Functional energetics of CD4+-cellular immunity in monoclonal antibody-associated progressive multifocal leukoencephalopathy in autoimmune disorders

Background

Progressive multifocal leukoencephalopathy (PML) is an opportunistic central nervous system- (CNS-) infection that typically occurs in a subset of immunocompromised individuals. An increasing incidence of PML has recently been reported in patients receiving monoclonal antibody (mAb) therapy for the treatment of autoimmune diseases, particularly those treated with natalizumab, efalizumab and rituximab. Intracellular CD4⁺-ATP-concentration (iATP) functionally reflects cellular immunocompetence and inversely correlates with risk of infections during immunosuppressive therapy. We investigated whether iATP may assist in individualized risk stratification for opportunistic infections during mAb-treatment.

Methodology/Principal Findings

iATP in PHA-stimulated, immunoselected CD4⁺-cells was analyzed using an FDA-approved assay. iATP of mAb-associated PML (natalizumab (n = 8), rituximab (n = 2), efalizumab (n = 1)), or other cases of opportunistic CNS-infections (HIV-associated PML (n = 2), spontaneous PML, PML in a psoriasis patient under fumaric acids, natalizumab-associated herpes simplex encephalitis (n = 1 each)) was reduced by 59% (194.5±29 ng/ml, mean±SEM) in comparison to healthy controls (HC, 479.9±19.8 ng/ml, p<0.0001). iATP in 14 of these 16 patients was at or below 3^rd percentile of healthy controls, similar to HIV-patients (n = 18). In contrast, CD4⁺-cell numbers were reduced in only 7 of 15 patients, for whom cell counts were available. iATP correlated with mitochondrial transmembrane potential (ΔΨ_m) (iATP/ΔΨ_m−correlation:tau = 0.49, p = 0.03). Whereas mean iATP of cross-sectionally analysed natalizumab-treated patients was unaltered (448.7±12 ng/ml, n = 150), iATP was moderately decreased (316.2±26.1 ng/ml, p = 0.04) in patients (n = 7) who had been treated already during the pivotal phase III trials and had received natalizumab for more than 6 years. 2/92 (2%) patients with less than 24 months natalizumab treatment revealed very low iATP at or below the 3^rd percentile of HC, whereas 10/58 (17%) of the patients treated for more than 24 months had such low iATP-concentrations.

Conclusion

Our results suggest that bioenergetic parameters such as iATP may assist in risk stratification under mAb-immunotherapy of autoimmune disorders.     

The shock of the new: progress in schizophrenia genomics

A growing list of common and rare genetic risk variants are being implicated in schizophrenia susceptibility. As with other complex genetic disorders most of the variance in genetic risk is still to be attributed. What can be learned from progress to date? The available data challenges how we conceptualize schizophrenia and suggests strong aetiological links with other psychiatric and developmental disorders. With the identification of rare copy number risk variants implicating specific genes (e.g. VIPR2 and NRXN1) it is increasingly possible to investigate molecular aetiology in patient subgroups to establish whether schizophrenia represents one or many different disease processes. This review summarizes recent research progress and suggests how the tools of modern genomics and neuroscience can be applied to best understand this devastating disorder.     

Solution-based targeted genomic enrichment for precious DNA samples

ABSTRACT: BACKGROUND: Solution-based targeted genomic enrichment (TGE) protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1) modifying or eliminating time consuming steps; 2) increasing yield to reduce input DNA and excessive PCR cycling; and 3) enhancing reproducible. RESULTS: We developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company. CONCLUSIONS: This solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.     

Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes

The role of the dicarboxylic acid transport (dct) system in the Rhizobium meliloti-Alfalfa symbiosis was investigated. Mutants of R. meliloti CM2 unable to grow on medium containing succinate as the sole carbon source were isolated following chemical and transposon mutagenesis. These mutants were also unable to utilize malate or fumarate as the sole source of carbon. Transport studies with ¹⁴C-labelled succinate showed that the mutants were specifically defective in succinate transport. Revertants of both chemical and transposon mutants were obtained at a frequency of 10^-5–10^-6. The R. meliloti dct mutants were able to nodulate Alfalfa plants but the nodules formed were unable to fix nitrogen. Revertants of the mutants were fully effective on plants. The mutants unable to transport succinate were used to isolate dct genes from a R. meliloti gene bank. Two plasmids containing a common 26.5 Mdal insert were found to complement some of the mutants. The presence of this DNA insert in the complementing mutant strains restored their effectivenss of plants. This DNA fragment encoding succinate transport function(s) was used to produce genetically engineered R. meliloti strains with an increased rate of succinate uptake.Abbreviation dct dicarboxylic acid transport     

Effects of an active warm-up on variation in bench press and back squat (upper and lower body measures)

