Mireia Ferrer

The role of the spleen in malaria

The spleen is a complex organ that is perfectly adapted to selectively filtering and destroying senescent red blood cells (RBCs), infectious microorganisms and Plasmodium‐parasitized RBCs. Infection by malaria is the most common cause of spleen rupture and splenomegaly, albeit variably, a landmark of malaria infection. Here, the role of the spleen in malaria is reviewed with special emphasis in lessons learned from human infections and mouse models.

Exosomes from Plasmodium yoelii-Infected Reticulocytes Protect Mice from Lethal Infections

Exosomes are 30–100-nm membrane vesicles of endocytic origin that are released after the fusion of multivesicular bodies (MVBs) with the plasma membrane. While initial studies suggested that the role of exosomes was limited to the removal of proteins during the maturation of reticulocytes to erythrocytes, recent studies indicate that they are produced by different types of cells and are involved in promoting inter-cellular communication and antigen presentation. Here, we describe the isolation and characterization of exosomes from peripheral blood of BALB/c mice infected with the reticulocyte-prone non-lethal Plasmodium yoelii 17X strain. Importantly, proteomic analysis revealed the presence of parasite proteins in these vesicles. Moreover, immunization of mice with purified exosomes elicited IgG antibodies capable of recognizing P. yoelii-infected red blood cells. Furthermore, lethal challenge of immunized mice with the normocyte-prone lethal P. yoelii 17XL strain caused a significant attenuation in the course of parasitaemia, increased survival time, and altered the cell tropism to reticulocytes. These results were obtained also when the exosomes were isolated from a P. yoelii-infected reticulocyte culture indicating that reticulocyte-derived exosomes carry antigens and are involved in immune modulation. Moreover, inclusion of CpG ODN 1826 in exosome immunizations elicited IgG2a and IgG2b antibodies and promoted survival, clearance of parasites and subsequent sterile protection of 83% of the animals challenged with P. yoelli 17XL. To our knowledge, this is the first report of immune responses elicited by exosomes derived from reticulocytes opening new avenues for the modulation of anti-malaria responses.

Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor

The subcellular localization and function of variant subtelomeric multigene families in Plasmodium vivax remain vastly unknown. Among them, the vir superfamily is putatively involved in antigenic variation and in mediating adherence to endothelial receptors. In the absence of a continuous in vitro culture system for P. vivax, we have generated P. falciparum transgenic lines expressing VIR proteins to infer location and function. We chose three proteins pertaining to subfamilies A (VIR17), C (VIR14) and D (VIR10), with domains and secondary structures that predictably traffic these proteins to different subcellular compartments. Here, we showed that VIR17 remained inside the parasite and around merozoites, whereas VIR14 and VIR10 were exported to the membrane of infected red blood cells (iRBCs) in an apparent independent pathway of Maurers clefts. Remarkably, VIR14 was exposed at the surface of iRBCs and mediated adherence to different endothelial receptors expressed in CHO cells under static conditions. Under physiological flow conditions, however, cytoadherence was only observed to ICAM‐1, which was the only receptor whose adherence was specifically and significantly inhibited by antibodies against conserved motifs of VIR proteins. Immunofluorescence studies using these antibodies also showed different subcellular localizations of VIR proteins in P. vivax‐infected reticulocytes from natural infections. These data suggest that VIR proteins are trafficked to different cellular compartments and functionally demonstrates that VIR proteins can specifically mediate cytoadherence to the ICAM‐1 endothelial receptor.

Strain-specific spleen remodelling in Plasmodium yoelii infections in Balb/c mice facilitates adherence and spleen macrophage-clearance escape

Knowledge of the dynamic features of the processes driven by malaria parasites in the spleen is lacking. To gain insight into the function and structure of the spleen in malaria, we have implemented intravital microscopy and magnetic resonance imaging of the mouse spleen in experimental infections with non‐lethal (17X) and lethal (17XL) Plasmodium yoelii strains. Noticeably, there was higher parasite accumulation, reduced motility, loss of directionality, increased residence time and altered magnetic resonance only in the spleens of mice infected with 17X. Moreover, these differences were associated with the formation of a strain‐specific induced spleen tissue barrier of fibroblastic origin, with red pulp macrophage‐clearance evasion and with adherence of infected red blood cells to this barrier. Our data suggest that in this reticulocyte‐prone non‐lethal rodent malaria model, passage through the spleen is different from what is known in other Plasmodium species and open new avenues for functional/structural studies of this lymphoid organ in malaria.

