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.

[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.

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.

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.

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.

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.

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.

The role of microglia and macrophages in the pathophysiology of the CNS.

Microglia are a major ghal component of the central nervous system (CNS) and are extremely sessile. Only a subtype, the perivascular microglia, are regularly replaced from the bone marrow in adult animals. Microglia respond to virtually any, even minor pathological events in the CNS. In most pathological settings microglia are aided by infiltrating hematogenous macrophages. Upon activation microglia and macrophages share most phenotypical markers and can exert similar effector functions. After transection of a CNS fibre tract microglia are insufficiently activated and hematogenous macrophages do not significantly enter the degenerating nerve stump. Thereby myelin debris that contains neurite outgrowth inhibiting activity persists for long time. This is in sharp contrast to the peripheral nervous system in which hematogenous macrophages are rapidly recruited in response to axotomy and clear myelin debris allowing regrowth of axons from the proximal stump. However, CNS lesion paradigms with breakdown of the blood-brain barrier such as cerebral ischemia, brain abscesses and stab wounds elicit prompt microglial activation, macrophage recruitment and debris clearance. There is increasing evidence that microglia play an active part in degenerative CNS diseases. In Alzheimer's disease activated microglia appear to be involved in plaque formation. In experimental globoid cell dystrophy T-cell independent induction of major histocompatibility complex class II molecules on microglia accelerates demyelination. In autoimmune diseases microglia probably have dual functions. Microglia present antigen to infiltrating T cells and exert effector functions thereby locally augmenting immune responses. On the other hand, microglia have the capacity to downregulate T cell responses. In the human acquired immunodeficiency syndrome (AIDS) virus infected macrophages probably introduce the virus to the CNS and in concert with microglia are involved in the pathophysiology of the AIDS dementia complex.

Inflammation and glial responses in ischemic brain lesions.

Focal cerebral ischemia elicits a strong inflammatory response involving early recruitment of granulocytes and delayed infiltration of ischemic areas and the boundary zones by T cells and macrophages. Infiltration of hematogenous leukocytes is facilitated by an upregulation of the cellular adhesion molecules P-selectin, intercellular adhesion molecule-1 and vascular adhesion molecule-1 on endothelial cells. Blocking of the leukocyte/endothelial cell adhesion process significantly reduces stroke volume after transient, but not permanent middle cerebral artery occlusion. In the infarct region microglia are activated within hours and within days transform into phagocytes. Astrocytes upregulate intermediate filaments, synthesize neurotrophins and form glial scars. Local microglia and infiltrating macrophages demarcate infarcts and rapidly remove debris. Remote from the lesion no cellular infiltration occurs, but astroglia and microglia are transiently activated. Astrocytic activation is induced by spreading depression. In focal ischemia neurons die acutely by necrosis and in a delayed fashion by programmed cell death, apoptosis. Proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1 beta are upregulated within hours in ischemic brain lesions. Either directly or via induction of neurotoxic mediators such as nitric oxide, cytokines may contribute to infarct progression in the post-ischemic period. On the other hand, inflammation is tightly linked with rapid removal of debris and repair processes. At present it is unclear whether detrimental effects of inflammation outweigh neuroprotective mechanisms or vice versa. In global ischemia inflammatory responses are limited, but micro- and astroglia are also strongly activated. Glial responses significantly differ between brain regions with selective neuronal death and neighbouring areas that are more resistent to ischemic damage.

The role of macrophages in immune-mediated damage to the peripheral nervous system.

