Structure-function analysis on the level of individual synapses

Excitatory synapses in the mammalian brain are made on small protrusions of the postsynaptic cell called dendritic spines. Dendritic spines are highly variable in their morphology and in their microanatomy (e.g. presence of subsynaptic organelles). It is unclear whether and how variability in spine morphological and anatomical properties translates into differences in synaptic function. Using two photon imaging, we analyzed how spine properties can affect synaptic signals and the potential for synaptic plasticity at single identified spine synapses. We show that synaptic signals can be tightly regulated on the level of individual synapses and that differences in spine morphology and microanatomy regulate synaptic function. We also provide evidence for the existence of functionally distinct populations of synapses in regard to their potential for synaptic plasticity. The present thesis is subdivided into three main sections. The first section is dedicated to the analysis of the function of specialized subsynaptic organelles in regulating synaptic plasticity. In the second section we studied the impact of spine morphology on synaptic signals and in the third section we examined whether critical proteins can be tagged to individual synapses in response to plasticity inducing stimuli. In pyramidal cells, only a subset of dendritic spines contains endoplasmic reticulum (ER). Spine ER often forms a ‘spine apparatus’, a specialized organelle with unknown function. It is unclear whether these specialized subsynaptic structures can affect the function of the synapse on the spine head. The possible involvement of spine ER in shaping spine calcium transients, a key trigger for synaptic plasticity, raises the possibility that spine ER could modulate the potential of a given synapse to undergo activity dependent modifications. Using a genetic approach to label the ER in living neurons, we find that the ER preferentially localizes to spines containing strong synapses. We demonstrate that spine ER represents a specialized calcium signaling machinery required for the induction of metabotropic glutamate receptor dependent long term depression at individual synapses. We demonstrate that different subsets of synapses exist in regard to their potential to undergo specific forms of plasticity. Spine ER represents the anatomical correlate for a mechanism by which strong synapses can be retuned in an activity dependent manner. Dendritic spines are separated from their parent dendrite by a thin spine neck. The spine neck slows down diffusion of molecules from the spine head to the parent dendrite, allowing spine-specific action of second messengers and activated enzymes. The resistance of the spine neck is crucial in determining whether spines can also be considered electrical compartments. Only a high enough spine neck resistance leads to electrical compartmentalization and activation of voltage gated channels in the spine in response to synaptic stimulation. We show that spine neck resistance can change in an activity dependent manner. Using single spine calcium imaging as a reporter of NMDA receptor activation and spine head depolarization, we show that spines can indeed act as electrical compartments. Using pharmacological experiments and modeling, we demonstrate that different voltage dependent channels cooperatively participate in shaping spine head depolarization and spine calcium transients. We also show that in vivo the spine neck resistance is higher compared to the situation in acutely sliced brain tissue, demonstrating that in the living animal a higher fraction of spines can be considered electrical compartments compared to the in vitro situation. We provide strong evidence that the spine neck can profoundly affect synaptic calcium signals. Biochemical and electrical compartmentalization is dynamically regulated in an activity dependent way. Spine calcium signals can activate key signaling cascades responsible for the induction of synaptic plasticity. Long term potentiation (LTP) has been shown to require the activity of CaMKII, a serine/ threonine kinase. A chemical protocol leading to LTP has been shown to induce translocation of CaMKII to dendritic spines. It is however unclear whether this molecule acts at single synapses or whether it can spread and modulate neighboring synapses in response to more physiological protocols. Using a new optical approach to induce LTP at single visualized synapses, we show that LTP induction is accompanied by a long-lasting increase of CaMKII at the stimulated synapse. This increase was specific to the stimulated spine and did not spread to neighboring spines. We provide evidence that CaMKII acts locally, on the micrometer scale, to regulate plasticity. We show that the concentration of proteins involved in regulating synaptic plasticity can be tightly regulated at the level of single synapses

