Progressive multifocal leukoencephalopathy in a patient post allo-HCT successfully treated with JC virus specific donor lymphocytes.

BACKGROUND: Progressive multifocal leukoencephalopathy is a demyelinating CNS disorder. Reactivation of John Cunningham virus leads to oligodendrocyte infection with lysis and consequent axonal loss due to demyelination. Patients usually present with confusion and seizures. Late diagnosis and lack of adequate therapy options persistently result in permanent impairment of brain functions. Due to profound T cell depletion, impairment of T-cell function and potent immunosuppressive factors, allogeneic hematopoietic cell transplantation recipients are at high risk for JCV reactivation. To date, PML is almost universally fatal when occurring after allo-HCT. METHODS: To optimize therapy specificity, we enriched JCV specific T-cells out of the donor T-cell repertoire from the HLA-identical, anti-JCV-antibody positive family stem cell donor by unstimulated peripheral apheresis [1]. For this, we selected T cells responsive to five JCV peptide libraries via the Cytokine Capture System technology. It enables the enrichment of JCV specific T cells via identification of stimulus-induced interferon gamma secretion. RESULTS: Despite low frequencies of responsive T cells, we succeeded in generating a product containing 20 000 JCV reactive T cells ready for patient infusion. The adoptive cell transfer was performed without complication. Consequently, the clinical course stabilized and the patient slowly went into remission of PML with JCV negative CSF and containment of PML lesion expansion. CONCLUSION: We report for the first time feasibility of generating T cells with possible anti-JCV activity from a seropositive family donor, a variation of virus specific T-cell therapies suitable for the post allo transplant setting. We also present the unusual case for successful treatment of PML after allo-HCT via virus specific T-cell therapy.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

This corrects the article DOI: 10.1038/cdd.2016.22.

[Immune-mediated neuropathies]. / Immunneuropathien.

The Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are the most common immune-mediated polyneuropathies, which can show variable clinical and electrophysiological manifestations. Rarer immune-mediated neuropathies encompass paraproteinemic neuropathies (PPN), multifocal motor neuropathy (MMN) and vasculitic neuropathies. The diagnosis usually relies on the history of symptom evolution, distribution of nerve dysfunction and particularly on characteristic features in nerve conduction studies, aided by cerebrospinal fluid (CSF) examination and nerve biopsy findings. The therapeutic toolbox encompasses corticosteroids, immunoglobulins and plasmapheresis often accompanied by long-term immunosuppression. It is important to note that immune-mediated neuropathies selectively respond to treatment and contraindications need to be considered. Despite treatment a considerable number of patients suffer from permanent neurological deficits.

A novel treatment of cystic fibrosis acting on-target: cysteamine plus epigallocatechin gallate for the autophagy-dependent rescue of class II-mutated CFTR.

We previously reported that the combination of two safe proteostasis regulators, cysteamine and epigallocatechin gallate (EGCG), can be used to improve deficient expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in patients homozygous for the CFTR Phe508del mutation. Here we provide the proof-of-concept that this combination treatment restored CFTR function and reduced lung inflammation (P<0.001) in Phe508del/Phe508del or Phe508del/null-Cftr (but not in Cftr-null mice), provided that such mice were autophagy-competent. Primary nasal cells from patients bearing different class II CFTR mutations, either in homozygous or compound heterozygous form, responded to the treatment in vitro. We assessed individual responses to cysteamine plus EGCG in a single-centre, open-label phase-2 trial. The combination treatment decreased sweat chloride from baseline, increased both CFTR protein and function in nasal cells, restored autophagy in such cells, decreased CXCL8 and TNF-α in the sputum, and tended to improve respiratory function. These positive effects were particularly strong in patients carrying Phe508del CFTR mutations in homozygosity or heterozygosity. However, a fraction of patients bearing other CFTR mutations failed to respond to therapy. Importantly, the same patients whose primary nasal brushed cells did not respond to cysteamine plus EGCG in vitro also exhibited deficient therapeutic responses in vivo. Altogether, these results suggest that the combination treatment of cysteamine plus EGCG acts 'on-target' because it can only rescue CFTR function when autophagy is functional (in mice) and improves CFTR function when a rescuable protein is expressed (in mice and men). These results should spur the further clinical development of the combination treatment.

