A conformation-selective monoclonal antibody against a small molecule-stabilised signalling-deficient form of TNF.

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.

Primary angiitis of the central nervous system: diagnosis and treatment.

Primary angiitis of the central nervous system (PACNS) represents a rare inflammatory disease affecting the brain and spinal cord. Stroke, encephalopathy, headache and seizures are major clinical manifestations. The diagnosis of PACNS is based on the combination of clinical presentation, imaging findings (magnetic resonance imaging and angiography), brain biopsy, and laboratory and cerebral spinal fluid (CSF) values. PACNS can either be confirmed by magnetic resonance angiography (MRA)/conventional angiography or tissue biopsy showing the presence of typical histopathological patterns. Identification of PACNS mimics is often challenging in clinical practice, but crucial to avoid far-reaching treatment decisions. In view of the severity of the disease, with considerable morbidity and mortality, early recognition and treatment initiation is necessary. Due to the rareness and heterogeneity of the disease, there is a lack of randomized data on treatment strategies. Retrospective studies suggest the combined administration of cyclophosphamide and glucocorticoids as induction therapy. Immunosuppressants such as azathioprine, methotrexate or mycophenolate mofetil are often applied for maintenance therapy. In addition, the beneficial effects of two biological agents (anti-CD20 monoclonal antibody rituximab and tumour necrosis factor-α blocker) have been reported. Nevertheless, diagnosis and treatment is still a clinical challenge, and further insights into the immunopathogenesis of PACNS are required to improve the diagnosis and management of patients. The present review provides a comprehensive overview of diagnostics, differential diagnoses, and therapeutic approaches of adult PACNS.

Immune cell profiling in the cerebrospinal fluid of patients with primary angiitis of the central nervous system reflects the heterogeneity of the disease.

Primary angiitis of the central nervous system (PACNS) is a rare and heterogeneous inflammatory disease of the CNS vasculature with poorly understood pathophysiology. Comprehensive immune-cell phenotyping revealed increased frequencies of leukocytes in the cerebrospinal fluid (CSF) of PACNS patients compared to patients with multiple sclerosis, ischemic stroke, and somatoform disorders (n = 18 per group). Changes in the intrathecal immune-cell profile were heterogeneous in PACNS. While proportions of T-cell subsets remained unaltered, some PACNS patients showed a shift toward NK- or B cells. Intrathecal immunoglobulin synthesis was observed in a subgroup of PACNS patients with an increased frequency of antibody producing plasma cells.

Beyond body size: consistent decrease of traits within orthopteran assemblages with elevation.

Morphological traits provide the interface between species and their environment. For example, body size affects the fitness of individuals in various ways. Yet especially for ectotherms, the applicability of general rules of interspecific clines of body size and even more so of other morphological traits is still under debate. Here we tested relationships between elevation (as a proxy for temperature) and productivity with four ecologically relevant morphological traits of orthopteran assemblages that are related to fecundity (body size), dispersal (wing length), jumping ability (hind femur length), and predator detection (eye size). We measured traits of 160 orthopteran species that were sampled along an extensive environmental gradient at Mt. Kilimanjaro (Tanzania), spanning elevations from 790 to 4,410 m above sea level (a.s.l.) with different levels of plant productivity. For traits other than body size, we calculated the residuals from a regression on body length to estimate the variation of traits irrespective of body size. Bayesian analyses revealed that mean body size of assemblages, as well as the means of relative wing length, hind femur length, and eye size, decreased with increasing elevation. Body size and relative eye size also decreased with increasing productivity. Both phylogenetic relationships, as well as species-specific adaptations, contributed to these patterns. Our results suggest that orthopteran assemblages had higher fecundity and better dispersal and escape abilities, as well as better predator detection at higher temperatures (low elevations) than at low temperatures (high elevations). Large body sizes might be advantageous in habitats with low productivity because of a reduced risk of starvation. Likewise, large eye size might be advantageous because of the ability to detect predators in habitats with low vegetation cover, where hiding possibilities are scarce. Our study highlights that changes in temperature and productivity not only lead to interspecific changes in body size but are also related to independent changes of other morphological traits that influence the ecological fit of organisms in their environment.

Defining mechanisms of neural plasticity after brainstem ischemia in rats.