Lymphocyte counts in patients with ANCA-associated vasculitis

How lymphocyte counts relate to treatment-response in patients with ANCA-associated vasculitis (AAV) is controversial, and data on short-term variability of lymphocyte counts are lacking. Retrospective single center evaluation of disease activity and lymphocyte counts in patients with AAV, and of lymphocyte counts in kidney transplant-recipients, were done; both at the University Hospital Basel, Switzerland. Twenty-three patients with AAV were included. Remission was achieved in all patients. Ten patients experienced a relapse after a median of 66weeks (range 15-189weeks). Median lymphocyte counts at diagnosis were significantly higher than at remission (1.38×109/L vs. 0.99×109/L; P=0.007). By contrast, median lymphocyte counts at remission and relapse did not differ significantly. However, intra-individual variability of lymphocyte counts early after diagnosis was high [median lymphocyte variability-range during the first 3weeks of treatment 1.57 (range 0.27-3.95), n=17]. This variability was not specific to patients with AAV, but was also observed in patients after kidney transplantation [variability of 1.76 (range 0.74-3.95, n=31)]. The significantly higher median lymphocyte counts at diagnosis of AAV make lymphocyte counts a valuable surrogate for the treatment-efficiency in clinical studies. By contrast, on a patient-level, variability of lymphocyte counts impedes meaningful interpretation of individual measurement

Genotypic and phenotypic resistance testing of HIV-1 in routine clinical care

Data on genotypic and phenotypic resistance testing of HIV-1 in the routine clinical setting are lacking. In a retrospective single-center study, all patients (n=102) for whom genotypic resistance typing (GRT) and phenotypic resistance typing (PRT) were performed during the calendar year 2002 were examined. GRT and PRT results were concordant for 79% of the drugs, being highest for nevirapine (92%) and lowest for didanosine (57%). Concordance of results for protease inhibitors was lowest for lopinavir (78%) and highest for indinavir (88%). Discordant results for lamivudine were observed in 16% of patients; 90% of these results corresponded to high-level resistance by PRT and susceptibility by GRT. Overall, HIV loads were lower and CD4+ cell counts higher after therapy following resistance testing, but a significant association with the number of active drugs as predicted by GRT or PRT could not be identified. In a subgroup of 43 patients with virological failure under antiretroviral therapy and sufficient follow-up data, HIV loads were significantly lower after 3 and 6months. More patients with HIV loads <400/ml had 2 or more active drugs according to PRT (21/29 [75%]) than according to GRT ([15/29 [52%]; p=0.109. This was also found for HIV loads <50/ml (PRT 16/22 [72%], GRT 10/22 [42%]; p=0.103), although the differences were not statistically significant. There was no discernable difference between GRT and PRT in the clinic-based population, but the numbers of resistance tests performed are not sufficient to draw definitive conclusion

Inhibition of the different complement pathways has varying impacts on the serum bactericidal activity and opsonophagocytosis against Haemophilus influenzae type b

Defense against Haemophilus influenzae type b (Hib) is dependent on antibodies and complement, which mediate both serum bactericidal activity (SBA) and opsonophagocytosis. Here we evaluated the influence of capsule-specific antibodies and complement inhibitors targeting the central component C3, the alternative pathway (AP; fB, fD), the lectin pathway (LP; MASP-2) and the terminal pathway (C5) on both effector functions. Findings may be relevant for the treatment of certain diseases caused by dysregulation of the complement system, where inhibitors of complement factors C3 or C5 are used. Inhibitors against other complement components are being evaluated as potential alternative treatment options that may carry a reduced risk of infection by encapsulated bacteria. Serum and reconstituted blood of healthy adults were tested for bactericidal activity before and after vaccination with the Hib capsule-conjugate vaccine ActHIB. Most sera had bactericidal activity prior to vaccination, but vaccination significantly enhanced SBA titers. Independently of the vaccination status, both C3 and C5 inhibition abrogated SBA, whereas inhibition of the LP had no effect. AP inhibition had a major inhibitory effect on SBA of pre- vaccination serum, but vaccination mitigated this inhibition for all disease isolates tested. Despite this, SBA-mediated killing of some Hib isolates remained retarded. Even for the most serum-resistant isolate, SBA was the dominating defense mechanism in reconstituted whole blood, as addition of blood cells to the serum did not enhance bacterial killing. Limited Fc receptor-mediated opsonophagocytosis was unmasked when bacterial killing by the membrane attack complex was blocked. In the presence of C3 or C5 inhibitors, addition of post-vaccination, but not of pre-vaccination serum to the blood cells triggered opsonophagocytosis, leading to suppression of bacterial multiplication. Taken together, our data indicate that for host defense against Hib, killing by SBA is more efficient than by blood cell opsonophagocytosis. However, additional defense mechanisms, such as bacterial clearance by spleen and liver, may play an important role in preventing Hib-mediated sepsis, in particular for Hib isolates with increased serum-resistance. Results indicate potentially improved safety profile of AP inhibitors over C3 and C5 inhibitors as alternative therapeutic agents in patients with increased susceptibility to Hib infection