Redundant functions of phospholipases D1 and D2 in platelet α-granule release.

BACKGROUND: Platelet activation and aggregation are crucial for primary hemostasis, but can also result in occlusive thrombus formation. Agonist-induced platelet activation involves different signaling pathways leading to the activation of phospholipases, which produce second messengers. The role of phospholipase C (PLC) in platelet activation is well established, but less is known about the relevance of phospholipase D (PLD). OBJECTIVE AND METHODS: The aim of this study was to determine a potential function of PLD2 in platelet physiology. Thus, we investigated the function of PLD2 in platelet signaling and thrombus formation, by generating mice lacking PLD2 or both PLD1 and PLD2. Adhesion, activation and aggregation of PLD-deficient platelets were analyzed in vitro and in vivo. RESULTS: Whereas the absence of PLD2 resulted in reduced PLD activity in platelets, it had no detectable effect on the function of the cells in vitro and in vivo. However, the combined deficiency of both PLD isoforms resulted in defective α-granule release and protection in a model of FeCl3 -induced arteriolar thrombosis, effects that were not observed in mice lacking only one PLD isoform. CONCLUSION: These results reveal redundant roles of PLD1 and PLD2 in platelet α-granule secretion, and indicate that this may be relevant for pathologic thrombus formation.

Fast CPMG-based Bloch-Siegert B(1)+ mapping.

A novel method for B(1)+ mapping based on the Bloch-Siegert (BS) shift was recently presented. This method applies off-resonant pulses before signal acquisition to encode B(1) information into the signal phase. BS-based methods possess significant advantages in measurement time and accuracy compared to magnitude-based B(1)+ methods. This study extends the idea of BS B(1)+ mapping to Carr, Purcell, Meiboom, Gill (CPMG)-based multi-spin-echo (BS-CPMG-MSE) and turbo-spin-echo (BS-CPMG-TSE) imaging. Compared to BS-based spin echo imaging (BS-SE), faster acquisition of the B(1)+ information was possible using the BS-CPMG-TSE sequence. Furthermore, signal loss by T(2)* effects could be minimized using these spin echo-based techniques. These effects are critical for gradient echo-based BS methods at high field strengths. However, multi-spin-echo-based BS B(1) methods inherently possess high specific absorption rates. Thus, the relative specific absorption rate of BS-CPMG-TSE sequences was estimated and compared with the specific absorption rate produced by BS-SE sequences.

Improved encoding strategy for CPMG-based Bloch-Siegert B(1)(+) mapping.

Bloch-Siegert (BS) based B(1)(+) mapping methods use off-resonant pulses to encode quantitative B(1)(+) information into the signal phase. It was recently shown that the principle behind BS-based B(1)(+) mapping can be expanded from spin echo (BS-SE) and gradient-echo (BS-FLASH) based BS B(1)(+) mapping to methods such as Carr, Purcell, Meiboom, Gill (CPMG)-based turbo-spin echo (BS-CPMG-TSE) and multi-spin echo (BS-CPMG-MSE) imaging. If CPMG conditions are preserved, BS-CPMG-TSE allows fast acquisition of the B(1)(+) information and BS-CPMG-MSE enables simultaneous mapping of B(1)(+), M(0), and T(2). To date, however, two separate MRI experiments must be performed to enable the calculation of B(1)(+) maps. This study investigated a modified encoding strategy for CPMG BS-based methods to overcome this limitation. By applying a "bipolar" off-resonant BS pulse before the refocusing pulse train, the needed phase information was able to be encoded into different echo images of one echo train. Thus, this technique allowed simultaneous B(1)(+) and T(2) mapping in a single BS-CPMG-MSE experiment. To allow single-shot B(1)(+) mapping, this method was also applied to turbo-spin echo imaging. Furthermore, the presented modification intrinsically minimizes phase-based image artifacts in BS-CPMG-TSE experiments.