OBJECTIVE: Neurological recovery after stroke mainly depends on the location of the lesion. A substantial portion of strokes affects the brainstem. However, patterns of neural plasticity following brainstem ischemia are almost unknown. METHODS: Here, we established a rat brainstem ischemia model that resembles key features of the human disease and investigated mechanisms of neural plasticity, including neurogenesis and axonal sprouting as well as secondary neurodegeneration. RESULTS: Spontaneous functional recovery was accompanied by a distinct pattern of axonal sprouting, for example, an increased bilateral fiber outgrowth from the corticorubral tract to the respective contralesional red nucleus suggesting a compensatory role of extrapyramidal pathways after damage to pyramid tracts within the brainstem. Using different markers for DNA replication, we showed that the brainstem displays a remarkable ability to undergo specific plastic cellular changes after injury, highlighting a yet unknown pattern of neurogenesis. Neural progenitor cells proliferated within the dorsal brainstem and migrated toward the lesion, whereas neurogenesis in classic neurogenic niches, the subventricular zone of the lateral ventricle and the hippocampus, remained, in contrast to what is known from hemispheric stroke, unaffected. These beneficial changes were paralleled by long-term degenerative processes, that is, corticospinal fiber loss superior to the lesion, degeneration of spinal tracts, and a decreased neuron density within the ipsilesional substantia nigra and the contralesional red nucleus that might have limited further functional recovery. INTERPRETATION: Our findings provide knowledge of elementary plastic adaptions after brainstem stroke, which is fundamental for understanding the human disease and for the development of new treatments. Ann Neurol 2018;83:1003-1015.

Antibody Concentrations Decrease 14-Fold in Children With Celiac Disease on a Gluten-Free Diet but Remain High at 3 Months.

BACKGROUND & AIMS: Celiac disease can be identified by a serologic test for IgA against tissue transglutaminase (IgA-TTG) in a large proportion of children. However, the increased concentrations of antibody rarely normalize within the months after children are placed on a gluten-free diet (GFD). Early serologic predictors of sufficient adherence to GFD are required for optimal treatment. METHODS: In a prospective study, we observed the response to a GFD in 345 pediatric patients (67% girls; mean age, 8.4 y) who underwent duodenal biopsy to confirm or refute celiac disease from October 2012 through December 2015. Baseline serum samples were tested centrally for IgA-TTG and IgG against deamidated gliadin. Follow-up serologic analyses of children on a GFD were performed about 3 months later. RESULTS: The geometric mean concentration of IgA-TTG decreased from 72.4-fold to 5.2-fold the upper limit of normal (ULN), or by a factor of 14.0 (95% CI, 12.0-16.4). A substantial response (defined as a larger change than the typical variation in patients not on a GFD) was observed in 80.6% of the children. Only 28.1% of patients had a substantial response in the concentration of IgG against deamidated gliadin. Concentration of IgA-TTG remained above 1-fold the ULN in 83.8% of patients, and above 10-fold the ULN in 26.6% of patients with a substantial response. CONCLUSIONS: Serum concentration of IgA-TTG decreases substantially in most children with celiac disease within 3 months after they are placed on a GFD, but does not normalize in most. This information on changes in antibody concentrations can be used to assess patient response to the diet at short-term follow-up evaluations. Patients with a substantial response to a GFD often still have high antibody levels after 3 months. German Clinical Trials Registry no. DRKS00003854.

Effects of Different Exercise Strategies and Intensities on Memory Performance and Neurogenesis.

It is well established that physical exercise affects both hippocampal neurogenesis and memory functions. Until now, distinctive effects of controlled and voluntary training (VT) on behavior and neurogenesis as well as interactions between exercise intensity, neurogenesis and memory performance are still elusive. The present study tested the impact of moderate controlled and VT on memory formation and hippocampal neurogenesis and evaluated interactions between exercise performance, learning efficiency and proliferation of progenitor cells in the hippocampus. Our data show that both controlled and VT augmented spatial learning and promoted hippocampal neurogenesis. Regression analysis revealed a significant linear increase of the amount of new hippocampal neurons with increased exercise intensity. Regression analysis of exercise performance on retention memory performance revealed a quadratic, inverted u-shaped relationship between exercise performance and retention of spatial memory. No association was found between the amount of newborn neurons and memory performance. Our results demonstrate that controlled training (CT), if performed with an appropriate combination of speed and duration, improves memory performance and neurogenesis. Voluntary exercise elevates neurogenesis dose dependently to high levels. Best cognitive improvement was achieved with moderate exercise performance.

