Experimental Models Flashcards
Why is malaria still a problem?
- environmental conditions
- poverty, poor housing, lack of infrastructure, economics, wars etc
- vector (mosquito) hard to control
- parasites have short generation time
- rapid parasite evolution in response to intervention eg drug resistance, potential for vaccine escape mutants
How are malaria infections studied in humans?
controlled human malaria infection (CHMI)
used as treatment for neurosyphilis in early 1900 (p. vivax and p. falciparum)
– high fevers caused by malaria killed bacteria pre antibiotics
now used to test drugs and vaccines, and research into malaria immunobiology
infection of human volunteers via infected-mosquito bite or blood passage
– infections are cured by drugs at low parasite density (severe malaria cannot be studied)
What are the advantages of CHMI?
infections are highly controlled and monitored
parasites used are well characterised
detailed evaluation of anti-malarial drug kinetics, accessibility, toxicity, and parasite-killing rate
proof of concept efficacy data for vaccines, including titre and longevity of Ab response and degree of protection conferred
detailed data on immune responses and parasite adaptations
possibility of long follow up, re-infections etc
What are the limitations of CHMI?
hosts differ from main affected global population (african children)
– chmi mostly carried out in naive, healthy adult volunteers
– few trials in malaria endemic regions
little genetic diversity in parasite inoculum (<5 parasite genotypes available for CHMI)
infections can only be studied at v low parasitaemia (outcome if left untreated?)
– no severe malaria
– no chronic infections
obvs cannot study liver stage
What is the course of untreated P. falciparum infection?
- high initial numbers (>10 000 parasite/microlitre)
- numbers soon decrease
- threshold for detection through good RDT/microscopy = >100 parasite/microlitre
– numbers after every burst = threshold for microscopy - threshold for detection through PCR/LAMP = >10 parasite/microlitre
– <10 parasite/microlitre = asymptomatic
What are the advantages of rodent models?
fewer ethical problems than using humans or primates
in vivo
defined genetic background of hosts
- genetics more easily manipulated to test particular cell types/cytokines
direct imaging of host-parasite interactions possible
potential of humanised mice
can study liver infection
What are the limitations of rodent models?
course of infection different to human malaria (lack of or limited recrudescence (reactivation of malaria due to incomplete eradication))
clinical features differ: hypothermia, heart failure
major difference is pathological features: no widespread sequestration of infected RBCs in tissues/organs
differences in immune responses
differences in liver infection compared to humans
– P. berghei can potentially complete cycle in skin fibroblasts at site of inoculation without requiring hepatic cycle
In the context of detection by the immune system, what advantage do mice have over humans?
mice express TLR 11 and 12, which recognises profilin, a protein expressed in protozoan parasites including plasmodium
profilin responsible for motility through skin using actin filaments
Give some info on P. chabaudi.
seen as most similar to human infection; acute infection followed by a resolving chronic infection
– in susceptible mouse strains, pathology/death associated w severe malaria
– pathological features different (limited sequestration of infected RBCs)
many genetically distinct strains of P. chabaudi are available
- investigation of strain specific immunity
- effect of co-infection on development of immune responses
Give some info on P. berghei.
forms acute infection that does not resolve and is lethal
– death due to severe anaemia/organ failure called ‘experimental cerebral malaria’
– pathology is completely different to human cerebral malaria (controversial?)
– sequestration does not occur in mice
easiest malaria parasite to genetically manipulate
Why is P. berghei easy to genetically manipulate?
It has a high transfection efficiency
– DNA only needs to cross the erythrocyte membrane, the parasite plasma membrane, and the nuclear membrane to be transfected
(how is this different from other species?)
How has P. berghei been used in gene identification and vaccine research?
– widely used for analysis of gene function throughout life cycle
– identification of essential genes including those for transmission and development in liver
– preliminary work leading to design of transmission-blocking and attenuated-sporozoite vaccines in humans
What Old World primate is used as a NHPIM, and what strains are used as models for human malaria?
macaque
many basic elements of the immune system are shared
the four known human malaria species (P. falciparum,P. vivax,P. malariaeandP. ovale) do not develop in the blood of macaques
– P. coatneyimodel for P. falciparum
– P. cynomolgias a model forP. vivax
- is also v genetically manipulated
– P. knowlesias a model for both
What Old World primate is used as a NHPIM, and what strains are used as models for human malaria?
