Severe malaria infections cause almost 1 million deaths annually, mostly among non-immune African children. The pathogenesis of severe malaria is poorly understood. It is increasingly appreciated that while host innate immune responses such as inflammation and phagocytosis are critical for control of parasite replication, they can become dysregulated and contribute to severe disease. The goals of this work were: (1) to characterize inflammatory responses to malaria by defining their relationship to phagocytosis and identifying novel molecular mediators, and (2) to evaluate the utility of biomarkers of inflammation and other host responses for predicting outcome in severe malaria infection. Using an in vitro model of the malaria-macrophage interaction, inflammatory and phagocytic responses to Plasmodium falciparum were found to be partially coupled. Activation of Toll-like receptors (TLRs) by purified parasite components increased internalization of parasitized erythrocytes, but uptake of parasitized erythrocytes did not require TLRs, nor did it trigger cytokine production via TLRs or other receptors. Two candidate molecules – Triggering receptor expressed on myeloid cells-1 (TREM-1) and Chitinase-3 like-1 (CHI3L1) – did not appear to critically modulate inflammation to malaria in vitro or in murine models. However, exogenous TREM-1 activation enhanced the pro- inflammatory nature of the response to P. falciparum, with potential implications for malarial-bacterial co-infection. CHI3L1-deficient mice showed a trend towards earlier death in experimental cerebral malaria, suggesting that CHI3L1 may protect against severe malaria; however, further investigation in more informative models is required. Admission levels of plasma TREM-1, CHI3L1, and other biomarkers of inflammation and endothelial activation were increased in Ugandan children with severe malaria. Simple combinations of these biomarkers predicted mortality among severe malaria patients with high accuracy, warranting larger validation studies. Taken together, these findings identify host responses as putative targets for adjunctive therapies, and suggest the utility of host biomarker combinations as prognostic tests for severe malaria.

Severe malaria infections cause almost 1 million deaths annually, mostly among non-immune African children. The pathogenesis of severe malaria is poorly understood. It is increasingly appreciated that while host innate immune responses such as inflammation and phagocytosis are critical for control of parasite replication, they can become dysregulated and contribute to severe disease. The goals of this work were: (1) to characterize inflammatory responses to malaria by defining their relationship to phagocytosis and identifying novel molecular mediators, and (2) to evaluate the utility of biomarkers of inflammation and other host responses for predicting outcome in severe malaria infection. Using an in vitro model of the malaria-macrophage interaction, inflammatory and phagocytic responses to Plasmodium falciparum were found to be partially coupled. Activation of Toll-like receptors (TLRs) by purified parasite components increased internalization of parasitized erythrocytes, but uptake of parasitized erythrocytes did not require TLRs, nor did it trigger cytokine production via TLRs or other receptors. Two candidate molecules – Triggering receptor expressed on myeloid cells-1 (TREM-1) and Chitinase-3 like-1 (CHI3L1) – did not appear to critically modulate inflammation to malaria in vitro or in murine models. However, exogenous TREM-1 activation enhanced the pro- inflammatory nature of the response to P. falciparum, with potential implications for malarial-bacterial co-infection. CHI3L1-deficient mice showed a trend towards earlier death in experimental cerebral malaria, suggesting that CHI3L1 may protect against severe malaria; however, further investigation in more informative models is required. Admission levels of plasma TREM-1, CHI3L1, and other biomarkers of inflammation and endothelial activation were increased in Ugandan children with severe malaria. Simple combinations of these biomarkers predicted mortality among severe malaria patients with high accuracy, warranting larger validation studies. Taken together, these findings identify host responses as putative targets for adjunctive therapies, and suggest the utility of host biomarker combinations as prognostic tests for severe malaria.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 204-240).

