Jean-Luc Murk

Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography

Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze‐substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high‐pressure frozen and aldehyde‐fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.

Endosomal compartmentalization in three dimensions: implications for membrane fusion.

Endosomes are major sorting stations in the endocytic route that send proteins and lipids to multiple destinations in the cell, including the cell surface, Golgi complex, and lysosomes. They have an intricate architecture of internal membrane structures enclosed by an outer membrane. Recycling proteins remain on the outer membrane, whereas proteins that are destined for degradation in the lysosome are sorted to the interior. Recently, a retrograde pathway was discovered whereby molecules, like MHC class II of the immune system, return from the internal structures to the outer membrane, allowing their further transport to the cell surface for T cell activation. Whether this return involves back fusion of free vesicles with the outer membrane, or occurs via the continuity of the two membrane domains, is an unanswered question. By electron tomography of cryo-immobilized cells we now demonstrate that, in multivesicular endosomes of B-lymphocytes and dendritic cells, the inner membranes are free vesicles. Hence, protein transport from inner to outer membranes cannot occur laterally in the plane of the membrane, but requires fusion between the two membrane domains. This implies the existence of an intracellular machinery that mediates fusion between the exoplasmic leaflets of the membranes involved, which is opposite to regular intracellular fusion between cytoplasmic leaflets. In addition, our 3D reconstructions reveal the presence of clathrin-coated areas at the cytoplasmic face of the outer membrane, known to participate in protein sorting to the endosomal interior. Interestingly, profiles reminiscent of inward budding vesicles were often in close proximity to the coats.

The plasticity of multivesicular bodies and the regulation of antigen presentation.

Multivesicular bodies (MVBs) are ubiquitous endocytic organelles containing numerous 50-80 nm vesicles. MVBs are very dynamic in shape and function. In antigen presenting cells (APCs), MVBs play a central role in the loading of major histocompatibility complex class II (MHC II) with antigenic peptides. How MHC II is transported from MVBs to the cell surface is only partly understood. One way involves direct fusion of MVBs with the plasma membrane. As a consequence, their internal vesicles are secreted as so-called exosomes. An alternative has been illustrated in maturing dendritic cells (DCs). Here, MVBs are reshaped into long tubules by back fusion of the internal vesicles with the MVB limiting membrane. Vesicles derived from the tips of these tubules then carry MHC II to the cell surface.

Acute influenza virus-associated encephalitis and encephalopathy in adults: a challenging diagnosis

Background: Acute influenza-associated encephalopathy/encephalitis (IAE) in adults is a rare but well-known complication of influenza virus infection. The diagnosis is difficult to make due to the absence of distinctive clinical symptoms and validated diagnostic criteria. We present an illustrative case and a case review on acute IAE in adults. Methods: We performed a Medline search of the English literature using the terms influenz*, encephal* and adult, and constructed a database of detailed descriptions of patients with influenza virus infection with influenza-like symptoms at the onset of neurological symptoms. Results: A total of 44 patients were included. Confusion and seizures were the most prevalent neurological symptoms, present in 12 (27 %) and 10 (23 %) patients, respectively. Magnetic resonance imaging (MRI) was performed in 21 patients and anomalies were found in 13 (62 %), with lesions located throughout the brain. Influenza virus RNA was detected in cerebrospinal fluid (CSF) in 5 (16 %) of 32 patients. Eight (18 %) of the forty-four patients died. The benefits of antiviral and immunomodulatory therapy have not been well studied. Discussion: Our results show that many different neurological symptoms can be present in patients with acute onset IAE. Therefore, the diagnosis should be considered in patients with fever and neurological symptoms, especially during the influenza season. Laboratory diagnosis consists of demonstration of influenza virus RNA in brain tissue, CSF or respiratory samples, and demonstration of intrathecal antibody production against influenza virus. The presence of brain lesions in MRI and influenza virus in CSF appear to be of prognostic value.

