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Pathology- Experimental and clinical evidence. Hilary term 2014 Malaria:?It is caused by four different types of PLASMODIA: vivax, ovale, malariae, and falciparum. The first and last are the most common causes. The VECTOR for the plasmodia is the FEMALE ANOPHELES MOSQUITO. Clinical: MOST infections with malaria-causing agents are CLINICALLY SILENT, reflecting the ability of adaptive immune mechanisms to prevent disease. In NONIMMUNE individuals, however, infections are more clinically overt, and a minority of these can become SEVERE OR LIFE THREATENING, manifesting a range of discrete and overlapping disease syndromes of complex aetiologies. Those dying of malaria can have multiple-organ or systemic involvement. ? Overall patterns of disease depend markedly on the AGE and the PREVIOUS IMMUNOLOGICAL EXPERIENCE of the host, and TRANSMISSION DYNAMICS: in areas of high malaria transmission, the burden of disease is borne by YOUNG CHILDREN (who have NOT YET DEVELOPED IMMUNITY); life-threatening disease in this setting typically consists of metabolic acidosis (which leads to respiratory distress), cerebral malaria and severe malarial anaemia. However, in areas of lower transmission, primary infections might occur in ADULTHOOD. ? Moreover, HOST GENETICS and IMMUNOLOGICAL RESPONSES determine the individual OUTCOME.??
UNCOMPLICATED MALARIA symptoms can be non-specific and easily missed by clinicians. ? Fever (spike can reach 41C), chills headache, weakness, vomiting and diarrhoea. ? The FEVER, CHILLS and SWEATS are experienced PERIODICALLY in a sort of cyclical cycle. INCUBATION TIME is about 2 weeks. SPLENOMEGALY seen in most patients, HEPATOMEGALY in about a third, and ANAEMIA is common. If left UNTREATED, malaria caused by P. falciparum is potentially lifethreatening as a result of extensive BRAIN (cerebral malaria) and KIDNEY damage, SEVERE ANAEMIA and ACUTE RESPIRATORY DISTRESS SYNDROME. ? Malaria caused by the OTHER 3 SPECIES is usually self-limited.People who are HETEROZYGOUS for SICKLE CELL ANAEMIA are PROTECTED because their RBCs have too little ATPase activity.Life-cycle: there are TWO PHASES. 1) The sexual cycle which occurs primarily in the MOSQUITOES and 2) the asexual cycle which occurs in
HUMANS (these are the intermediate hosts). However, the sexual cycle is INITIATED in humans due to the formation of the gametocytes within the RBCs- however this is COMPLETED in
? This INTERDEPENDENCE means that the mosquito is not a "flying syringe"; there is an OBLIGATORY LIFE-CYCLE within the vector. ? The parasite that goes in in the vector is NOT THE SAME as the one that come out. The parasite enters the BLOOD from a mosquito bite- it originates from the mosquitoes SALIVARY GLANDS. At this stage it is in a SPECIFICALLY DIFFERENTIATED FORM that is "injectable" - sporozoite; i.e. it has gone through the obligatory stage of the life-cycle WITHIN THE MOSQUITO. It then makes its way IMMEDIATELY to the liver. These liver-stage parasites, schizonts, then undergo a SERIES OF ASEXUAL MULTIPLICATIONS (extraerythrocytic schizogony) over the next 14 days, resulting in thousands of merozoites which BURST from the hepatocyte. The released merozoites have an ACTIVE MECHANSIM for INJECTION into RBCs, and infect them. The organism then DIFFERENTIATES into a ringshaped trophozoite; this grows into an AMEBOID FORM, and an EQUIVALENT series of ASEXUAL multiplication cycles (erythrocytic schizogony) are initiated within these cells, which produces a SCHIZONT filled with new INFECTIVE MEROZOITES, at which point the cells burst and the infective cycle begins anew.
? The PERIODIC RELEASE of merozoites from RBCs (the erythrocytic stage takes about 48 hours) in this way causes the typical RECURRENT symptoms of chills, fever and sweats described above. I.e. the blood-stage parasites are responsible for the CLINICAL MANIFESTATIONS of the disease. Some merozoites in RBCs then DIFFERENTIATE into MALE GAMETOCYTES and others into FEMALE GAMETOCYTES. ? These have no other function other than hanging around in the blood WAITING TO BE TAKEN UP in a MOSQUITO BITE. Once both the male and female gametocytes and INGESTED by the female anopheles mosquito, the male one undergoes a RAPID NUCLEAR DIVISION, producing 8 flagellated microgametocytes which PENETRATE the female gametocyte in the mosquito GUT, generating zygotes. The zygote (or once MOTILE and ELONGATED known as ookinetes) then ENCYSTS on the exterior of the gut wall, at this stage being known as an oocyst. ? It then RUPTURES, releasing hundreds of sporozoites into the mosquito body cavity, where they eventually migrate to the mosquito SALIVARY GLAND. ? This part of the life-cycle is called the sporogenic cycle.
