGS and NMD are linked, relying on the same mechanism for PTC recognition, and that the NMTGS pathway branches from the NMD pathway at a step after Upf1 function.

Influence of inhibitory killer immunoglobulin-like receptors and their HLA-C ligands on resolving hepatitis C virus infection

An estimated 2%-3% of the world's population is chronically infected with hepatitis C virus (HCV) and this is a major cause of liver disease worldwide. Following acute infection, outcome is variable with acute HCV successfully resolved in some individuals (20%-30%), but in the majority of cases the virus is able to persist. Co-infection with human immunodeficiency virus has been associated with a negative impact on the course of HCV infection. The host's immune response is an important correlate of HCV infection outcome and disease progression. Natural killer (NK) cells provide a major component of the antiviral immune response by recognising and killing virally infected cells. NK cells modulate their activity through a combination of inhibitory and activatory receptors such as the killer immunoglobulin-like receptors (KIRs) that bind to human leukocyte antigen (HLA) Class I molecules. In this workshop component, we addressed the influence of KIR genotypes and their HLA ligands on resolving HCV infection and we discuss the implications of the results of the study of Lopez-Vazquez et al. on KIR and HCV disease progression.

Modeling of transfer kinetics at the serum-cerebrospinal fluid barrier in rabbits with experimental meningitis: application to grepafloxacin

The goals of the present study were to model the population kinetics of in vivo influx and efflux processes of grepafloxacin at the serum-cerebrospinal fluid (CSF) barrier and to propose a simulation-based approach to optimize the design of dose-finding trials in the meningitis rabbit model. Twenty-nine rabbits with pneumococcal meningitis receiving grepafloxacin at 15 mg/kg of body weight (intravenous administration at 0 h), 30 mg/kg (at 0 h), or 50 mg/kg twice (at 0 and 4 h) were studied. A three-compartment population pharmacokinetic model was fit to the data with the program NONMEM (Nonlinear Mixed Effects Modeling). Passive diffusion clearance (CL(diff)) and active efflux clearance (CL(active)) are transfer kinetic modeling parameters. Influx clearance is assumed to be equal to CL(diff), and efflux clearance is the sum of CL(diff), CL(active), and bulk flow clearance (CL(bulk)). The average influx clearance for the population was 0.0055 ml/min (interindividual variability, 17%). Passive diffusion clearance was greater in rabbits receiving grepafloxacin at 15 mg/kg than in those treated with higher doses (0.0088 versus 0.0034 ml/min). Assuming a CL(bulk) of 0.01 ml/min, CL(active) was estimated to be 0.017 ml/min (11%), and clearance by total efflux was estimated to be 0.032 ml/min. The population kinetic model allows not only to quantify in vivo efflux and influx mechanisms at the serum-CSF barrier but also to analyze the effects of different dose regimens on transfer kinetic parameters in the rabbit meningitis model. The modeling-based approach also provides a tool for the simulation and prediction of various outcomes in which researchers might be interested, which is of great potential in designing dose-finding trials.

Lactate and glucose concentrations in brain interstitial fluid, cerebrospinal fluid, and serum during experimental pneumococcal meningitis

Metabolic abnormalities during bacterial meningitis include hypoglycorrhachia and cerebrospinal fluid (CSF) lactate accumulation. The mechanisms by which these alterations occur within the central nervous system (CNS) are still incompletely delineated. To determine the evolution of these changes and establish the locus of abnormal metabolism during meningitis, glucose and lactate concentrations in brain interstitial fluid, CSF, and serum were measured simultaneously and sequentially during experimental pneumococcal meningitis in rabbits. Interstitial fluid samples were obtained from the frontal cortex and hippocampus by using in situ brain microdialysis, and serum and CSF were directly sampled. There was an increase of CSF lactate concentration, accompanied by increased local production of lactate in the brain, and a decrease of CSF-to-serum glucose ratio that was paralleled by a decrease in cortical glucose concentration. Brain microdialysate lactate concentration was not affected by either systemic lactic acidosis or artificially elevated CSF lactate concentration. These data support the hypothesis that the brain is a locus for anaerobic glycolysis during meningitis, resulting in increased lactate production and perhaps contributing to decreased tissue glucose concentration.