Macrophage-mediated segmental demyelination is the pathological hallmark of autoimmune demyelinating polyneuropathies, including the demyelinating form of Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy. Macrophages serve a multitude of functions throughout the entire pathogenetic process of autoimmune neuropathy. Resident endoneurial macrophages are likely to act as local antigen-presenting cells by their capability to express major histocompatibility complex antigens and costimulatory B7-molecules, and may thus be critical in triggering the autoimmune process. Hematogenous infiltrating macrophages then find their way into the peripheral nerve together with T-cells by the concerted action of adhesion molecules, matrix metalloproteases and chemotactic signals. Within the nerve, macrophages regulate inflammation by secreting several pro-inflammatory cytokines including IL-1, IL-6, IL-12 and TNF-alpha. Autoantibodies are likely to guide macrophages towards their myelin or primarily axonal targets, which then attack in a complement-dependent and receptor-mediated manner. In addition, non-specific tissue damage occurs through the secretion of toxic mediators and cytokines. Later, macrophages contribute to the termination of inflammation by promoting T-cell apoptosis and expressing anti-inflammatory cytokines including TGF-beta1 and IL-10. During recovery, they are tightly involved in allowing Schwann cell proliferation, remyelination and axonal regeneration to proceed. Macrophages, thus, play dual roles in autoimmune neuropathy, being detrimental in attacking nervous tissue but also salutary, when aiding in the termination of the inflammatory process and the promotion of recovery.

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.

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 characterization of Ia expression during Sindbis virus encephalitis in normal and athymic nude mice.

To characterize the expression of Ia systemically and locally on mononuclear cells during acute viral encephalitis, weanling mice were inoculated intracerebrally with Sindbis virus (SV), an alphavirus. Peripheral blood mononuclear cells, splenocytes and perivascular inflammatory cells in frozen brain sections were examined immunocytochemically for the presence of Ia. Ia expression increased in the spleen, blood and brain during SV encephalitis. The majority of the cells in the central nervous system (CNS) expressing Ia were perivascular mononuclear cells but Ia was also found on stellate parenchymal cells. Using one micrometer cryopreserved serial sections we identified these parenchymal cells as macrophages and microglia but not astrocytes. We also identified rare Ia-positive cells resembling endothelial cells. Frozen brain sections of SV-infected T cell-deficient nu/nu mice were also examined for Ia expression. The number and percentage of Ia-positive cells in perivascular inflammatory cells were markedly decreased compared to normal mice and Ia-positive stellate parenchymal cells were less numerous. This suggests that immunocompetent T cells are necessary for "normal" infiltration of inflammatory cells and for Ia expression in the CNS during SV encephalitis.

Nerve injury, axonal degeneration and neural regeneration: basic insights.

Axotomy or crush of a peripheral nerve leads to degeneration of the distal nerve stump referred to as Wallerian degeneration (WD). During WD a microenvironment is created that allows successful regrowth of nerve fibres from the proximal nerve segment. Schwann cells respond to loss of axons by extrusion of their myelin sheaths, downregulation of myelin genes, dedifferentiation and proliferation. They finally aline in tubes (Büngner bands) and express surface molecules that guide regenerating fibres. Hematogenous macrophages are rapidly recruited to the distal stump and remove the vast majority of myelin debris. Molecular changes in the distal stump include upregulation of neurotrophins, neural cell adhesion molecules, cytokines and other soluble factors and their corresponding receptors. Axonal injury not only induces muscle weakness and loss of sensation but also leads to adaptive responses and neuropathic pain. Regrowth of nerve fibres occurs with high specificity with formerly motor fibres preferentially reinnervating muscle. This involves recognition molecules of the L2/HNK-1 family. Nerve regeneration occurs at a rate of 3-4 mm/day after crush and 2-3 mm/day after sectioning a nerve. Nerve regeneration can be fostered pharmacologically. Upon reestablishment of axonal contact Schwann cells remyelinate nerve sprouts and downregulate surface molecules characteristic for precursor/premyelinating or nonmyelinating Schwann cells. At present it is unclear whether axonal regeneration after nerve injury is impeded in neuropathies.

Differential recruitment of CD8+ macrophages during Wallerian degeneration in the peripheral and central nervous system.