? At some points during the life-cycle it's ONLY A FEW CELLS that establish the next stage of the infection whereas at other points it's MANY MORE. ? These "pinch points" are relevant for VACCINE DEVELOPMENT. the mosquitoes as this is the site where the two games FUSE.????Pathogenesis and immune evasion: The HETEROGENEITY of the syndromes might arise from: the SITE-SPECIFIC LOCALIZATION of
parasitized red blood cells among target organs; the local and systemic action of BIOACTIVE PARASITE PRODUCTS (e.g. toxins); the local and systemic production of PRO-INFLAMMATORY cytokines and chemokines by the innate and adaptive immune systems in response to PARASITE PRODUCTS; and the resultant activation and recruitment of INFLAMMATORY CELLS. ? According to this view, diverse organ-specific or systemic disease syndromes are end-stage processes of ATYPICAL INFLAMMATORY CASCADES that are initiated in target organs by pathogen products and are maintained by infiltrating cells through positive-feedback cycles. In most cases, immune homeostasis through Treg cells and antiinflammatory mediators corrects the cascade effect, and responses are adequately downregulated. ? Indeed, the CHRONIC TOLERANCE built up in areas of endemic exposure causes INCREASED SUSCEPTIBILITY to other parasites e.g. Salmonella and Epstein-Barr, indirect burden of malaria. In severe disease, however, a 'run-away' effect can ensue, with FATAL CONSEQUENCES. Appropriate regulation of immune responses might therefore be a key to healthy outcomes, and UNDERSTANDING these processes might aid in the development of VACCINE-BASED INTERVENTIONS. The parasite forms KNOBS on the surface of RBCs, these are formed from Plasmodium falciparum erythrocyte membrane protein 1 (EMP1). ? This acts to ADHERE to both cell-surface RECEPTORS and ADHESIONAL MOLECULES (such as ICAM1, CD36, CD31) as well as ECM COMPONENTS. ? It appears that it is REMODELLING the surface of the RBC such that it can ADHERE to capillaries and thus stay in and accumulate in the PERIPHERAL CAPILLARIES (indeed in CEREBRAL MALARIA the infected red blood cells can OBSTRUCT the capillaries in the brain. ? By doing this, the parasite can AVOID host 'CLEANSING MECHANISMS' e.g. in the SPLEEN which would otherwise REMOVE RBCs with compromised deformability or altered antigenicity. ? Can lead to OBSTRUCTION of the MICROCIRCULATION.
? These are clearly going to be ANTIGENIC as remodelling the RBC membrane involves some of the proteins being EXPOSED; thus the pathogen is not just "hiding away" from the immune system. INSTEAD the pathogen deals with this problem by evolving 50 TYPES of the genes encoding PFEMP1, and ONLY ONE IS EXPRESSED at any one time. ANTIGENIC VARIATION. ? Thus this is a NEAT WAY in which the pathogen deals with the TRADEOFF apparent within its life-cycle strategy ? avoiding the cleansing action of the SPLEEN and simultaneously avoiding being OVERLY ANTIGEN in the blood either.
? The parasite lifecycle has evolved to allow it to rapidly select for advantageous mutations. All of the HUMAN infective stages are HAPLOID - immediate expression of all change of function mutations. Inside-host replication of blood stage parasites occurs INTRACELLULARLY and allows
RAPID EXPANSION of favourable strains. The SEXUAL stage of the parasite which is obligatory therefore allows for EXTENSIVE RECOMBINATION, especially combined with the fact that many infections are mixed-clone. (Except in extreme cases of population bottlenecking).
? Despite this antigenic variation, those antigens expressed on the parasite SEXUAL STAGES within the mosquito are minimally POLYMORPHISTIC ?
presumably reflects the less advanced immune mechanisms of the mosquito. The SPEED of the life cycle also makes it difficult for the immune system to KEEP UP. In between the main stages of the life-cycle, which are INTRACELLULAR and so offer ANTIBODY PROTECTION, the transition between each of these stages, during which time the parasite is EXPOSED to humoral factors, is very rapid. ? It takes the parasite 10-15 mins to migrate from the SKIN TO the LIVER, whilst a new generation of MEROZOITES invade a RBC in less than 30s. ? This is VERY SHORT in relation to the 7-14 days it takes to develop an effective, antigen-specific immune response. RBCs are a NIFTY CHOICE since they DO NOT express MHC class I, meaning that the CTL-response is essentially REDUNDANT during the erythrocytic stage. Most of the PATHOLOGICAL findings in malaria relate to the DESTRUCTION of RBCs - these are destroyed both during their RUPTURE and release of merozoites but also by the action of the SPLEEN.