Targeting Different Monocyte/Macrophage Subsets Has No Impact on Outcome in Experimental Stroke.

BACKGROUND AND PURPOSE: Peripheral immune cell infiltration contributes to neural injury after ischemic stroke. However, in contrast to lymphocytes and neutrophils, the role of different monocyte/macrophage subsets remains to be clarified. Therefore, we evaluated the effects of selective and unselective monocyte/macrophage depletion and proinflammatory (M1-) and anti-inflammatory (M2-) macrophage transfer on the outcome after experimental cerebral ischemia. METHODS: To assess short-term effects of monocytes/macrophages in acute ischemic stroke, mice underwent transient middle cerebral artery occlusion and received either clodronate liposomes for unselective macrophage depletion, MC-21-antibody for selective depletion of proinflammatory Ly-6Chigh monocytes, or proinflammatory (M1-) or anti-inflammatory (M2-) macrophage transfer. In addition, the impact of MC-21-antibody administration and M2-macrophage transfer on long-term neural recovery was investigated after photothrombotic stroke. Neurobehavioral tests were used to analyze functional outcomes, infarct volumes were determined, and immunohistochemical analyses were performed to characterize the postischemic inflammatory reaction. RESULTS: Selective and unselective monocyte/macrophage depletion and M1- and M2-macrophage transfer did not influence tissue damage and neurobehavioral outcomes in the acute phase after middle cerebral artery occlusion. Beyond, selective depletion of Ly-6Chigh monocytes and M2-macrophage transfer did not have an impact on neural recovery after photothrombotic stroke. CONCLUSIONS: Targeting different monocyte/macrophage subsets has no impact on outcome after ischemic stroke in mice. Altogether, our study could not identify monocytes/macrophages as relevant therapeutic targets in acute ischemic stroke.

Neutrophil granulocytes in cerebral ischemia - Evolution from killers to key players.

Neutrophil granulocytes (or polymorphonuclear cells, PMNs) have long been considered as crude killing machines, particularly trained to attack bacterial or fungal pathogens in wounds or infected tissues. That perspective has fundamentally changed over the last decades, as PMNs have been shown to exert a livery exchange between other cells of the innate and adaptive immune system. PMNs do provide major immunomodulatory contribution during acute inflammation and subsequent clearance. Following sterile inflammation like cerebral ischemia, PMNs are among the first hematogenous cells attracted to the ischemic tissue. As inflammation is a crucial component within stroke pathophysiology, several studies regarding the role of PMNs following cerebral ischemia have been carried out. And indeed, recent research suggests a direct connection between PMNs' influx and brain damage severity. This review highlights the latest research regarding the close interconnection between PMNs and co-working cells following cerebral ischemia. We describe how PMNs are attracted to the site of injury and their tasks within the inflamed brain tissue and the periphery. We further report of new findings regarding the interaction of PMNs with resident microglia, immigrating macrophages and T cells after stroke. Finally, we discuss recent research results from experimental studies in the context with current clinical trials and point out potential new therapeutic applications that could emerge from this new knowledge on the action and interaction of PMNs following cerebral ischemia.

Combining Growth Factor and Bone Marrow Cell Therapy Induces Bleeding and Alters Immune Response After Stroke in Mice.

