Multiple B-cell clones producing antibodies directed to the spacer and disintegrin/thrombospondin type-1 repeat 1 (TSP1) of ADAMTS13 in a patient with acquired thrombotic thrombocytopenic purpura

BACKGROUND: The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP. METHODS: Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains. RESULTS: Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13. CONCLUSION: Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.

Dissociation of periodic leg movements from arousals in restless legs syndrome

The purpose of this study was to characterize the nature of the relation between periodic leg movements during sleep (PLMS) and cortical arousals to contribute to the debate on the clinical significance and treatment of PLMS.

Identification and quantification of a new family of peptide endocannabinoids (Pepcans) showing negative allosteric modulation at CB1 receptors

The α-hemoglobin-derived dodecapeptide RVD-hemopressin (RVDPVNFKLLSH) has been proposed to be an endogenous agonist for the cannabinoid receptor type 1 (CB(1)). To study this peptide, we have raised mAbs against its C-terminal part. Using an immunoaffinity mass spectrometry approach, a whole family of N-terminally extended peptides in addition to RVD-Hpα were identified in rodent brain extracts and human and mouse plasma. We designated these peptides Pepcan-12 (RVDPVNFKLLSH) to Pepcan-23 (SALSDLHAHKLRVDPVNFKLLSH), referring to peptide length. The most abundant Pepcans found in the brain were tested for CB(1) receptor binding. In the classical radioligand displacement assay, Pepcan-12 was the most efficacious ligand but only partially displaced both [(3)H]CP55,940 and [(3)H]WIN55,212-2. The data were fitted with the allosteric ternary complex model, revealing a cooperativity factor value α < 1, thus indicating a negative allosteric modulation. Dissociation kinetic studies of [(3)H]CP55,940 in the absence and presence of Pepcan-12 confirmed these results by showing increased dissociation rate constants induced by Pepcan-12. A fluorescently labeled Pepcan-12 analog was synthesized to investigate the binding to CB(1) receptors. Competition binding studies revealed K(i) values of several Pepcans in the nanomolar range. Accordingly, using competitive ELISA, we found low nanomolar concentrations of Pepcans in human plasma and ∼100 pmol/g in mouse brain. Surprisingly, Pepcan-12 exhibited potent negative allosteric modulation of the orthosteric agonist-induced cAMP accumulation, [(35)S]GTPγS binding, and CB(1) receptor internalization. Pepcans are the first endogenous allosteric modulators identified for CB(1) receptors. Given their abundance in the brain, Pepcans could play an important physiological role in modulating endocannabinoid signaling.

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Dissociation between overt and unconscious face processing in fusiform face area

The precise role of the fusiform face area (FFA) in face processing remains controversial. In this study, we investigated to what degree FFA activation reflects additional functions beyond face perception. Seven volunteers underwent rapid event-related functional magnetic resonance imaging while they performed a face-encoding and a face-recognition task. During face encoding, activity in the FFA for individual faces predicted whether the individual face was subsequently remembered or forgotten. However, during face recognition, no difference in FFA activity between consciously remembered and forgotten faces was observed, but the activity of FFA differentiated if a face had been seen previously or not. This demonstrated a dissociation between overt recognition and unconscious discrimination of stimuli, suggesting that physiological processes of face recognition can take place, even if not all of its operations are made available to consciousness.

Dissociation of epileptic and inflammatory activity in Rasmussen Encephalitis

SUMMARY: Multimodal imaging was performed in Rasmussen Encephalitis (RE) during episodes of complex-partial and focal motor status epilepticus including independent component analysis of BOLD-fMRI, arterial spin labeling perfusion imaging and diffusion tensor imaging. The active epileptic network and topographically independent brain areas showed regional hyperperfusion and progressive atrophy. The results suggest that hyperperfusion outside of the epileptic network represent active inflammation in RE and the imaging protocol presented here, allows assessing thereby the disease activity non-invasively.

Dynamic repair of scapholunate dissociation with dorsal extensor carpi radialis longus tenodesis

This study investigates the results of a technique using an extensor carpi radialis longus (ECRL) tenodesis for symptomatic scapholunate instability. Symptomatic scapholunate instability has been corrected so far either by limited wrist fusion or by various techniques of soft tissue repair. Limited wrist fusion greatly reduces wrist motion and increases the probability of osteoarthritis in the remaining mobile wrist segments. On the other hand, most types of soft tissue repair are technically difficult to perform and have disappointing results due to the inherent laxity. The presented dynamic approach was used in 20 wrists of 19 patients with static scapholunate instability. Preoperative evaluation included in all patients clinical examination, radiologic evaluation, and arthroscopy for establishing the diagnosis of static scapholunate instability. The technique involves the fixation of the ECRL tendon on the dorsal aspect of the scaphoid by means of a cancellous screw and a special washer. Dynamic ECRL tenodesis of the scaphoid is a safe and simple procedure that enhances the extension forces on the scaphoid in all wrist positions. The results of this preliminary report in 20 wrists showed dynamic ECRL tenodesis to be an effective treatment option for treating symptomatic static scapholunate instability.