PlasmodiaMesquitoHuman Life Cycle

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Genetics in the news.
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Malaria

• Malaria has long been recognized as an important
  parasitic disease of humans, having been
  described by the early Egyptians in the third
  millennium B.C., 
• Despite the introduction of control programs in
  many parts of the world over the past few
  decades, the impact of malaria on human
  populations continues to increase.  Recent
  estimates suggest,
• (1) that 1.5 billion persons live in areas of the
  world where malaria is an endemic disease,
• (2) that the number of infected humans exceeds
  500,000,000, and,
• (3) that 1-3 million persons die each year.

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Malarias Range(cross hatching)
Science Volume 289, Number 5485 (2000), p. 1763
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LINKS BETWEEN MALARIA AND POVERTY

• Poverty affects malaria. Communities with low
  incomes, limited education and poor access to
  health care are least able to engage in malaria
  control activities.
• Malaria affects poverty. In poor households, a
  greater proportion of income is likely to be
  spent on malaria treatment than in affluent
  households,
• malaria illness causes absenteeism from work and
  school, poor scholastic performance, lack of
  labor for cultivation, and a decline in child
  care, etc.
• Hence, a negative spiral can develop with malaria
  causing and deepening poverty which, in turn,
  exacerbates inequalities in societies.

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General Malaria Pathologymore later.

• Host (human) inflammatory response resulting in
  severe chills and fever,
• paroxysms,
• Anemia due to loss of red blood cells,
• Recurrence of paroxysms at periodic intervals.

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Causative Agent Plasmodium sp. (Protozoa
Haemosporina)

• These single celled eukaryotes have heteroxenous
  life cycles with both vertebrate and invertebrate
  hosts,
• Humans,
• Mosquitos.
• Macro- and micro- gametes develop independently
  and the resultant zygote is motile and is called
  the Ookinete.

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Malarial Plasmodium

• There are four important species that infect
  humans, causing malaria
• P. vivax - benign tertian malaria - accounts
  for 43 of cases
• P. falciparum - malignant tertian malaria
  accounts for 50 cases
• P. malariae - quartan malaria - accounts for
  7 of cases
• P. ovale - mild tertian malaria - accounts for
  

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P. vivax - benign tertian malaria

• The disease gets its name from the time between
  paroxysms, which occur every 48 hours,
• from the Roman custom to call the first day of
  the event day 1 and therefore 48 hours later
  would be day 3,
• Tertian malaria accounts for 43 of all cases of
  malaria.
• This species is mainly found in Asia and relapses
  have been known to occur up to 8 years after the
  first infection due to the presence of the
  dormant exoerythrocytic stage also known as
  hypnozoites.
• P. vivax are only capable of infecting red blood
  cells via genetically determined receptor sites.
  Only people exhibiting these antigenic sites are
  susceptible to the disease.

P. ovale - mild tertian malaria similar
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Duffy Blood Group

• Human populations express two dominant alleles,
  signified as Fya and Fyb,
• the expressed glycoproteins are recognized by
  their respective antigens on the erythocyte cell
  membranes,
• A third allele Fy has no antigen associated with
  it.
• The Fy/Fy genotype appears in about 40 of the
  African population as opposed to less than 0.1
  in European populations.
• Caucasians have Fya//Fyb genotypes and therefore
  express the antigen on the surface of the red
  cell to which the plasmodium can bind, allowing
  it to infect the cell.

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Tertian Malaria

• The paroxysms commence with the patient having a
  feeling of intense cold (15 min - 1 hour),
• this is due to a rapid rise in body temperature
  to 104-106o F,
• accompanied with violent shivering,
• often with nausea and vomiting.
• Followed by the hot phase, which includes
  headache and often delirium (2 - 6 hours).
• The fever breaks with the copious production of
  perspiration. The body temperature drops back to
  normal after 2 - 4 hours. The patient may sleep
  for 8-12 hours and feel well until the next
  paroxysm.

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P. falciparum - malignant tertian malaria

• The time between the onset of paroxysms is 48
  hours, but the period of fever is prolonged,
  lasting from 24-36 hours,
• The course of this disease is very rapid and it
  is not uncommon to see more than 60 of a
  patient's red blood cells infected.
• When the number of infected cells rises above 25
  the disease is usually fatal, in spite of
  treatment.

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Falciparum Malaria

• In Falciparum malaria the paroxysms last longer
  20-36 hours and so the patient does not recover
  before the onset of the next bout.
• In 10 of P. falciparum cases patients develop
  cerebral malaria, 80 of which result in death.
• characterized by headaches followed by the
  patient falling into a coma, often with
  convulsions.
• This is particularly common in children.
• Ultimately death ensues with the patients
  temperature reaching up to 108 F.

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P. malariae - quartan malaria

• This species of malaria has a 72 hour cycle of
  paroxysms and is found in both the New and Old
  World.
• Recrudescence has been reported up to 53 years
  after the first infection.
• It is the principal cause of malaria episodes as
  a result of blood transfusion.

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Anopheline Mosquitoes

• Anopheline mosquito are the only vector (except
  blood transfusions, and unprotected sex)
• Out of the 380 species of Anopheline mosquito, 60
  can transmit malaria.
• Only female mosquitos are involved as the males
  do not feed on blood.