[Acute ischemic stroke. New approaches to antithrombotic treatment]. / Akuter ischämischer Schlaganfall. Neue Ansätze in der Antithrombosetherapie.

The only recommended therapy in the acute phase of ischemic stroke is thrombolysis within 4.5-(6) h after symptom onset. For secondary stroke prevention platelet inhibitors or, in cases of cardiac embolism, anticoagulants are used. However, these substances bear significant limitations: either they show only moderate efficacy (platelet inhibitors), or they are associated with a considerable bleeding risk (rt-PA, anticoagulants). Although the majority of strokes are caused by embolic or thrombotic vessel occlusion, strikingly little is known about the pathophysiological role of platelets and their local function in the brain vasculature. The recent development of novel transgenic mouse lines paved the way for the in-depth analysis of the different molecular steps of thrombus formation involving platelets and the plasma coagulation cascade in models of acute ischemic stroke. It was demonstrated that prevention of early platelet adhesion to the damaged vessel wall by blocking the platelet surface receptors GPIbα or GPVI dramatically protects against experimental stroke without increasing the frequency of intracranial hemorrhage. Moreover, the critical involvement of the blood coagulation factor XII (FXII)-driven intrinsic coagulation cascade in thrombus formation during the course of ischemic brain damage could be unraveled thereby disproving established concepts of hemostasis. Based on these findings novel pharmacological blockers of GPIbα and FXIIa were designed that likewise proved to be safe and effective in animal stroke studies. Those compounds now lay the groundwork for a novel and intriguing concept in ischemic stroke and other thromboembolic diseases: antithrombosis devoid of any bleeding complications. Further preclinical testing is currently ongoing.

In vivo imaging of inflammation in the peripheral nervous system by (19)F MRI.

Visualization of neuroinflammation is still a major task in neuroscience and neurology since inflammatory processes play a central pathophysiological role in many disorders of the nervous system but are not yet covered by conventional imaging techniques. Recently, (19)F magnetic resonance imaging (MRI) was introduced as a new cellular imaging technology. In the present study, we established (19)F high field MRI for cell tracking in the peripheral nervous system (PNS) of rats using dedicated MR coils. To mimic focal neuroinflammation, lysolecithin was locally injected into the left sciatic nerve inducing demyelination followed by severe infiltration of monocytes/macrophages from the circulation. Systemic administration of perfluorocarbons (PFC) led to a fluorine signal along the proximal stretch of the affected sciatic nerves in in vivo(19)F MRI which was not seen on the right healthy side. The preferential in vivo uptake of PFC by circulating mononuclear cells was confirmed by density gradient centrifugation of the blood. Removal of nerves with consecutive ex vivo(19)F MRI and additional (19)F spectroscopy for quantification corroborated the localization of the (19)F marker within the injured nerves (1.07×10(18)±1.00×10(18) mean detectable fluorine spins) while contralateral naive nerves did not exhibit any detectable fluorine signal. Histological assessment confirmed the presence of numerous ED1-positive macrophages within the nerve lesions. Control experiments showed that intraneural application of saline led to an inflammatory reaction restricted to the perineurium which could also be detected by (19)F MRI. In conclusion, we show that (19)F MRI is a promising new technology to visualize hematogenous macrophage responses in the nervous system.

Application of compressed sensing to in vivo 3D ¹9F CSI.