Η κινάση εστιακής προσκόλλησης (FAK) είναι μια πρωτεϊνική κινάση τυροσίνης (nRPTK) με κυτταροπλασματική κατανομή στην περιφέρεια του κυττάρου. Εντοπίζεται κυρίως σε περιοχές του κυττάρου γνωστές ως εστίες προσκόλλησης, με τις οποίες το κύτταρο προσκολλάται στην εξωκυτταρική ύλη. Συμμετέχει σε σημαντικές κυτταρικές διεργασίες όπως στην κυτταρική κίνηση και μετανάστευση, στον κυτταρικό πολλαπλασιασμό, στην κυτταρική επιβίωση και απόπτωση. Η FAK ενεργοποιείται από πολλά ερεθίσματα που μπορούν να επάγουν φωσφορυλίωση της σε αμινοξέα τυροσίνης (Υ397, Υ576, Υ577, Υ861, Υ925) όπως για παράδειγμα αυξητικοί παράγοντες, νευροπεπτίδια, παράγοντες που ενεργοποιούν υποδοχείς συνδεδεμένους με G-πρωτεΐνες και μηχανικά ερεθίσματα αλλά κυρίως μετά από τη διέγερση των ιντεγκρινικών υποδοχέων των κυττάρων. Ο ρόλος της FAK στην μεταγωγή μηνυμάτων, στο πλαίσιο της κυτταρικής επικοινωνίας είναι διττός: δρα ως κινάση φωσφορυλιώνοντας τα υποστρώματα της αλλά και ως πρωτεΐνη συνδετήρας (adaptor protein) δημιουργώντας πολυπρωτεϊνικά σηματοδοτικά σύμπλοκα. Δεδομένης, λοιπόν, της ιδιαίτερης φύσης του μορίου, η FAK θα μπορούσε να χαρακτηριστεί ως ενεργοποιούμενη πρωτεΐνη ικρίωμα (activable scaffold protein). Στην παρούσα εργασία μελετήσαμε την ενεργοποίηση της FAK κατά την κυτταροφαγία του βακτηρίου E. coli. Έτσι, χρησιμοποιήθηκαν φαρμακολογικοί αναστολείς για να ανιχνευθεί η συμμετοχή τελεστών της μεταγωγής σημάτων στην ενεργοποίηση της FAK. Επιπλέον, διερευνήθηκε με ανοσοκατακρημνίσεις η συμπλοκοποίηση της FAK με πρωτεΐνες-κλειδιά σημαντικών κυτταρικών μονοπατιών σε κύτταρα που βρίσκονταν σε εναιώρημα. / Cells respond to extracellular stimuli with the recruitment of multiple cytoskeletal and regulatory proteins, which form specialized complexes. These complexes are usually involved in the promotion of integrin-mediated signals to the nucleus. In this study, we investigated whether any signal transduction complexes are constitutively present, in Medfly hemocyte suspension, in the absence of external stimuli. Hemocytes from the 3rd instar larvae were isolated and protein crude extracts were prepared. In hemocyte lysates, the presence of FAK (focal adhesion kinase), Integrin β1 and the adaptor protein Paxillin was identified with immunoprecipitation and immunoblot analysis. The presence of these proteins was also confirmed with immunofluorescence microscopy, in attached hemocytes on glass slides. Co-immunoprecipitation with FAK and immunoblot analysis with anti-Paxillin, anti-tubulin, anti-actin and anti-ERK revealed the complex formation of FAK with Paxillin, Tubulin, Actin and ERK in hemocyte suspensions. The profiles of this complex and of each one of these proteins, separately, varies, during development and phagocytosis of E.coli. Consequently, it was demonstrated that FAK forms complex with cytoskeletal proteins like paxillin, tubulin, actin and signalling proteins such as ERK, in Medfly hemocytes, without the influence of any extracellular stimuli.

H κινάση εστιών προσκόλλησης (FAK) συμμετέχει στη μεταγωγή μηνυμάτων κατά την κυτταροφαγία. Στα αιμοκύτταρα των εντόμων υπάρχει σε διαφορετικές ποσότητες κατά την ανάπτυξη. Ενεργοποιείται με φωσφορυλίωση στην Tyr-397. Kατά την κυτταροφαγία του βακτηρίου Ε. coli ενεργοποιείται άμεσα. Ανάμεσα στα μόρια που σχηματίζουν σύμπλοκο με την FAK είναι και οι πρωτείνες pinch, Src και οι ΜΑΡΚ. Κάποια από τα παραπάνω σχηματίζουν σύμπλοκο κατά την κυτταροφαγία ενώ για άλλα το σύμπλοκο προυπάρχει. Ο κυτταροσκελετός ακτίνης και τουμπουλίνης χρειάζονται για την κυτταροφαγία, όπως χρειάζεται και η έκκριση. Τέλος διαπιστώθηκε πως κατά την κυτταροφαγία του βακτηρίου Ε. coli και του πεπτιδίου RGD, συμμετέχουν οι πρωτείνες Src, Ras, Rho και JNK. / Focal adhesion kinase (FAK) participates in signal transduction at phagocytosis. Insect hemocytes have FAK in different mounts during development. FAK becomes activated after phosphorylation at Tyr-397. During phagocytosis of E. coli, FAK becomes immediately activated. Pinch, Src and MAPK are some of the molecules which are in complex with FAK. Some of these molecules are in complex with FAK during phagocytosis, but some others there are in complex at any time. Phagocytosis, in order to happen, needs actin and tubulin cytoskeleton. Secretion is also needed for this purpose. Finally, we found that the proteins Src, Rho, Ras and JKN participate in phagocytosis of the RGD peptide and in phagocytosis of the microbe Escherichia coli.