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Malaria Cycle Three Main (Human) Stages

• Stage I Upon infection by the mosquito, the
  malaria parasites move rapidly into the liver
  (within 30 minutes ),
• and reproduce rapidly (mitosis) for 5 days or
  more, depending on the species ( P. falciparum or
  P. vivax) ,
• Stage II The malaria parasite exits the liver,
  enters the bloodstream, and within minutes invade
  red blood cells, where they grow and divide,
• every 48-72 hours (time differences depend on
  the species) the red blood cells rupture,
• dispersing more parasites along with waste
  products/toxins into the blood stream,
• this step causes fever, chills and anaemia in the
  victim,
• the released parasites then invade other red
  blood cells, beginning the cycle again.
• Stage III Some parasites invade red blood cells
  and develop into sexual forms,
• may be ingested by uninfected biting mosquitoes,
  
• inside the mosquito they mate and begin to
  reproduce,
• the zygotes (plasmodia) make their way to the
  salivary glands of the mosquito, ready to move on
  to another victim when the mosquito takes its
  next blood meal.

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Plasmodia/Mesquito/Human Life Cycle

• 1. Sporozoite (schizonts)/liver,
• 2-5. Mitosis, liver cell lysis,
• 6. Trophozoite (schizonts)/red blood cell,
• 7-11 (mitosis),
• 12. Gametocytes (via meiosis),

• 13,14. Gametes via mitosis (midgut),
• 15,16. Ookinetes, zygotes via conjugation,
• cross the midgut epithilium,
• 17. Oocysts, mitosis,
• 18-20. Cross salivary epithelium.

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Fighting Malaria

• Insecticides (mosquitoes develop resistance
  quickly),
• most effective (DDT) banned,
• Bed nets (often coated with insecticides),
• Post infection drugs, even the best (chloroquine
  and sulfadoxine-pyrimethamine) generally lose
  effectiveness over time.

• New Chinese remedy Qinghaosu plant, used in
  fever remedies for 2,000 years,
• Artemisia annua, known as sweet wormwood or
  Chinese wormwood, grows wild, even in the United
  States.
• Incredibly effective, although long term
  effectiveness and side effects are not known.

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Plasmodium Mosquito Life Cycle
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Receptor Mediated Transfer
Ookinete
Ookinete
Ross Cell Mosquito epithelium
Ross Cell Mosquito epithelium
normal/infection
no infection
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The Experiment

• Try to find a transgene that produces a peptide
  that will effectively block the receptor mediated
  transfer of ookinetes,
• a transgene is an introduced gene.

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Preliminary Work
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Phage Display
T7 phage
recombinant phage express the sequence as part
of the gIII protein.
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Recombinatorial Library
Insert DNA coding for dodecapeptides (XCX8CX)
into T7 genome.
Allow recombinant DNAs to replicate and repackage
themselves.
Run T7 phage over the receptor.
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Procedure

• 109 different phages in the population,
• XCX8CX yields 2010 different possible peptides,
• Mosquitoes were injected with 1011 phages.

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SM1

• PCQRAIFQSICN protein sequence bound both
  salivary gland and midgut epithelia,
• Injection of the peptide into the mosquitos body
  cavity inhibited oocyst formatin by roughly 90.

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SM1 Injected into Mosquitoes
A mouse model system has been established in
order to provide a clinical vertebrate host.
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Delivery

• We cant hope to hand-inject every mosquito in
  the world,
• or, even feed them all SM1 peptide,
• How can we deliver the 12 amino acid peptide into
  the correct cells in mosquitoes?

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Transgenic Construct
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Transformation

• Embryos are injected with the transgene
  construct, and a helper plasmid,
• Surviving embryos are raised and crossed with
  virgin wt mosquitos,
• Subsequent offspring are scored for GFP eyes.

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trans (top)
wt (top)
trans (bottom)
wt (front)
trans (front)
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Southern Blot
till a NotI or BglII site.
Probe 3xP3-EGFP-SV40 and SM1 DNA,
Blot Genomic DNA from different transgenic
individuals
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Northern Blot
Probes SM1 DNA (mt rRNA for control)
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Transgenic SM1 Results
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Other Controls

• wt Phage infection no anti-ookinete function,
• GFP transgene only no anti-ookinete function,
• Transposons ditto,
• Other peptides ditto.

Further SM1 was inserted into 4 different
genomic regions, all had the same
phenotype, This suggests that the anti-ookinete
function wasnt due to a disrupted gene.
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Epitope Tagging

• Fluorescent antibodies are available for a
  portion of a virus protein (HA1)...

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Trangenic
Wt
Midguts
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Does SM1 Stop New Infections?

• Transgenic and wt mosquitoes are fed blood meals
  on malaria infected mice,
• Then are placed with uninfected mice,
• These mice are then assayed for malaria.

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And its Only a Heterozygote

• The authors conjecture that the possibility
  exists that homozygous SM1 expressing mosquitoes
  might display stronger anti-ookinete function.

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But(t)

• SM1 transgenic mosquitoes dont seem to kill all
  plasmodium, thus, the plasmodium may be able to
  mutate and maybe find a way around the
  SM1-blocked receptor,
• SM1 transgenic mosquitoes
• healthy?
• how do you replace natural populations?
• are there unknown environmental consequences of
  the transgene?
• how do you test for this?
• are all of the anopholines similar?
• maybe the receptor differs between species?

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Biotechnology in General
Scenario 1
Scenario 2
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To Know

• Understand the basic life cycle of Plasmodium,
• Understand the Figures and Tables in this
  experiment,
• Understand the general rational of the experiment.

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Friday

• Assigned Questions 6.8, 6.13, 6.18, 6.19, 11.6,
  11.7,
• Chapter 7 (7.1 - 7.5),