This study shows how applying compressed sensing (CS) to (19)F chemical shift imaging (CSI) makes highly accurate and reproducible reconstructions from undersampled datasets possible. The missing background signal in (19)F CSI provides the required sparsity needed for application of CS. Simulations were performed to test the influence of different CS-related parameters on reconstruction quality. To test the proposed method on a realistic signal distribution, the simulation results were validated by ex vivo experiments. Additionally, undersampled in vivo 3D CSI mouse datasets were successfully reconstructed using CS. The study results suggest that CS can be used to accurately and reproducibly reconstruct undersampled (19)F spectroscopic datasets. Thus, the scanning time of in vivo(19)F CSI experiments can be significantly reduced while preserving the ability to distinguish between different (19)F markers. The gain in scan time provides high flexibility in adjusting measurement parameters. These features make this technique a useful tool for multiple biological and medical applications.

The role of glycoprotein Ibalpha and von Willebrand factor interaction in stroke development.

Ischaemic stroke is a devastating disease with limited treatment options due to numerous uncertainties regarding the underlying pathophysiology. The contribution of glycoprotein (GP)Ibalpha and von Willebrand factor (VWF) in stroke development has only recently been established in mice. Complete blockade of GPIbalpha led to a significant reduction of infarct volumes in mice undergoing one hour of transient middle cerebral artery occlusion (tMCAO). High shear-induced changes in VWF confirmation are a prerequisite for VWF binding to collagen and GPIbalpha expressed on platelets. Importantly, transgenic VWF-/- mice were similarly protected against ischemic stroke after tMCAO, and hydrodynamic injection of a VWF-encoding plasmid restored VWF serum levels and the susceptibility towards stroke. Secreted VWF is rapidly cleaved by ADAMTS13. Accordingly, ADAMTS13 deficient mice developed larger infarction after tMCAO, while infusion of recombinant ADAMTS13 into wild-type mice was stroke-protective. In conclusion, there is compelling evidence that GPIbalpha/VWF interactions and downstream signaling via phospholipase D1 (PLD1) provide new therapeutic targets in ischemic stroke.

Enhanced cortical reperfusion protects coagulation factor XII-deficient mice from ischemic stroke as revealed by high-field MRI.

Intrinsic coagulation factor XII deficient (FXII(-/-)) mice are protected from ischemic stroke. To elucidate underlying mechanisms we investigated the early ischemic period in vivo by multimodal magnetic resonance imaging (MRI) at 17.6 Tesla. Cerebral ischemia was induced by either transient (60 min) or permanent occlusion of the middle cerebral artery (t/pMCAO). 10 FXII(-/-) mice underwent t- , 10 FXII(-/-) mice p- and 10 Wildtype (Wt) mice tMCAO. Cerebral blood flow (CBF), diffusion-weighted-imaging (DWI) and T2-relaxometry were measured at 2 h and 24 h after MCAO. Outcome measures were evaluated after motion correction and normalization to atlas space. 2 h after tMCAO CBF reduction was similar in FXII(-/-) and Wt mice extending over cortical (CBF (ml/100 g/min) 33.6+/-6.9 vs. 35.3+/-4.6, p=0.42) and subcortical regions (25.7+/-4.5 vs. 31.6+/-4.0, p=0.17). At 24 h, recovery of cortical CBF by +36% was observed only in tMCAO FXII(-/-) mice contrasting a further decrease of -30% in Wt mice after tMCAO (p=0.02, F((1,18))=6.24). In FXII(-/-) mice in which patency of the MCA was not restored (pMCAO) a further decrease of -75% was observed. Cortical reperfusion in tMCAO FXII(-/-) mice was related to a lower risk of infarction of 59% vs. 93% in Wt mice (p=0.04). Subcortical CBF was similarly decreased in both tMCAO groups (Wt and FXII(-/-)) relating to a similar risk of infarction of 89% (Wt) vs. 99% (FXII(-/-), p=0.17). Deficiency of FXII allows neocortical reperfusion after tMCAO and rescues brain tissue by this mechanism. This study supports the concept of FXII as a promising new target for stroke prevention and therapy.