[Endocarditis prophylaxis with amoxicillin, clindamycin or erythromycin? Serum bacteriostatic and bactericidal effect against Streptococcus viridans]

The effectiveness of antibiotic prophylaxis against endocarditis was assessed by testing the bactericidal and bacteriostatic action in serum of 12 healthy volunteers who had taken the recommended antibiotics according to laid down guidelines. Blood was obtained from these subjects every two hours for 12 hours after oral intake of amoxicillin (3 g as a single dose), clindamycin (600 mg a single dose) or erythromycin (1.5 g plus another 0.5 g after six hours), the serum being tested against three Strep. viridans strains. Two of the three strains were "tolerant", i.e. in relation to the minimal inhibition concentration (MIC) their growth was inhibited, but--in relation to the minimal bactericidal concentration (MBC)--they were not killed. A bacteriostatic effect by amoxicillin and clindamycin was demonstrated by a micromethod against all three strains during the 12-hour period. But erythromycin did not achieve bacteriostasis in all serum samples. A bactericidal effect was demonstrated only in those samples that contained amoxicillin, and then only against the non-tolerant of the three strains. These results support the view that amoxicillin and clindamycin are effective in the prophylaxis against Strep. viridans bacteraemia because of their constant bacteriostatic effect, as measured in serum.

Serum lipoproteins during treatment with the antihypertensive agent indapamide

Considering the documented, potentially undesirable influence of various thiazide-type or loop diuretics on serum lipoproteins, we prospectively investigated in 69 men (mean age +/- SEM, 32 +/- 1 years) the metabolic effects of the new diuretic-antihypertensive compound indapamide. Compared to placebo, indapamide (2.5 mg/day) given for 6 to 8 weeks lowered (p less than 0.02 to less than 0.001) blood pressure (supine values from 148/98 +/- 3/2 to 137/93 +/- 3/2) in 29 men with mild to moderate essential hypertension, but not in 40 healthy men. In both groups, significant (p less than 0.05 to less than 0.001) decreases in body weight (-0.8 kg) and plasma potassium (-0.6 mmol/L), and increases in plasma uric acid (+20%), renin activity (+200%), and aldosterone documented good compliance. There were no significant changes in total cholesterol (in all subjects, from 208 +/- 6 to 213 +/- 6 mg/dl), low- or very low-density lipoprotein (VLDL) cholesterol (127 +/- 6 to 129 +/- 6 and 21 +/- 1 to 21 +/- 2 respectively), high-density lipoprotein cholesterol (50 +/- 1 to 51 +/- 1 mg/dl), total triglycerides (Tg) (108 +/- 5 to 112 +/- 6 mg/dl), VLDL-Tg, apoproteins A1 and A2, plasma glucose, epinephrine, norepinephrine, sodium, calcium, magnesium, and creatinine; apoprotein B (84 +/- 2 to 88 +/- 3 mg/dl) and plasma insulin after glucose loading dose tended to be increased minimally. The absence of distinct lipoprotein alterations after short-term indapamide treatment may be of clinical and epidemiological interest.