BACKGROUND AND PURPOSE: Bone marrow cell (BMC)-based therapies, either the transplantation of exogenous cells or stimulation of endogenous cells by growth factors like the granulocyte colony-stimulating factor (G-CSF), are considered a promising means of treating stroke. In contrast to large preclinical evidence, however, a recent clinical stroke trial on G-CSF was neutral. We, therefore, aimed to investigate possible synergistic effects of co-administration of G-CSF and BMCs after experimental stroke in mice to enhance the efficacy compared with single treatments. METHODS: We used an animal model for experimental stroke as paradigm to study possible synergistic effects of co-administration of G-CSF and BMCs on the functional outcome and the pathophysiological mechanism. RESULTS: G-CSF treatment alone led to an improved functional outcome, a reduced infarct volume, increased blood vessel stabilization, and decreased overall inflammation. Surprisingly, the combination of G-CSF and BMCs abrogated G-CSFs' beneficial effects and resulted in increased hemorrhagic infarct transformation, altered blood-brain barrier, excessive astrogliosis, and altered immune cell polarization. These increased rates of infarct bleeding were mainly mediated by elevated matrix metalloproteinase-9-mediated blood-brain barrier breakdown in G-CSF- and BMCs-treated animals combined with an increased number of dilated and thus likely more fragile vessels in the subacute phase after cerebral ischemia. CONCLUSIONS: Our results provide new insights into both BMC-based therapies and immune cell biology and help to understand potential adverse and unexpected side effects.

Promoting recovery from ischemic stroke.

Over recent decades, experimental and clinical stroke studies have identified a number of neurorestorative treatments that stimulate neural plasticity and promote functional recovery. In contrast to the acute stroke treatments thrombolysis and endovascular thrombectomy, neurorestorative treatments are still effective when initiated days after stroke onset, which makes them applicable to virtually all stroke patients. In this article, selected physical, pharmacological and cell-based neurorestorative therapies are discussed, with special emphasis on interventions that have already been transferred from the laboratory to the clinical setting. We explain molecular and structural processes that promote neural plasticity, discuss potential limitations of neurorestorative treatments, and offer a speculative viewpoint on how neurorestorative treatments will evolve.

Defining a conformational consensus motif in cotransin-sensitive signal sequences: a proteomic and site-directed mutagenesis study.

The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.

Progressive cognitive deficits in a mouse model of recurrent photothrombotic stroke.

BACKGROUND AND PURPOSE: In spite of its high disease burden, there is no specific treatment for multi-infarct dementia. The preclinical evaluation of candidate drugs is limited because an appropriate animal model is lacking. Therefore, we aimed to evaluate whether a mouse model of recurrent photothrombotic stroke is suitable for the preclinical investigation of multi-infarct dementia. METHODS: Recurrent photothrombotic cortical infarcts were induced in 25 adult C57BL/6 mice. Twenty-five sham-operated animals served as controls. The object recognition test and the Morris water maze test were performed >6 weeks to assess cognitive deficits. Afterward, histological analyses were performed to characterize histopathologic changes associated with recurrent photothrombotic infarcts. RESULTS: After the first infarct, the object recognition test showed a trend toward an impaired formation of recognition memories (P=0.08), and the Morris Water Maze test revealed significantly impaired spatial learning and memory functions (P<0.05). After recurrent infarcts, the object recognition test showed significant recognition memory deficits (P<0.001) and the Morris water maze test demonstrated persisting spatial learning and memory deficits (P<0.05). Histological analyses revealed remote astrogliosis in the hippocampus. CONCLUSIONS: Our results show progressive cognitive deficits in a mouse model of recurrent photothrombotic stroke. The presented model resembles the clinical features of human multi-infarct dementia and enables the investigation of its pathophysiological mechanisms and the evaluation of treatment strategies.

Granulocyte colony-stimulating factor (G-CSF) treatment in combination with transplantation of bone marrow cells is not superior to G-CSF treatment alone after cortical stroke in spontaneously hypertensive rats.

Granulocyte-colony stimulating factor (G-CSF) and bone marrow derived mononuclear cells (BM-MNCs) have both been shown to improve functional outcome following experimental stroke. These effects are associated with increased angiogenesis and neurogenesis. In the present study, we aimed to determine synergistic effects of G-CSF and BM-NMC treatment on long-term structural and functional recovery after photothrombotic stroke. To model the etiology of stroke more closely, we used spontaneously hypertensive (SH) rats in our experiment. Bone marrow derived mononuclear cells transplantation was initiated 1 h after the onset of photothrombotic stroke. Repeated G-CSF treatment commenced immediately after BM-MNC treatment followed by daily injections for five consecutive days. The primary endpoint was functional outcome after ischemia. Secondary endpoints included analysis of neurogenesis and angiogenesis as well as determination of infarct size. Granulocyte-colony stimulating factor treated rats, either in combination with BM-MNC or alone showed improved somatosensory but not gross motor function following ischemia. No beneficial effect of BM-MNC monotherapy was found. Infarct volumes were comparable in all groups. In contrast to previous studies, which used healthy animals, post-stroke neurogenesis and angiogenesis were not enhanced by G-CSF. In conclusion, the combination of G-CSF and BM-MNC was not more effective than G-CSF alone. The reduced efficacy of G-CSF treatment and the absence of any beneficial effect of BM-MNC transplantation might be attributed to hypertension-related morbidity.