Saimiri and Aotus
– P. simium,which is genetically and biologically similar toP. vivax,
– P. brasilianum, which is genetically and biologically similar toP. malariae
new world can be infected w P. falciparum
What are the benefits to using NHPs as models for malaria infection?
able to study relapsing malaria caused by P. vivax hypnozoites (P. cynomolgi in NHPs)
all of the primate plasmodium species and NHP genomes have been sequenced, allowing studies of influences of infection on gene expression of both parasite and host
NHPs can be used to validate studies performed in rodents
* rodents –> NHPs –> humans
peripheral blood B cell subsets and T-cells can be phenotyped and classified similarly between humans and macaques
NHPs can develop severe malaria syndromes similar to humans, such as cerebral malaria and severe anemia
generation of many parasites for use in other experiments
study of CM via MRI brain scans
What are the downsides to using NHPs as models for malaria infection?
obvious ethical concerns
– high cognitive abilities and capacity for suffering
smaller sample sizes
expensive to take care for
New World monkey systems-based research limitations include repeated tissue sampling and volume limitations imposed by their small size (~ 1 kg)
studying chronic infections difficult as it would require monitoring of a single individual over a long period of time
fewer genetic tools and transgenic approaches are available
How was a CHMI study used to look at differences is gene expression between individuals after infection with malaria?
14 volunteers injected w P. falciparum-infected blood
followed for 7-11 days, blood collected every 2 days
3 sets of people found:
– inflammatory = increased gene expression
– suppressive = decreased gene expression
– unresponsive = no change in gene expression
clinical symptoms also varied and matched transcriptional responses
who would go on to develop severe malaria?
Do host immune cells contribute to severe malaria pathology?
no widespread presence of inflammatory cells such as neutrophils and monocytes at sites of sequestration
limited evidence for monocytes or T cells at sites of brain sequestration in fatal human CM
– pronounced feature in experimental CM mouse model
How does the spleen restructure in malaria infection?
mouse models
during infection, open circulation turns into a closed system
– forms closed fibroblast barrier associated w fibronectin and collagen
white pulp become disorganised
– causing loss of B cells in marginal zones
– T and B cells scattered due to loss of GCs
How does haematopoiesis change during malaria infection?
increasing demand for myeloid cells, recuing BM capacity to produce erythrocytes and lymphocytes
loss of B cell progenitors during peak parasitaemia is associated w increased B cell development in spleen
– mouse models show the development of atypical B cells
Can gametocytes be studied in experimental models?
it is common for lab isolates to lose the ability to produce gametocytes in vitro after prolonged cultivation due to the absence of selection pressure to transmit
What are humanised mice?
mice that express human genes or have been engrafted with human tissues
How are humanised mice generated?
transplantation with either primary human hepatocytes or red blood cells under immunodeficiency conditions to prevent xenorejection
What are the benefits to humanised mice?
becoming attractive and suitable models as a potential substitute to NHPs
various organs and or tissues can be transplanted into mice, becoming a more convenient approach to modelling several aspects of malaria parasite biology as well as disease pathogenesis
for example, recent experiments have shown in vivo P. falciparum cytoadhesion
Human liver chimaeric mice are currently available to examine the biology of sporozoite invasion of hepatocytes
What are the current downsides to humanised mice?
still lack sufficient tissue or organ penetration of the engrafted cells due to the hostile mouse microenvironment for foreign human cells to survive and proliferate
the use of immunocompromised mice (SCID) limits the study of certain aspects of host–pathogen interactions such as host immune responses
What is the P. berghei ANKA strain?
an experimental cerebral malaria (ECM) model, in which mice develop similar neurological features to human CM
– paralysis, coma, and death
What are the differences between ECM and human CM?
sequestration of iRBCs can occur in the lung but accumulation in the brain does not correlate w pathogenesis
– use of a different P. berghei strain (NK65) shows occlusion of brain micro-vessels
However, desth in human CM is caused by brain swelling depressing brain stem which regulates breathing
– not been shown in ECM
ECM also appears to be largely an inflammatory syndrome
How has the liver stage in mice been used to come up w new treatments?
Recently, it has been demonstrated that immunization of mice with late-stage arresting sporozoites that lack a type II fatty acid synthesis enzyme (Pyfabb/f−) result in robust humoral as well as CD4 T-cells immune responses capable of blocking follow-up challenges with wild-type sporozoite
What type of research are humanised mice used in?
used for pathogenesis rather than immune response
– however, mice transplanted with immune system components (CD4 and CD8 T cells, B cells) are being developed to combat this limitation
How have in vitro assays been used?
used in exploratory evaluation of inhibitory activities of immune sera raised against sporozoites infecting cultured human hepatocytes
absence of the complex three-dimensional architecture of the ideal liver microenvironment always limit their physiological relevance
Can humanised mice replicate human disease pathology?
Liver chimaeric mice which can sustain a low-level parasitaemia in the blood if transfused with human red blood cells
– parasites demonstrate tissue sequestration as hypnozoites
parasites are only maintained for short time due to rapid clearance of hRBCs