Prognosis in pediatric hematologic malignancies is associated with serum concentration of mannose-binding lectin-associated serine protease-2 (MASP-2)

BACKGROUND: Mannose-binding lectin (MBL) and MBL-associated serine protease-2 (MASP-2) are key components of the lectin pathway of complement activation. Their serum concentrations show a wide interindividual variability. This study investigated whether the concentration of MBL and MASP-2 is associated with prognosis in pediatric patients with cancer. METHODS: In this retrospective multicenter study, MBL and MASP-2 were measured by commercially available ELISA in frozen remnants of serum taken at diagnosis. Associations of overall survival (OS) and event-free survival (EFS) with MBL and MASP-2 were assessed by multivariate Cox regression accounting for prognostically relevant clinical variables. RESULTS: In the 372 patients studied, median serum concentration of MBL was 2,808 microg/L (range, 2-10,060) and 391 microg/L (46-2,771) for MASP-2. The estimated 4-year EFS was 0.60 (OS, 0.78). In the entire, heterogeneous sample, MBL and MASP-2 were not significantly associated with OS or EFS. In patients with hematologic malignancies, however, higher MASP-2 was associated with better EFS in a significant and clinically relevant way (hazard ratio per tenfold increase (HR), 0.22; 95% CI, 0.09-0.54; P = 0.001). This was due to patients with lymphoma (HR, 0.11; 95% CI, 0.03-0.47; P = 0.003), but less for those with acute leukemia (HR, 0.35; 95% CI, 0.11-1.15; P = 0.083). CONCLUSION: In this study, higher MASP-2 was associated with better EFS in pediatric patients with hematologic malignancies, especially lymphoma. Whether MASP-2 is an independent prognostic factor affecting risk stratification and anticancer therapy needs to be assessed in prospective, disease-specific studies.

Performance of Interleukin-6 and Interleukin-8 serum levels in pediatric oncology patients with neutropenia and fever for the assessment of low-risk

BACKGROUND: Patients with chemotherapy-related neutropenia and fever are usually hospitalized and treated on empirical intravenous broad-spectrum antibiotic regimens. Early diagnosis of sepsis in children with febrile neutropenia remains difficult due to non-specific clinical and laboratory signs of infection. We aimed to analyze whether IL-6 and IL-8 could define a group of patients at low risk of septicemia. METHODS: A prospective study was performed to assess the potential value of IL-6, IL-8 and C-reactive protein serum levels to predict severe bacterial infection or bacteremia in febrile neutropenic children with cancer during chemotherapy. Statistical test used: Friedman test, Wilcoxon-Test, Kruskal-Wallis H test, Mann-Whitney U-Test and Receiver Operating Characteristics. RESULTS: The analysis of cytokine levels measured at the onset of fever indicated that IL-6 and IL-8 are useful to define a possible group of patients with low risk of sepsis. In predicting bacteremia or severe bacterial infection, IL-6 was the best predictor with the optimum IL-6 cut-off level of 42 pg/ml showing a high sensitivity (90%) and specificity (85%). CONCLUSION: These findings may have clinical implications for risk-based antimicrobial treatment strategies.

Low serum alpha-tocopherol and selenium are associated with accelerated apoptosis in severe sepsis

During sepsis, a severe systemic disorder, micronutrients often are decreased. Apoptosis is regarded as an important mechanism in the development of often significant immunosuppression in the course of the disease. This study aimed to investigate alpha-tocopherol and selenium in reference to apoptosis in patients with sepsis. 16 patients were enrolled as soon as they fulfilled the criteria of severe sepsis. 10 intensive care patients without sepsis and 11 healthy volunteers served as controls. alpha-Tocopherol, selenium and nucleosomes were measured in serum. Phosphatidylserine externalization and Bcl-2 expression were analyzed in T-cells by flow cytometry. Serum alpha-tocopherol and selenium were decreased in severe sepsis but not in non-septic critically ill patients (p < 0.05). Conversely, markers of apoptosis were increased in sepsis but not in critically ill control patients: Nucleosomes were found to be elevated 3 fold in serum (p < 0.05) and phosphatidylserine was externalized on an expanded subpopulation of T-cells (p < 0.05) while Bcl-2 was expressed at lower levels (p < 0.05). The decrease of micronutrients correlated with markers of accelerated apoptosis. Accelerated apoptosis in sepsis is associated with low alpha-tocopherol and selenium. The results support the investigation of micronutrient supplementation strategies in severe sepsis.