High interindividual variability in the CD4/CD8 T cell ratio and natalizumab concentration levels in the cerebrospinal fluid of patients with multiple sclerosis.

Strongly decreased leucocyte counts and a reduced CD4/CD8 T cell ratio in the cerebrospinal fluid (CSF) of natalizumab (NZB)-treated multiple sclerosis (MS) patients may have implications on central nervous (CNS) immune surveillance. With regard to NZB-associated progressive multi-focal leucoencephalopathy, we aimed at delineating a relationship between free NZB, cell-bound NZB, adhesion molecule (AM) expression and the treatment-associated shift in the CSF T cell ratio. Peripheral blood (PB) and CSF T cells from 15 NZB-treated MS patients, and CSF T cells from 10 patients with non-inflammatory neurological diseases and five newly diagnosed MS patients were studied. Intercellular adhesion molecule-1 (ICAM-1), leucocyte function antigen-1 (LFA-1), very late activation antigen-4 (VLA-4), NZB saturation levels, and T cell ratios were analysed by flow cytometry. NZB concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Lower NZB saturation levels (P<0.02) and a higher surface expression of ICAM-1 and LFA-1 (P<0.001) were observed on CSF CD8 T cells. CSF T cell ratios (0.3-2.1) and NZB concentrations (0.01-0.42 µg/ml) showed a pronounced interindividual variance. A correlation between free NZB, cell-bound NZB or AM expression levels and the CSF T cell ratio was not found. Extremely low NZB concentrations and a normalized CSF T cell ratio were observed in one case. The differential NZB saturation and AM expression of CSF CD8 T cells may contribute to their relative enrichment in the CSF. The reduced CSF T cell ratio appeared sensitive to steady-state NZB levels, as normalization occurred quickly. The latter may be important concerning a fast reconstitution of CNS immune surveillance.

Application of serum natalizumab levels during plasma exchange in MS patients with progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a severe complication of natalizumab treatment. Restoring immune function by plasmapheresis/immunoadsorption (PLEX/IA) is important for the outcome of PML. We report on four multiple sclerosis (MS) patients whom developed PML during natalizumab treatment, in whom we measured serum natalizumab concentrations before and during PLEX. Depending on the serum natalizumab concentration at the time of PML diagnosis, the number of PLEX treatments necessary to reach subtherapeutic serum natalizumab concentrations is variable. Measuring serum natalizumab concentrations before and during PLEX is helpful to determine the optimum number of PLEX treatments in individual MS patients with PML.

Lower etanercept levels are associated with high disease activity in ankylosing spondylitis patients at 24 weeks of follow-up.

BACKGROUND: Previous data have shown that etanercept levels are associated with clinical response in rheumatoid arthritis. However, for ankylosing spondylitis (AS), data regarding this topic are inconclusive. OBJECTIVES: To investigate the relationship between etanercept levels and clinical response in patients with AS. METHODS: Observational prospective cohort study of 162 patients with AS =treated with etanercept, monitored during 24 weeks of treatment. Etanercept trough levels were determined, retrospectively, using an ELISA. Disease activity was measured using AS Disease Activity Score (ASDAS), including C-reactive protein (CRP) and Bath AS Disease Activity index (BASDAI). Active disease was defined as ASDAS≥2.1. Since etanercept is a drug administered at home there might have been some variation in trough level sampling. RESULTS: At 24 weeks etanercept levels were significantly higher in patients with ASDAS<2.1, (3.8 mg/L; IQR 2.5-5.2) compared with patients with ASDAS≥2.1 (2.3 mg/L; IQR 1.2-3.4; p≤0.001). Generalised estimating equation analysis demonstrated a statistically significant association between etanercept levels and ASDAS, BASDAI, CRP and erythrocyte sedimentation rate (all p<0.001). When patients were categorised into quartiles according to etanercept levels, the lowest quartile (etanercept<1.80 mg/L) comprised 35% of all patients with ASDAS≥2.1 while the highest quartile comprised only 14%. CONCLUSIONS: Disease activity and inflammation are associated with etanercept levels in patients with AS at 24 weeks of treatment. Measuring etanercept levels might help in identifying overtreatment and undertreatment and optimise etanercept therapy in AS.

Drug levels, anti-drug antibodies, and clinical efficacy of the anti-TNFα biologics in rheumatic diseases.