Electrophysiological aspects of sensory conduction velocity in healthy adults. 2. Ratio between the amplitude of sensory evoked potentials at the wrist on stimulating different fingers in both hands.

The normal ratio between the amplitude of the sensory evoked potential (SEP) at the wrist on stimulating digits 1, 2, 3, and 5 was determined in 44 healthy adult subjects. The first digit had the larger amplitude, and the fifth digit the smallest SEP. The amplitude expresses the density of sensory innervation in each finger. The ratio between the amplitude of different fingers varied according to the age of the subject. The amplitude of the SEP from a digit innervated by the median nerve decreased in the elderly more than the SEP amplitude of the digit innervated by the ulnar nerve, probably because of a chronic compression in the carpal tunnel. The changes in the normal amplitude ratio can be applied to the topographic diagnosis of radicular and brachial plexus lesions if a fixed segmental sensory innervation of the hand is accepted. In 44 right handed subjects the amplitude of the sensory evoked potentials at the wrist was significantly larger in the left hand. This asymmetry of sensory innervation between hands could be physiological, and suggests a greater density of sensory innervation in the left hand of right handed subjects.

Spleen rupture in a case of untreated Plasmodium vivax infection.

1 Fundac¸a˜o de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), Manaus, Brazil, 2Universidade do Estado do Amazonas, Manaus, Brazil, 3Barcelona Centre forInternational Health Research (CRESIB, Hospital Clinic–Universitat de Barcelona), Barcelona, Spain, 4 Laboratory of Pathology, Barcelona, Spain, 5 Institucio´ Catalana deRecerca I Estudis Avanc¸ats, Barcelona, Spain

On cytoadhesion of Plasmodium vivax : raison d'être?

It is generally accepted that Plasmodium vivax, the most widely distributed human malaria parasite, causes mild disease and that this species does not sequester in the deep capillaries of internal organs. Recent evidence, however, has demonstrated that there is severe disease, sometimes resulting in death, exclusively associated with P. vivax and that P. vivax-infected reticulocytes are able to cytoadhere in vitro to different endothelial cells and placental cryosections. Here, we review the scarce and preliminary data on cytoadherence in P. vivax, reinforcing the importance of this phenomenon in this species and highlighting the avenues that it opens for our understanding of the pathology of this neglected human malaria parasite.

Intravital microscopy of the spleen: quantitative analysis of parasite mobility and blood flow.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes, the complete development of the parasite in the skin, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints. Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. ETHICS STATEMENT: All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

Chronic Partial Denervation Is More Widespread than Is Suspected Clinically in Paralytic Poliomyelitis

Clinical evaluation, quantitative analysis of the EMG, and motor unit fiber density were carried out on 34 selected patients that suffered paralytic poliomyelitis. 50% of the subjects developed a late and slowly progressive weakness. Automatic analysis of the electromyogram showed a great increase in mean amplitude in weak muscles but also in hypertrophic ones, and in other muscles that had normal strength. Increase in mean amplitude and in motor unit fiber density was greater in the weaker muscles. The increased amplitude ad motor unit fiber density found in clinically unaffected muscles confirms that neurogenic atrophy is more widespread than is suspected clinically. Thus, the late deterioration of function developed in some of the patients always takes place in muscles which are previously damaged and partially depleted in motor units. Widespread neurogenic involvement of the muscles can play an important role in the late deterioration of these patients.

Single fibre electromyography in central core disease

Single fibre electromyography in the extensor digitorum communis muscle was studied in five patients with central core disease. The average number of muscle fibre action potentials belonging to the same motor unit was higher in patients than in healthy subjects of the same age. The increase in motor unit fibre density is consistent with increased terminal innervation ratio described in other papers about central core disease.