? Note that the AGGREGATION of RBCs obstructing the MICROVASCULATURE (and thus the virulence factor mentioned above) is unique to Plasmodium falciparum infection ? this causes ANAEMIC and INFLAMMATORY symptoms, and is particularly pathological since it CONCENTRATES the parasites IN VARIOUS TARGET ORGANS (depending on the type of EMP expressed).
??????????CHLOROQUINE is the drug of choice for ACUTE MALARIA caused by SENSITIVE STRAINS- it targets the pathogen in its BLOOD STAGE. As a WEAK BASE, it is uncharged at neutral pH whilst carrying a positive charge at acidic pH ? owing to this property it is SELECTIVELY ACCUMULATED inside lysosomes. The intracellular trophozoite feeds on the HAEMOGLOBIN of the RBC that serves as a source of AAs. Digestion of the globin protein takes place inside the Plasmodium lysosome resulting in the generation of FREE HAEM. Normally this is insoluble and precipitates in the form of a black pigment inside the lysosomes; however CHLOROQUINE in the lysosome INTERFERES with pigment formation leading to the accumulation of free haem which is HIGHLY TOXIC to the parasite. ? However, RESISTANCE has become a problem.
??????????RESISTANCE to CHLORQUINE by P. falciparum is conferred by mutations in the conserved gene for the the P. falciparum chloroquine resistance transporter (PfCRT) that is expressed on the DIGESTIVE VACUOLE MEMBRANE. Drug efflux by the mutated molecule represents an EXPANSION OF THE NORMAL PHYSIOLOGICAL FUNCTION of PfCRT, which may include transport of amino acids or short polypeptides from the digested hemoglobin.
??????????? Malaria caused by this species is INSTEAD treated using ARTEMISININCOMBINATION THERAPIES (the 'combination' acting to prevent the onset of RESISTANCE): there is controversy as to its true MECHANISM of ACTION, but
some consensus attributes it to a DISRUPTION of REDOX HOMEOSTASIS in the parasites.
??????????Clinical episodes of SEVERE malaria have recently been associated with expression of var genes that encode specific PfEMP1s. The same var genes were INDEPENDENTLY OBSERVED to bind brain endothelium in vitro (role in cerebral malaria?). Once the crucial var LIGAND and its endothelial RECEPTOR have been identified, screening could be used to IDENTIFY small molecules that BLOCK infected erythrocytes from BINDING TO OR ACTIVATING MICROVASCULAR ENDOTHELIUM through this pathway.
? ????????? The vaccine challenge and potential solutions:Vaccine developed needs ideally to be SIMPLE, CHEAP and STABLE as the places where it is needed: most are POOR and RURAL with poor infrastructures.The various stages of the malaria parasite lifestyle elicit differing protective immune mechanisms as each is associated with different polymorphic antigens.
? The PRECISE MECHANISM of natural immunity to malaria (in response to chronic infection) is unclear: it is thought to be against the blood borne stages of the pathogen, but T CELL RESPONSES against the LIVER-BORNE initial infection are also seen. Moreover, is the naturally acquired immunity WORTH EMULATING? ? It develops SLOWLY and DOES NOT result in sterile protection, suggesting that maybe we should look to eliciting OTHER TYPES of RESPONSE.
? Overall, this makes vaccine development harder as the exact type of response that should be generated for best protection is unclear.What is the relative importance of different arms of the immune system during infection, and does this BALANCE CHANGE on subsequent infections? ? Each of the different stages may present as different windows of opportunity for VACCINES; however each intervention in isolation may INCREASE the SUSCEPTIBILTY of the host to a stage further down the life-cycle. ? Thus replicating the immune response as a WHOLE should be a priority.? We know that acute, proinflammatory, cytokine-mediated effector responses from both the innate and effector arms of the immune system can LIMIT THE INITIAL REPLICATION PHASE of blood-stage parasites and reduce direct damage to the host, such as hemolysis and erythrocyte degradation. But these responses need to be QUICKLY CONTROLLED by anti-inflammatory mechanisms to prevent immunopathology, notably by IL-10-secreting CD4 + T cells. EVENTUAL CONTROL or CLEARANCE of the PARASITAEMIA, however, depends on antibody-mediated responses, which become more effective over time, presumably because of the gradual acquisition and affinity maturation of ANTIBODIES recognizing prevalent parasite genotypes. Generating this precise TEMPORAL PRECISION in the immune response with a vaccine, including an antibody response of SUFFICIENT QUANTITY and QUALITY to provide longterm protection is a challenge. ? This will probably only be resolved when we have a better understanding of the precise role that T H cells have in shaping antimalarial B cell responses and B cell memory, as well as the factors that are important for the generation of LONG-LIVED PLASMA CELLS.Could induce a CTL RESPONSE at the PRE-ERYTHROCYTIC stage where the parasite infects the HEPATOCYTES. However, this would require the parasite to GET OUT of the vacuole and reach the CYTOSOL so that its peptides could be presented.? Proof-of-concept of VACCINE POTENTIAL at PRE-ERYTHROCYTIC STAGE: if you irradiate mosquitoes, the parasites inside become ATTENUATED, though because they can still INFECT they can elicit IMMUNITY. ? This worked-
"SANARIA" - however the problem was that it had to be stored in LIQUID NITROGEN and given INTRAVENOUSLY, meaning it was DIFFICULT to DEPLOY in the developed countries where it was most needed. The RTS,S vaccine was engineered using the CIRCUMSPOROZOITE PROTEIN of Plasmodium falciparum malaria parasite as an antigen, in addition to HBsAg (which SELF-ASSEMBLES into a virus-like particle, enabling CSP to be expressed on the surface) and a chemical adjuvant to boost the immune system response. This aims to PREVENT INFECTION by inducing high ANTIBODY TITERS that block the parasite from infecting the liver. ? Was found to elicit 30% PROTECTION, but this only lasted a YEAR. DNA VIRUSES - where you immunize with a VECTOR whereby the protein antigen is then EXPRESSED IN VIVO. Needs a STRONG PROMOTOR. ? Elicits CTL and antibody responses. Could these SYNERGISE with a SPOROZOATE VACCINE?