Alternative complement pathway inhibition does not abrogate meningococcal killing by serum of vaccinated individuals

Dysregulation of complement activation causes a number of diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. These conditions can be treated with monoclonal antibodies (mAbs) that bind to the complement component C5 and prevent formation of the membrane attack complex (MAC). While MAC is involved in uncontrolled lysis of erythrocytes in these patients, it is also required for serum bactericidal activity (SBA), i.e. clearance of encapsulated bacteria. Therefore, terminal complement blockage in these patients increases the risk of invasive disease by Neisseria meningitidis more than 1000-fold compared to the general population, despite obligatory vaccination. It is assumed that alternative instead of terminal pathway inhibition reduces the risk of meningococcal disease in vaccinated individuals. To address this, we investigated the SBA with alternative pathway inhibitors. Serum was collected from adults before and after vaccination with a meningococcal serogroup A, C, W, Y capsule conjugate vaccine and tested for meningococcal killing in the presence of factor B and D, C3, C5 and MASP-2 inhibitors. B meningococci were not included in this study since the immune response against protein-based vaccines is more complex. Unsurprisingly, inhibition of C5 abrogated killing of meningococci by all sera. In contrast, both factor B and D inhibitors affected meningococcal killing in sera from individuals with low, but not with high bactericidal anti-capsular titers. While the anti-MASP-2 mAb did not impair SBA, inhibition of C3 impeded meningococcal killing in most, but not in all sera. These data provide evidence that vaccination can provide protection against invasive meningococcal disease in patients treated with alternative pathway inhibitors

In vaccinated individuals serum bactericidal activity against B meningococci is abrogated by C5 inhibition but not by inhibition of the alternative complement pathway

INTRODUCTION: Several diseases caused by the dysregulation of complement activation can be treated with inhibitors of the complement components C5 and/or C3. However, complement is required for serum bactericidal activity (SBA) against encapsulated Gram-negative bacteria. Therefore, C3 and C5 inhibition increases the risk of invasive disease, in particular by Neisseria meningitidis. As inhibitors against complement components other than C3 and C5 may carry a reduced risk of infection, we compared the effect of inhibitors targeting the terminal pathway (C5), the central complement component C3, the alternative pathway (FB and FD), and the lectin pathway (MASP-2) on SBA against serogroup B meningococci. METHODS: Serum from adults was collected before and after vaccination with the meningococcal serogroup B vaccine 4CMenB and tested for meningococcal killing. Since the B capsular polysaccharide is structurally similar to certain human polysaccharides, 4CMenB was designed to elicit antibodies against meningococcal outer membrane proteins. RESULTS: While only a few pre-vaccination sera showed SBA against the tested B meningococcal isolates, 4CMenB vaccination induced potent complement-activating IgG titers against isolates expressing a matching allele of the bacterial cell surface-exposed factor H-binding protein (fHbp). SBA triggered by these cell surface protein-specific antibodies was blocked by C5 and reduced by C3 inhibition, whereas alternative (factor B and D) and lectin (MASP-2) pathway inhibitors had no effect on the SBA of post-4CMenB vaccination sera. DISCUSSION: Compared to the SBA triggered by A,C,W,Y capsule polysaccharide conjugate vaccination, SBA against B meningococci expressing a matching fHbp allele was remarkably resilient against the alternative pathway inhibition

Microscopic simulation of membrane molecule diffusion on corralled membrane surfaces