Colchicine aggravates coxsackievirus B3 infection in mice

BACKGROUND There is a clinical need for immunosuppressive therapy that can treat myocarditis patients in the presence of an active viral infection. In this study we therefore investigated the effects of colchicine, an immunosuppressive drug which has been used successfully as treatment for pericarditis patients, in a mouse model of coxsackievirus B3(CVB3)-induced myocarditis. METHODS Four groups of C3H mice were included: control mice (n=8), mice infected with CVB3 (1×10(5) PFU, n=10), mice with colchicine administration (2mg/kg i.p, n=5) and mice with combined CVB3 infection and colchicine administration (n=10). After three days, the heart, pancreas and spleen were harvested and evaluated using (immuno)histochemical analysis and CVB3 qPCR. RESULTS Mice were terminated at day 3 post-virus infection as colchicine treatment rapidly resulted in severe illness and mortality in CVB3-infected mice. Colchicine significantly decreased the number of macrophages in the heart in CVB3-infected mice (p<0.01) but significantly increased the number of neutrophils (p<0.01). In the pancreas, colchicine caused complete destruction of the acini in the CVB3-infected mice and also significantly decreased macrophage (p<0.01) and increased neutrophil numbers (p<0.01). In the spleen, colchicine treatment of CVB3-infected mice induced massive apoptosis in the white pulp and significantly inhibited the virus-induced increase of megakaryocytes in the spleen (p<0.001). Finally, we observed that colchicine significantly increased CVB3 levels in both the pancreas and the heart. CONCLUSIONS Colchicine treatment in CVB3-induced myocarditis has a detrimental effect as it causes complete destruction of the exocrine pancreas and enhances viral load in both heart and pancreas.

Lymphocytes Infiltrate the Quadriceps Muscle in Lymphocytic Myocarditis Patients: A Potential New Diagnostic Tool

BACKGROUND Diagnosing lymphocytic myocarditis (LM) is challenging because of the large variation in clinical presentation and the limitations inherent in current diagnostic tools. The objective of this study was to analyze infiltration of inflammatory cells in quadriceps skeletal muscle of LM patients and investigate the potential diagnostic value of assaying infiltrating inflammatory cells. METHODS Quadriceps muscle tissue, obtained at autopsy from control patients (n = 9) and LM patients (n = 21), was analyzed using immunohistochemistry for infiltration of lymphocytes (CD45), macrophages (CD68), neutrophilic granulocytes (myeloperoxidase), and several lymphocyte subtypes (CD3, CD4, CD8, CD20) and using polymerase chain reaction for a panel of myocarditis-associated viruses. Additionally, quadriceps muscle from mice with acute coxsackievirus B3-induced myocarditis and control mice was analyzed for presence of lymphocytes and virus. RESULTS In quadriceps muscle of LM patients the number of infiltrating lymphocytes were significantly increased and LM was diagnosed with specificity of 100% and sensitivity of 71%. Parvovirus B19 was the primary virus found in our patient groups, found in quadriceps tissue of 3 LM patients (although it was also found in 1 control patient). In the mice, enteroviral RNA was present in the quadriceps muscle, although enteroviral capsid proteins and lymphocyte infiltration were found primarily in the adipose tissue within and directly adjacent to the myocyte tissue, rather than in the myocyte tissue itself. CONCLUSIONS LM is associated with lymphocyte infiltration and viral presence in quadriceps muscle. This indicates that skeletal muscle biopsy/lymphocyte quantification might be a potential diagnostic tool for LM patients.