CONTROLLED HUMAN MALARIA INFECTION (CHMI): this is done with a falciparum strain that is chloroquine-sensitive, and the BLOOD-STAGE PARASITAEMIA is monitored by highly sensitive RT-PCR; as soon as the parasite is detected in the blood it is treated with ANTI-MALARIALS The problem with an ANTI-MEROZOITE VACCINE is that extremely high antibody concentrations are needed: this is thought to be due to there ONLY BEING A MINUTE AVAILIABLE before the parasite infects the next RBC. Also there is ANTIGENIC POLYMORPHISM. TRANSMISSION-BLOCKING VACCINES - point being the mosquito TAKES UP THE ANITBODIES directed against antibodies expressed during the SEXUAL STAGE during the blood meal. ? Thus could BLOCK the transmission-cycle but wouldn't stop the person from contracting malaria. Balance of priorities between STOPPING TRANSMISSION and PROTECTING THE INDIVIDUAL.
?VECTOR-BORNE DISEASE transmitted by SANDFLIES and caused by the obligate intracellular protozoa of the genus Leishmania. Clinical features: -- CUTANEOUS FORMS where skin ulcers can form on exposed areas such as the face, arm and legs. - DIFFUSE cutaneous leishmaniasis produced disseminated and chronic skin lesions resembling those of lepromatous leprosy. MUCOCUTANEOUS FORMS can cause lesions that partially/totally DESTROY the mucous membranes of the nose, mouth and throat cavities. VISCERAL LEISHMANIASIS is characterised by high-fever, substantial weight-loss, swelling of the SPLEEN and LIVER, and anaemia. (If left untreated this last form has almost a 100% FATALITY RATE within two years. (Patients with VL should be evaluated for HIV co-infection). The strategy of the pathogens like Leishmania to avoid destruction by the immune system and thrive is by 'hiding' inside a cell. This location enables it to avoid the action of the humoral immune response (because the pathogen is safely inside a cell and outside the open bloodstream), and furthermore it may prevent the immune system from destroying its host through nondanger surface signals which discourage apoptosis. The primary cell types Leishmania infiltrates are phagocytotic cells such as neutrophils and macrophages. Usually, a phagocytotic immune cell like a macrophage will ingest a pathogen within an enclosed endosome and then fill this endosome with enzymes which digest the pathogen. However, in the case of Leishmania, these enzymes have no effect, allowing the parasite to multiply rapidly. This uninhibited growth of parasites eventually overwhelms the host macrophage or other immune cell, causing it to die. Transmitted by the sandfly, the protozoan parasites of the genus Leishmania major may switch the strategy of the first immune defense from eating/inflammation/killing to eating/no inflammation/no killing of their host phagocyte' and corrupt it for their own benefit. They use the willingly phagocytosing polymorphonuclear neutrophil granulocytes (PMN) rigorously as a tricky hideout, where they proliferate unrecognized from the immune system and enter the long-lived macrophages to establish a "hidden" infection. By a microbial infection PMN move out from the bloodstream and through the vessels' endothelial layer, to the site of the infected tissue (dermal tissue after fly bite). They immedia tely start their business there as the first immune response and phagocytize the invader because of the foreign and activating surfaces. In that processes an inflammation emerges. Activated PMN secrete chemokines, IL-8 particularly, to attract further granulocytes and stimulate them to phagocytosis. Furthermore Leishmania major increases the secretion of IL-8 by PMN. In the parasites case, that
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