Imaging of inflammation in the peripheral and central nervous system by magnetic resonance imaging.

Inflammation plays a central role in the pathophysiology of numerous disorders of the nervous system, but is also pivotal for repair processes like peripheral nerve regeneration. In this review we summarize recent advances in cellular magnetic resonance imaging (MRI) while nuclear imaging methods to visualize neuroinflammation are covered by Wunder et al. [Wunder A, Klohs J, Dirnagl U (2009) Non-invasive imaging of central nervous system inflammation with nuclear and optical imaging. Neuroscience, in press]. Use of iron oxide-contrast agents allows assessment of inflammatory processes in living organisms. Upon systemic application, circulating small (SPIO) and ultrasmall particles of iron oxide (USPIO) are preferentially phagocytosed by monocytes before clearance within the reticuloendothelial system of the liver, spleen and lymph nodes. Upon acute migration into the diseased nervous system these iron oxide-laden macrophages become visible on MRI by the superparamagnetic effects of iron oxide resulting in a signal loss on T2-w and/or bright contrast on T1-w MRI. There is an ongoing controversy, however, to what extent SPIO/USPIO also diffuses passively into the brain after disruption of the blood-brain barrier pretending macrophage invasion. Other confounding factors include circulating SPIO/USPIO particles within the blood pool, local hemorrhages, and intrinsic iron oxide-loading of phagocytes. These uncertainties can be overcome by in vitro preloading of cells with iron oxide contrast agents and consecutive systemic application into animals. Iron oxide-contrast-enhanced MRI allowed in vivo visualization of cellular inflammation during wallerian degeneration, experimental autoimmune neuritis and encephalomyelitis, and stroke in rodents, but also in patients with multiple sclerosis and stroke. Importantly, cellular MRI provides additional information to gadolinium-DTPA-enhanced MRI since cellular infiltration and breakdown of the blood-brain barrier are not closely linked. Coupling of antibodies to iron oxide particles opens new avenues for molecular MRI and has been successfully used to visualize cell adhesion molecules guiding inflammation.

On the parameterization of rigid base and basepair models of DNA from molecular dynamics simulations.

A method is described to extract a complete set of sequence-dependent material parameters for rigid base and basepair models of DNA in solution from atomistic molecular dynamics simulations. The method is properly consistent with equilibrium statistical mechanics, leads to effective shape, stiffness and mass parameters, and employs special procedures for treating spontaneous torsion angle flips and H-bond breaks, both of which can have a significant effect on the results. The method is accompanied by various analytical consistency checks that can be used to assess the equilibration of statistical averages, and different modeling assumptions pertaining to the rigidity of the bases and basepairs and the locality of the quadratic internal energy. The practicability of the approach is verified by estimating complete parameter sets for the 16-basepair palindromic oligomer G(TA)(7)C simulated in explicit water and counterions. Our results indicate that the method is capable of resolving sequence-dependent variations in each of the material parameters. Moreover, they show that the assumptions of rigidity and locality hold rather well for the base model, but not for the basepair model. For the latter, it is shown that the non-local nature of the internal energy can be understood in terms of a certain compatibility relation involving Schur complements.

Macrophage clustering as a diagnostic marker in sural nerve biopsies of patients with CIDP.