The current understanding of how receptors diffuse and cluster in the plasma membrane is limited. Data from single-particle tracking and laser tweezer experiments have suggested that membrane molecule diffusion is affected by the presence of barriers dividing the membrane into corrals. Here, we have developed a stochastic spatial model to simulate the effect of corrals on the diffusion of molecules in the plasma membrane. The results of this simulation confirm that a fence barrier (the ratio of the transition probability for diffusion across a boundary to that within a corral) on the order of 103–104 recreates the experimentally measured difference in diffusivity between artificial and natural plasma membranes. An expression for the macroscopic diffusivity of receptors on corralled membranes is derived to analyze the effects of the corral parameters on diffusion rate. We also examine whether the lattice model is an appropriate description of the plasma membrane and look at three different sets of boundary conditions that describe diffusion over the barriers and whether diffusion events on the plasma membrane may occur with a physically relevant length scale. Finally, we show that to observe anomalous (two-timescale) diffusion, one needs high temporal (microsecond) resolution along with sufficiently long (more than milliseconds) trajectories

Immunomodulation with romiplostim as a second-line strategy in primary immune thrombocytopenia: The iROM study.

Thrombopoietin receptor agonists (TPO-RAs) stimulate platelet production, which might restore immunological tolerance in primary immune thrombocytopenia (ITP). The iROM study investigated romiplostim's immunomodulatory effects. Thirteen patients (median age, 31 years) who previously received first-line treatment received romiplostim for 22 weeks, followed by monitoring until week 52. In addition to immunological data, secondary end-points included the sustained remission off-treatment (SROT) rate at 1 year, romiplostim dose, platelet count and bleedings. Scheduled discontinuation of romiplostim and SROT were achieved in six patients with newly diagnosed ITP, whereas the remaining seven patients relapsed. Romiplostim dose titration was lower and platelet count response was stronger in patients with SROT than in relapsed patients. In all patients, regulatory T lymphocyte (Treg) counts increased until study completion and the counts were higher in patients with SROT. Interleukin (IL)-4, IL-9 and IL-17F levels decreased significantly in all patients. FOXP3 (Treg), GATA3 (Th2) mRNA expression and transforming growth factor-β levels increased in patients with SROT. Treatment with romiplostim modulates the immune system and possibly influences ITP prognosis. A rapid increase in platelet counts is likely important for inducing immune tolerance. Better outcomes might be achieved at an early stage of autoimmunity, but clinical studies are needed for confirmation

Von Willebrand factor and the thrombophilia of severe COVID-19: in situ evidence from autopsies

BACKGROUND: COVID-19 is accompanied by a hypercoagulable state and characterized by microvascular and macrovascular thrombotic complications. In plasma samples from patients with COVID-19, von Willebrand factor (VWF) levels are highly elevated and predictive of adverse outcomes, especially mortality. Yet, VWF is usually not included in routine coagulation analyses, and histologic evidence of its involvement in thrombus formation is lacking. OBJECTIVES: To determine whether VWF, an acute-phase protein, is a bystander, ie, a biomarker of endothelial dysfunction, or a causal factor in the pathogenesis of COVID-19. METHODS: We compared autopsy samples from 28 patients with lethal COVID-19 to those from matched controls and systematically assessed for VWF and platelets by immunohistochemistry. The control group comprised 24 lungs, 23 lymph nodes, and 9 hearts and did not differ significantly from the COVID-19 group in age, sex, body mass index (BMI), blood group, or anticoagulant use. RESULTS: In lungs, assessed for platelets by immunohistochemistry for CD42b, microthrombi were more frequent in patients with COVID-19 (10/28 [36%] vs 2/24 [8%]; P = .02). A completely normal pattern of VWF was rare in both groups. Accentuated endothelial staining was found in controls, while VWF-rich thrombi were only found in patients with COVID-19 (11/28 [39%] vs 0/24 [0%], respectively; P < .01), as were NETosis thrombi enriched with VWF (7/28 [25%] vs 0/24 [0%], respectively; P < .01). Forty-six percent of the patients with COVID-19 had VWF-rich thrombi, NETosis thrombi, or both. Trends were also seen in pulmonary draining lymph nodes (7/20 [35%] vs 4/24 [17%]; P = .147), where the overall presence of VWF was very high. CONCLUSION: We provide in situ evidence of VWF-rich thrombi, likely attributable to COVID-19, and suggest that VWF may be a therapeutic target in severe COVID-19