The present study investigated the magnitude of diurnal variation in back squat and bench press using the MuscleLab linear encoder over three different loads and assessed the benefit of an active warm-up to establish whether diurnal variation could be negated. Ten resistance-trained males underwent (mean ± SD: age 21.0 ± 1.3?years, height 1.77 ± 0.06?m, and body mass 82.8 ± 14.9?kg) three sessions. These included control morning (M, 07:30?h) and evening (E, 17:30?h) sessions (5-min standardized warm-up at 150?W, on a cycle ergometer), and one further session consisting of an extended active warm-up morning trial (M_E, 07:30?h) until rectal temperature (T_rec) reached previously recorded resting evening levels (at 150?W, on a cycle ergometer). All sessions included handgrip, followed by a defined program of bench press (at 20, 40, and 60?kg) and back squat (at 30, 50, and 70?kg) exercises. A linear encoder was attached to an Olympic bar used for the exercises and average force (AF), peak velocity (PV), and time to peak velocity (tPV) were measured (MuscleLab software; MuscleLab Technology, Langesund, Norway) during the concentric phase of the movements. Values for T_rec were higher in the E session compared to values in the M session (Δ0.53?°C, P < 0.0005). Following the extended active warm-up in the morning (M_E), T_rec and T_m values were no different to the E values (P < 0.05). Values for T_m were lower in the M compared to the E condition throughout (P < 0.05). There were time-of-day effects for hand grip with higher values of 6.49% for left (P = 0.05) and 4.61% for right hand (P = 0.002) in the E compared to the M. Daily variations were apparent for both bench press and back squat performance for AF (3.28% and 2.63%), PV (13.64% and 11.50%), and tPV (?16.97% and ?14.12%, where a negative number indicates a decrease in the variable from morning to evening). There was a main effect for load (P < 0.0005) such that AF and PV values were larger at higher masses on the bar than lower ones and tPV was smaller at lower masses on the bar than at higher masses for both bench press and back squat. We established that increasing T_rec in the M–E values did not result in an increase of any measures related to bench press and back squat performance (P > 0.05) to increase from M to E levels. Therefore, MuscleLab linear encoder could detect meaningful differences between the morning and evening for all variables. However, the diurnal variation in bench press and back squat (measures of lower and upper body force and power output) is not explained by time-of-day oscillations in T_rec.     

Serum and cerebrospinal fluid concentrations of homoarginine,arginine, asymmetric and symmetric dimethylarginine,nitrite and nitrate in patients with multiple sclerosis and neuromyelitis optica

The pathogenic hallmarks of multiple sclerosis (MS) and neuromyelitis optica (NMO) are cellular and humoral inflammatory infiltrates and subsequent demyelination, or astrocytic cell death in NMO, respectively. These processes are accompanied by disruption of the blood–brain barrier as regularly observed by gadolinium enhancement on magnetic resonance imaging. The role of the l-arginine/nitric oxide (NO) pathway in the pathophysiology of neuroinflammatory diseases, such as MS and NMO, remains unclear. In the present study, we measured the concentrations of the nitric oxide (NO) metabolites nitrate and nitrite, the endogenous substrates of NO synthase (NOS) l-arginine (Arg) and l-homoarginine (hArg), and asymmetric dimethylarginine (ADMA), the endogenous inhibitor of NOS activity, in the serum and cerebrospinal fluid (CSF) of patients with MS, NMO or other neurologic diseases (OND). MS (551 ± 23 nM, P = 0.004) and NMO (608 ± 51 nM, P = 0.006) patients have higher ADMA concentrations in serum than healthy controls (HC; 430 ± 24 nM). For MS, this finding was confirmed in CSF (685 ± 100 nM in relapsing–remitting multiple sclerosis, RRMS; 597 ± 51 nM in secondary progressive multiple sclerosis, SPMS) compared with OND (514 ± 37 nM; P = 0.003). Serum concentrations of Arg (61.1 ± 9.7 vs. 63.6 ± 4.9 µM, P = 0.760), hArg (2.62 ± 0.26 vs. 2.52 ± 0.23 µM, P = 0.891), nitrate (38.1 ± 2.2 vs. 38.1 ± 3.0 µM) and nitrite (1.37 ± 0.09 vs. 1.55 ± 0.03 µM) did not differ between MS and OND. Also, CSF concentrations of hArg (0.685 ± 0.100 µM in RRMS, 0.597 ± 0.051 µM in SPMS, 0.514 ± 0.037 µM in OND), nitrate (11.3 ± 0.6 vs. 10.5 ± 0.3 µM) and nitrite (2.84 ± 0.32 vs. 2.41 ± 0.11 µM) did not differ between the groups. In NMO patients, however, serum Arg (117 ± 11 vs. 64 ± 4.9 μM, P = 0.004), nitrate (29 ± 2.1 vs. 38 ± 3 μM, P = 0.03), and nitrite (1.09 ± 0.02 vs. 1.55 ± 0.033 µM, P < 0.0001) were significantly different as compared to OND. Symmetric dimethylarginine (SDMA) concentration did not differ in serum between MS and HC (779 ± 43 vs. 755 ± 58 nM, P = 0.681) or in CSF between MS and OND patients (237 ± 11 vs. 230 ± 17 nM, P = 0.217). Our study suggests a potential role for ADMA and Arg in neuroinflammatory diseases with diverse functions in MS and NMO. Higher ADMA synthesis may explain reduced NO availability in NMO. hArg and SDMA seem not to play an important role in MS and NMO.     

Minimization of the Wilson’s Central Terminal voltage potential via a genetic algorithm

Objective

The Wilson Central Terminal (WCT) is an artificially constructed reference for surface electrocardiography, which is assumed to be near zero and steady during the cardiac cycle; namely it is the simple average of the three recorded limbs (right arm, left arm and left leg) composing the Einthoven triangle and considered to be electrically equidistant from the electrical center of the heart. This assumption has been challenged and disproved in 1954 with an experiment designed just to measure and minimize WCT. Minimization was attempted varying in real time the weight resistors connected to the limbs. Unfortunately, the experiment required a very cumbersome setup and showed that WCT amplitude could not be universally minimized, in other words, the weight resistors change for each person. Taking advantage of modern computation techniques as well as of a special ECG device that aside of the standard 12-lead Electrocardiogram (ECG) can measure WCT components, we propose a software minimization (genetic algorithm) method using data recorded from 72 volunteers.

Result

We show that while the WCT presents average amplitude relative to lead II of 58.85% (standard deviation of 30.84%), our minimization method yields an amplitude as small as 7.45% of lead II (standard deviation of 9.04%).