BACKGROUND: In adult patients with a slowly progressive demyelinating neuropathy, it may be difficult to distinguish between a hereditary neuropathy and chronic inflammatory demyelinating polyneuropathy (CIDP). The authors previously observed clustering of macrophages around endoneurial blood vessels in sural nerve biopsies from patients with CIDP. OBJECTIVES: To quantitate macrophage clustering around endoneurial blood vessels in CIDP vs hereditary neuropathies. METHODS: The authors studied 21 patients with CIDP, 18 patients with hereditary neuropathies, and 5 normal sural nerves. Numbers of macrophages, T-cells, and blood vessels were counted after immunohistochemical staining. The presence of three or more macrophages around one blood vessel was defined as a cluster. In a subsequent validation analysis, 65 stored biopsy specimens obtained from patients with a chronic neuropathy were re-evaluated for perivascular macrophage clustering according to criteria derived from the quantitative analysis of the first 221 biopsies in a blinded fashion. RESULTS: The percentage of endoneurial vessels with macrophage clusters was higher in CIDP than in hereditary neuropathies (CIDP median = 9.4, range 0 to 48; hereditary NP median = 0, range 0 to 7.7; p < 0.001). The evaluation of the 65 further biopsies showed that the presence of one perivascular macrophage cluster per fascicle proved to be a valid criterion to differentiate between inflammatory and other forms of neuropathy (chi2 test p = 0.0000025, sensitivity 75%, specificity 72%). CONCLUSION: The presence of clusters of macrophages around endoneurial vessels in sural nerve biopsies may serve as a useful additional marker for establishing the pathologic diagnosis of chronic inflammatory demyelinating polyneuropathy (CIDP).

Contralateral cytokine gene induction after peripheral nerve lesions: dependence on the mode of injury and NMDA receptor signaling.

There is increasing evidence that unilateral nerve injury evokes contralateral responses, but the underlying mechanisms are largely unknown. In the present investigation, we analyzed cytokine and chemokine gene induction in contralateral, non-lesioned nerves after sciatic nerve crush and chronic constriction injury (CCI) by quantitative reverse transcriptase polymerase chain reaction in mice. After sciatic nerve crush, contralateral changes in cytokine gene expression were restricted to interleukin (IL)-1beta, which showed a monophasic peak at the first postoperative day. Following CCI, contralateral transcripts for IL-1beta, IL-10 and monocyte chemoattractant protein-1 (MCP-1) were significantly increased already at day 1 and upregulation persisted over the next 4 weeks. In contrast, tumor necrosis factor alpha (TNF-alpha) levels remained unchanged. Contralateral gene induction was restricted to the homonymous opposite sciatic nerve, but spared the femoral nerve. NMDA receptor blockade completely abolished contralateral cytokine expression after CCI on the mRNA level. In contralateral dorsal root ganglia, only IL-10 mRNA levels were modified after nerve injury. Sham operation significantly increased the cytokine and chemokine gene expression at the ipsilateral side, but could not mediate contralateral effects. Our study confirms that nerve injury evokes contralateral responses and identifies NMDA-mediated signaling as one underlying mechanism.

The extent of cytokine induction in peripheral nerve lesions depends on the mode of injury and NMDA receptor signaling.

We compared cytokine and chemokine induction in mice after sciatic nerve crush and chronic constriction injury (CCI) by quantitative reverse transcriptase polymerase chain reaction. In both nerve lesion paradigms, transcripts for tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-1beta, IL-10, and monocyte chemoattractant protein-1 (MCP-1) were significantly increased in degenerating nerve stumps already at day 1, with a greater magnitude and longer duration in CCI. NMDA receptor blockade significantly reduced cytokine expression after CCI on the mRNA and protein level. In dorsal root ganglia, only IL-10 mRNA levels were modified after nerve injury. Our study indicates that the mode of nerve injury influences the extent of cytokine expression, and identifies NMDA-mediated signaling as one mechanism of cytokine induction in peripheral nerves.

Botulism type B presenting as pure autonomic dysfunction.

Botulism nowadays is a rare mostly food-borne disease caused by the toxin of Clostridium botulinum. On the other hand, botulinum toxin blocking cholinergic transmission has become a most powerful treatment option for many focal movement disorders and is increasingly used to treat autonomic disorders [2, 7, 8]. Although muscle weakness is the hallmark of botulism, autonomic dysfunction may be the leading clinical symptom in rare cases and should be included in the differential diagnosis of pure dysautonomia. Here, we describe a patient with autonomic dysfunction as the leading symptom of botulism type B.