No description available.

1. INTRODUCTION The incidence of diseases whose pathogenesis chain includes changes in the functioning of the immune system is currently on the increase. The increasing prevalence of primary and secondary immunodeficiencies is a relevant issue in immunopathology. Secondary immunodeficiencies, caused by such factors as bacteria, viruses, helminths, chemical substances, medications, radiation, stress, etc., result in higher morbidity. Immunosuppression occurs in cases of oncological or allergic diseases, bronchial asthma, etc. immunosuppression is relevant in cases of malignant tumors whose incidence in Lithuania has been increasing with every year during the recent period. Oncological treatment involves the application of radiotherapy and chemotherapy with cytostatics that are strong immunosuppressants, and thus the restoration of the immune system with immunostimulants is becoming a relevant issue. Insufficient nutrition, disturbed nutrient resorption, negative nitrogen balance, chronic alcoholism, and drug abuse – all these factors result in the deceleration of cell proliferation and decreased T-lymphocyte counts and function. Immunodeficiency increases morbidity with infectious diseases, since the organism becomes incapable of the rapid destruction of the causative agents. Such conditions increase the probability of the spreading of the infection, and condition the increased activity of the opportunistic infections caused by adenoviruses, cytomegaloviruses, etc... [to full text]

Mature dendritic cells (DC) isolated from lymphoid tissues initiate antigen-specific T-dependent responses even though they are non-phagocytic and weakly pinocytic, whereas Langerhans cells (LC; immature DC) can process protein antigens but are poorly immunostimulatory. Thus antigens may be acquired by cells of this lineage at an immature stage but, to our knowledge, there have been no studies on the phagocytic capacity of these cells in vitro. Using a newly-developed flow cytometric assay to measure the association between fluorescent markers and LC in epidermal cell cultures, and light and electron microscopy, we have observed phagocytosis of a variety of particles by freshly-isolated LC. The cells readily phagocytosed zymosan, heat-killed S. cerevisiae, bacteria (S. aureus and C. parvum) and fluorescent latex beads, but were unable to take up IgG- or complement-coated sheep erythrocytes, as opposed to MØ. Similarly, many freshly-isolated splenic DC had some phagocytic activity. However, the capacity of both LC and splenic DC to phagocytose zymosan, bacteria and fluorescent latex beads was markedly decreased after maturation in culture, consistently with the fact that mature DC are poorly phagocytic. Zymosan binding and uptake were much greater in fresh LC from C57BL/6 compared to BALB/c mice, and the loss of phagocytic capacity for zymosan during maturation followed different kinetics in the two strains. Two receptors mediating uptake of zymosan in LC were identified based on the effect of different inhibitors. Both of these receptors, recognising mannose and β-glucan residues, appear to be differentially regulated in the two mouse strains and during culture of LC. Our findings support the notion that DC are capable of acquiring particulate antigens for presentation at an immature stage, through recognition units for carbohydrate determinants common to a variety of potentially pathogenic organisms.

Malaria has exerted a major selective pressure for red blood cell (RBC) polymorphisms that confer protection to severe disease. There is a predominance of blood type O in malaria endemic regions, and several lines of evidence suggest that the outcome of Plasmodium falciparum infection may be influenced by ABO blood type antigens. Based on observations that enhanced phagocytosis of infected polymorphic RBCs is associated with protection to malaria in other red cell disorders, we hypothesized that infected type O RBCs may be more efficiently cleared by the innate immune system than infected type A and B RBCs. The present work demonstrates human macrophages in vitro and murine monocytes in vivo phagocytosed P. falciparum infected O RBCs more avidly than infected A and B RBCs independent of macrophage donor blood type. This difference in clearance may confer relative resistance to severe malaria in individuals with blood type O.

Malaria has exerted a major selective pressure for red blood cell (RBC) polymorphisms that confer protection to severe disease. There is a predominance of blood type O in malaria endemic regions, and several lines of evidence suggest that the outcome of Plasmodium falciparum infection may be influenced by ABO blood type antigens. Based on observations that enhanced phagocytosis of infected polymorphic RBCs is associated with protection to malaria in other red cell disorders, we hypothesized that infected type O RBCs may be more efficiently cleared by the innate immune system than infected type A and B RBCs. The present work demonstrates human macrophages in vitro and murine monocytes in vivo phagocytosed P. falciparum infected O RBCs more avidly than infected A and B RBCs independent of macrophage donor blood type. This difference in clearance may confer relative resistance to severe malaria in individuals with blood type O.