The strong macrophage response occurring during Wallerian degeneration in the peripheral but not central nervous system has been implicated in tissue remodeling and growth factor production as key requirements for successful axonal regeneration. We have previously identified a population of CD8+ phagocytes in ischemic brain lesions that differed in its recruitment pattern from CD4+ macrophages/microglia found in other lesion paradigms. In the present study we show that crush injury to the sciatic nerve induced strong infiltration by CD8+ macrophages both at the crush site and into the degenerating distal nerve stump. At the crush site, CD8+ macrophages appeared within 24 hours whereas infiltration of the distal nerve parenchyma was delayed to the second week. CD8+ macrophages were ED1+ and CD11b+ but always MHC class II-. Most CD8+ macrophages coexpressed CD4 while a significant number of CD4+/CD8-macrophages was also present. Expression of the resident tissue macrophage marker ED2 was largely restricted to the CD4+/CD8- population. Following intraorbital crush injury to the optic nerve, infiltration of CD8+ macrophages was strictly confined to the crush site. Taken together, our study demonstrates considerable spatiotemporal diversity of CD8+ macrophage responses to axotomy in the peripheral and central nervous system that may have implications for the different extent of axonal regeneration observed in both systems.

Differential regulation of Bax, Bcl-2, and Bcl-X proteins in focal cortical ischemia in the rat.

Focal ischemia in the parietal cortex of the rat results in massive neuronal death in the infarct zone and penumbra between 12 hours and 6 days after photothrombosis. To examine a possible role of Bcl-2 family proteins in this process of cell death, we investigated their expression by immunoblot assays and immunocytochemistry, and correlated expression patterns with TUNEL as well as morphological signs indicative of apoptosis. In the center of the lesion Bax immunostaining was increased in many degenerating neurons between 4 hours and 3 days after the induction of photothrombosis. At all time points examined, Bcl-2 and Bcl-X protein levels were markedly reduced in injured neurons as compared to the unlesioned side. At the border of the ischemic lesion, two areas were distinguished: 1 - 2 days after induction of photothrombosis, pyknotic cells located immediately adjacent to the lesion core displayed nuclear Bcl-X and Bax immunoreactivity. In contrast, large, morphologically intact neurons located more towards the healthy brain parenchyma displayed an increase in cytoplasmic Bcl-2 and Bcl-X proteins. Double staining for each of the Bcl-2 family proteins and TUNEL revealed that DNA strand breaks and nuclear fragmentation seen in cells located in the lesion core were often associated with increased levels of Bax, but not with elevated Bcl-2 or Bcl-X protein levels, suggesting a role for Bax in the induction of apoptotic death in these cells. The upregulation of Bcl-2 and Bcl-X expression in surviving neurons close to the penumbra might reflect an active survival mechanism that protects these neurons from cell death following a sublethal insult.

Expression of tissue factor in high-grade carotid artery stenosis: association with plaque destabilization.

BACKGROUND AND PURPOSE: The procoagulant protein tissue factor (TF) has been implicated in thromboembolic complications associated with advanced atherosclerosis. In this study, we investigated whether TF expression in high-grade stenoses of the internal carotid artery (ICA) is associated with clinical features of plaque destabilization and addressed the relationship between TF expression and plaque inflammation. METHODS: In 36 consecutive patients undergoing surgery for high-grade ICA stenosis, clinical evidence of plaque instability was provided by the recent occurrence of ischemic symptoms attributable to the stenosis and the detection of cerebral microembolism by means of transcranial Doppler ultrasound monitoring of the ipsilateral middle cerebral artery. Endarterectomy specimens were stained immunocytochemically for TF expression as well as macrophage (CD68) and T cell (CD3) infiltration. RESULTS: Morphologically, TF immunoreactivity was codistributed with plaque inflammation and predominantly localized to CD68+ macrophages. Accordingly, statistical analysis revealed a significant association of TF expression with plaque infiltration by macrophages (P<0.0001) and T cells (P=0.013). Plaques extensively stained for TF (median of TF+ total section area >40% in semiquantitative assessment) were more frequent in symptomatic (12/27) than in asymptomatic patients (1/9). Conversely, plaques exhibiting little TF expression (median of TF+ section area <20%) were more frequent in asymptomatic (3/9) than in symptomatic (1/27) patients (P=0.016). Likewise, we found a highly significant association of TF expression with the occurrence of cerebral microembolism (P=0.008). CONCLUSIONS: Induction of TF at sites of plaque inflammation may play an important role in the destabilization of high-grade ICA stenosis.