The objectives of this study are to evaluate the effect of anti-drug antibodies on the clinical efficacy and withdrawal rate of the anti-TNFα biologics in patients with rheumatic diseases. Consecutive patients with rheumatic diseases recently commenced on anti-TNFα biologics were recruited. Serum samples were collected for assay of drug level and antibody titer against the corresponding biologics. Comparison of the clinical efficacy and drug retention rate was performed between patients with and without anti-drug antibodies. Fifty-eight Chinese patients were studied (64 % women; age 47.8 ± 12.9 years; disease duration 6.7 ± 6.4 years). The proportion of patients using infliximab (IFX), adalimumab (ADA), and etanercept (ETN) was 41, 28, and 31 %, respectively. Antibodies against IFX, ADA, and ETN were demonstrated in 12(50 %), 5(31 %) and 0(0 %) patients, respectively. Patients who developed anti-drug antibodies had significantly lower levels of the corresponding drugs (IFX level: 0.004 ± 0.01 vs 3.81 ± 3.49 µg/ml; p = 0.002; ADA level: 0.0 vs 7.6 ± 8.3 µg/ml; p = 0.008). Anti-drug antibody-positive patients had a significantly higher cumulative drug withdrawal rate due to inefficacy (64.7 and 71.8 % vs 10.3 and 10.3 % at month 12 and month 24, respectively; p < 0.001). In rheumatoid arthritis and psoriatic arthritis, non-responders was significantly more frequent in antibody-positive patients (54 vs 13 %; p = 0.01). In spondyloarthritis, the improvement in ankylosing spondylitis disease activity score was significant in patients without antibodies (3.89 ± 0.82 to 2.22 ± 0.86; p = 0.01) but not in those with anti-drug antibodies (3.40 ± 1.67 to 3.23 ± 1.40; p = 0.73). We concluded that the presence of neutralizing antibodies is associated with lower serum levels of the anti-TNFα biologics, leading to lower efficacy and higher withdrawal rate.

Detection of infliximab levels and anti-infliximab antibodies: a comparison of three different assays.

BACKGROUND: Formation of antibodies to infliximab (ATI) inversely correlates with functional drug levels and clinical outcome. Comparison of drug levels and anti-drug antibody monitoring is hampered by lack of standardisation. AIM: To determine the correlation between three different assays for measuring infliximab and ATI. METHODS: Serum samples and spiked controls (total 62) were evaluated in a blinded way in infliximab and ATI assays developed by Sanquin Amsterdam, Netherlands (A), Laboratory for Pharmaceutical Biology, KU Leuven, Belgium (B) and a commercially available kit from Biomedical Diagnostics (BMD), Paris, France (C) performed by the University Medical Center Groningen (UMCG), Netherlands. RESULTS: All infliximab assays showed a linear quantitative correlation (Pearson r = 0.91 for A vs. B, 0.83 for A vs. C and 0.73 for B vs. C). Assay C detected infliximab in 11 samples (18%) not detected by A and B, including samples containing only ATI. All ATI assays showed a good linear correlation (Pearson r = 0.95 for A vs. B, 0.99 for A vs. C and 0.97 for B vs. C). Assay A detected ATI in five samples with low ATI that were not detected by assays B and C. Assay B did not detect ATI in three patient samples with low ATI according to assays A and C. CONCLUSIONS: There is a good correlation of infliximab and antibodies to infliximab measurements between these assays. Nevertheless, the Biomedical Diagnostics kit detected false positive infliximab levels in 18% of the samples.

A comprehensive study on the role of the Yersinia pestis virulence markers in an animal model of pneumonic plague.

We determined the role of Yersinia pestis virulence markers in an animal model of pneumonic plague. Eleven strains of Y. pestis were characterized using PCR assays to detect the presence of known virulence genes both encoded by the three plasmids as well as chromosomal markers. The virulence of all Y. pestis strains was compared in a mouse model for pneumonic plague. The presence of all known virulence genes correlated completely with virulence in the Balb/c mouse model. Strains which lacked HmsF initially exhibited visible signs of disease whereas all other strains (except wild-type strains) did not exhibit any disease signs. Forty-eight hours post-infection, mice which had received HmsF(-) strains regained body mass and were able to control infection; those infected with strains possessing a full complement of virulence genes suffered from fatal disease. The bacterial loads observed in the lung and other tissues reflected the observed clinical signs as did the cytokine changes measured in these animals. We can conclude that all known virulence genes are required for the establishment of pneumonic plague in mammalian animal models, the role of HmsF being of particular importance in disease progression.