Nitazoxanide May Modify the Course of Progressive Multifocal Leukoencephalopathy

To the editor, Progressive multifocal leukoencephalopathy (PML) caused by human neurotrophic polyomavirus (JCV) is a central nervous system (CNS) disease in immunodeficient patients. PML is associated with a poor prognosis and the only intervention leading to clearance of the virus is removal of iatrogenic immune suppression. In particular, the prognosis is dismal in patients with severe primary immunodeficiency (PID) [1]. However, our clinical experience described in this case report supports the possibility that nitazoxanide, a broad-spectrum antiparasitic and antiviral drug [2], may have helped in controlling the JCV in a single patient with PML associated with combined PID. This antiviral effect of nitazoxanide may have been produced by induction of innate immunity, downregulation of viral receptors, or interference with maturation of viral proteins [2, 3]. A female patient suffering from psychomotor retardation, bilateral optic nerve atrophy, and refractive amblyopia due to untreated hyperopia developed lymphopenia (0.5–0.9 × 10/l, reference range (ref) 1.2–3.5 × 10/l) in her adolescence. Despite a normal CD19 count (96–159 × 10/l, ref 80–616 × 10/l), B cell maturation was defective (marginal zone 0.48– 0.8 × 10/l, 0.5% of all CD19, ref 7.2–30.8%; switched memory B cells 0.67–1.1 × 10/l, 0.7%, ref 6.5–29.2%) with no response to pneumococcus antigens. She had low concentrations of IgA (0.6 g/l) and IgG (3.1 g/l), while IgE and IgMwere undetectable. CD3CD4 T cell counts were 256 × 10/l (ref

Progressive multifocal leukoencephalopathy and black fungus in a patient with rheumatoid arthritis without severe lymphocytopenia

Introduction: Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating brain infection caused by JC polyomavirus (JCV), primarily seen in patients with severely compromised cellular immunity. Clinical presentation varies depending on the affected white matter. PML prognosis is variable and effective treatments are lacking. Case presentation: A 75-year-old Chinese woman with type 2 diabetes mellitus, chronic kidney disease and rheumatoid arthritis, treated with low-dose methotrexate and prednisolone for 2.5 years, developed a Pleurostomophora richardsiae infection of her left arm. After 6 months of treating this rare black fungus infection with voriconazole, surgery and immunosuppression discontinuation, she presented with progressive afebrile encephalopathy with right-sided hemiparesis. There were no signs of inflammation or metabolic abnormalities. Brain magnetic resonance imaging revealed diffuse frontal white matter lesions and a cerebrospinal fluid PCR confirmed PML due to JC virus. Severe lymphopenia was never present, and at PML diagnosis, CD4 and CD8 T-cell counts were 454 mm−3 and 277 mm−3. CD8 T-cells were able to respond to JCV VP1 peptide stimulation with TNFα secretion. Peripheral B-cell count was only 8 mm−3. Mirtazapine and Maraviroc were started, but unfortunately, she rapidly deteriorated and died 5 weeks after PML diagnosis. Conclusion: Although peripheral lymphocyte counts were never low and CD4 T-cell count was close to normal, the persistent black fungus infection was a hallmark of severely compromised cellular immunity. The unexpected extremely low absolute B-cell count might suggest a protective role for B-cells. The paradoxical, clinical PML onset months after immunosuppressive discontinuation suggests that it was only discovered in the context of an immune reconstitution inflammatory syndrome.

Reply to the letter to the editor “Is colchicine really harmful in viral myocarditis?”

a Department of Pathology, VU University Medical Center, Postbox 7057, 1007 MB Amsterdam, The Netherlands b Department of Cardiology, VU University Medical Center, Postbox 7057, 1007 MB Amsterdam, The Netherlands c Department of Cardiac Surgery, VU University Medical Center, Postbox 7057, 1007 MB Amsterdam, The Netherlands d Institute for Cardiovascular Research of the Vrije Universiteit (ICaR-VU), VU University Medical Center, De Postbox 7057, 1007 MB Amsterdam, The Netherlands e Department of Immunopathology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, Postbox 9892, 1006 AN Amsterdam, The Netherlands f Department of Hematology, Academic Medical Center, Postbox 22660, 1100 DD Amsterdam, The Netherlands. g Department of Virology, University Medical Center Utrecht, Postbox 85500, 3508 GA Utrecht, The Netherlands, h Department of Cardiology, Maastricht University Medical Center, Postbox 5800, 6202 AZ Maastricht, The Netherlands