Use of Kaede and Kikume green-red fusions for live cell imaging of G protein-coupled receptors.

The fusion of fluorescent proteins to G protein-coupled receptors (GPCRs) is an important tool to study, e.g., trafficking and protein interactions of these important drug targets. In the past, the green fluorescent protein and its derivatives have been widely used as fluorescent tags. More recently, it was reported that photoconvertible fluorescent proteins (PCFPs) such as Kaede or Kikume green-red protein could also be used as fluorescent tags for GPCRs. These proteins have the obvious advantage that their fluorescence can be switched once the GPCR of interest has reached a specific subcellular compartment. Here, we summarize the recent progress for live cell imaging of GPCRs using these PCFPs for trafficking, biosynthesis, and protein/protein interaction studies.

Bone marrow-derived mononuclear cells do not exert acute neuroprotection after stroke in spontaneously hypertensive rats.

Bone marrow-derived mononuclear cells (BM-MNCs) were shown to improve the outcome in animal stroke models and clinical pilot studies on BM-MNCs for stroke patients were already conducted. However, relevant aspects of pre-clinical evaluation, such as the use of animals with comorbidities and dose-response studies, were not thoroughly addressed so far. We therefore investigated different BM-MNC doses in the clinical meaningful stroke model of spontaneously hypertensive (SH) rats. Three hours after the onset of transient middle cerebral artery occlusion (MCAO) animals received either one of three syngeneic BM-MNC doses or placebo intravenously. The primary endpoint was the infarct size. Secondary endpoints included functional outcome, mortality, inflammatory processes, and the dose-response relationship. In contrast to previous studies which used healthy animals no beneficial effect of BM-MNCs was found. Infarct volumes, mortality, behavioral outcomes, and the extent of the inflammatory response to cerebral ischemia were comparable in all groups. In conclusion, we could not demonstrate that early BM-MNC treatment improves the outcome after stroke in SH rats. Whether BM-MNCs improve neurological recovery after delayed treatment initiation was not investigated in the present study, but our data indicates that this should be determined in co-morbid animal stroke models before moving to large-scale clinical studies. Future preclinical stroke studies on co-morbid animals should also include groups of healthy animals in order to determine whether negative results can be attributed to the comorbid condition.

Meta-analysis of the efficacy of different training strategies in animal models of ischemic stroke.

BACKGROUND AND PURPOSE: Although several studies have shown beneficial effects of training in animal stroke models, the most effective training strategy and the optimal time to initiate training have not been identified. The present meta-analysis was performed to compare the efficacy of different training strategies and to determine the optimal time window for training in animal stroke models. METHODS: We searched the literature for studies analyzing the efficacy of training in animal models of ischemic stroke. Training was categorized into forced physical training, voluntary physical training, constraint-induced movement therapy, and skilled reaching training. Two reviewers independently extracted data on study quality, infarct size, and neurological outcome. Data were pooled by means of a meta-analysis. RESULTS: Thirty-five studies with >880 animals were included. A meta-analysis of all treatments showed that training reduced the infarct volume by 14% (95% confidence interval, 2%-25%) and improved the cognitive function by 33% (95% confidence interval, 8%-50%), the neuroscore by 13.4% (95% confidence interval, 1.5%-25.3%), and the running function by 6.6% (95% confidence interval, 1.4%-11.9%). Forced physical training reduced the infarct volume and enhanced the running function most effectively, whereas skilled reaching training improved the limb function most effectively. A meta-regression illustrated that training was particularly efficacious when initiated between 1 and 5 days after stroke onset. CONCLUSIONS: Our meta-analysis confirms that training reduces the infarct volume and improves the functional recovery in animal stroke models. Forced physical training and skilled reaching training were identified as particularly effective training strategies. The efficacy of training is time dependent.