Immunologic activity of schistosomal and bacterial TLR2 ligands in Gabonese children.

Schistosomes carry lipid moieties that interact with the immune system. To understand the consequence of interactions in terms of polarizing the cytokine profiles, the effect of two Toll-like receptor-2 (TLR2) activating schistosomal lipid fractions was studied on whole blood from Gabonese children living in a schistosomiasis endemic area. One fraction contained lysophosphatidylserine [monoacylglycerophosphoserine (lysoGPSer)] plus diacylphosphatidylserine [diacylglycerophosphoserine (GPSer)] while the other contained lysoGPSer and only a trace of GPSer. The effect of these schistosomal lipid fractions was compared with the known bacterial TLR2 ligands PAM3CSK4 and MALP-2. PAM3CSK4 and MALP-2 had preferential IL-10-activating capacities, while the fraction containing lysoGPSer plus GPSer had a strong TNF-alpha-inducing capacity. The fraction containing lysoGPSer was neutral with respect to pro- vs. anti-inflammatory effects. When Th1 and Th2 cytokines were analysed, the schistosomal lipid fraction containing lysoGPSer plus GPSer showed a stronger Th2 response compared to PAM3CSK4, MALP-2 and lysoGPSer alone. Therefore, the study indicates that not only TLR2 ligands derived from bacteria or from parasites can generate distinct cytokine profiles but also that the composition of lipid entities reaching the immune system can be important in leading to different immune outcomes. This information may be important for exploitation of immune modulatory molecules.

Antibody responses to Ascaris-derived proteins and glycolipids: the role of phosphorylcholine.

In addition to proteins, glycolipids can be targets of antibody responses and contribute to host-pathogen interaction. Following the structural analysis of Ascaris lumbricoides-derived glycolipids, the antibody responses of a group of children with no, light and heavy infections were analysed. The role of the phosphorylcholine moiety, present on Ascaris glycoproteins and glycolipids, in antibody reactivity of these infected individuals was determined. Children carrying heavy infections showed highest IgG reactivity to glycolipids compared to lightly or non-infected children. Substantial IgG antibody reactivity to both (glyco)proteins and glycolipids was found to be directed to the phosphorylcholine moiety as determined by either removal of this group or a competition assay. This was most pronounced for glycolipids, where removal of the phosphorylcholine moieties by hydrofluoric acid treatment abrogated IgG antibody reactivity. Measurement of IgG4 and IgE isotypes showed no IgG4 reactivity to Ascaris glycolipids, but raised IgE responses were detected in subjects with light or no Ascaris infections, suggesting that IgE responses to glycolipids may play a role in controlling parasite burden. Differences found in antibody profiles to glycolipids and (glyco)proteins, indicate that these different classes of compounds may have distinct roles in shaping of and interacting with humoral immune responses.

Recognition of schistosome glycolipids by immunoglobulin E: possible role in immunity.

To investigate the role of antibody responses to (glyco)lipids in immunity to schistosome infection, lipids extracted from Schistosoma mansoni eggs and adult worms were fractionated, and the antibody isotype profile reactive to the fractionated lipids in a well-characterized S. haematobium-infected population was investigated. In tests of 10 plasma samples it was found that immunoglobulin G (IgG) reactivity was highest to the fraction containing ceramidepolyhexosides, whereas IgE reactivity was most prominent to both cerebroside- and ceramidepolyhexoside-containing fractions. The fraction containing ceramidepolyhexosides was then tested for reactivity with IgG subclasses and IgE in plasma samples from 66 S. haematobium-infected patients. Considering IgG4 and IgE, isotypes of particular interest in helminth infections, we found that both isotypes recognized egg (glyco)proteins in more than 90% of the infected subjects. However, in the case of glycolipids, IgE reactivity was much more prominent than IgG4 reactivity (found in 80 and 41% of the subjects, respectively). Furthermore, worm glycolipid-specific IgE prior to treatment of the subjects with praziquantel was negatively correlated with egg counts at 2 years posttreatment, indicating that IgE directed towards glycolipids could play an important role in resistance to reinfection.